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string | predicted_class
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The nutritional goal of preterm infants in postnatal care is to achieve a growth rate that approximates the intrauterine growth and weight gain of a normal fetus of the same gestational age, without producing nutritional deficiencies, metabolic effects, toxicities or exaggerated nutritional supply (15,16).
|
other
| 99.9 |
Comparative analysis of the anthropometric measurements between the different study groups did not reveal significant differences in the variables at discharge from the hospital. Only length was found to be slightly more impaired at discharge in the group of infants receiving mixed feeding compared to those exclusively receiving formula. It should be considered, however, that length is difficult to measure with accuracy in neonates.
|
study
| 100.0 |
A study conducted in the United States from 1996 to 1998 on infants with a gestational age at birth of less than 33 weeks analyzed the growth and development to 1 year of corrected age of babies fed human milk or a milk formula. Growth was found to be inversely proportional to the consumption of human milk. However, assessment of neurological development revealed that infants fed maternal milk showed a better performance (17).
|
study
| 99.8 |
In a review published in 2014, British investigators who analyzed 9 trials comparing the risks and benefits of feeding preterm low birth weight babies with maternal milk from donors or with infant milk formula observed greater weight gain, length, and head circumference in the group of infants receiving formula during hospitalization. However, the risk of occurrence of necrotizing enterocolitis was higher in this group of infants (4).
|
review
| 99.9 |
Other studies did not observe greater weight and length gain among premature babies receiving a formula, with results similar to those detected in the present study. A review study conducted on 400 preterm babies with a gestational age ≤30 weeks observed a lower prevalence of necrotizing enterocolitis and retinopathy of prematurity, with no significant differences in weight gain in infants fed maternal milk compared to infants receiving a formula (21). Cristofalo et al. (22) studied 1979 premature infants with a mean gestational age of 27 weeks and observed no significant differences in weight-height gain or head circumference growth between infants fed maternal milk and infants receiving a formula.
|
review
| 99.9 |
A recently published study of newborns with less than 30 weeks of gestation and with birth weight <1250 g who were followed up for 7 years revealed that preterm infants predominantly breastfed during the neonatal period showed higher scores in tests for the evaluation of neuromotor development than infants receiving milk formulas (23). Another study of very low birth weight preterm infants also observed better cognitive and motor development in children fed maternal milk than in those receiving formulas (24).
|
review
| 98.44 |
Feeding premature babies with human milk, regardless of the weight gain, offers many advantages for the health of these patients. During hospitalization, human milk feeding is related to less occurrence of necrotizing enterocolitis, sepsis and urinary tract infection, decreased gastric pH, increased gastrointestinal motility, accelerated mucosal immunity, improved gut microflora, and decreased mucosal permeability leading to reduced bacterial translocation. The benefits of human milk remain after discharge as they improve indexes of neurodevelopment that persists into adolescence, avoiding obesity, precocious puberty and other problems (25 –28).
|
review
| 99.9 |
Today, despite the benefits reported in all studies, the incidence and duration of the use of maternal milk by preterm babies are usually lower than recommended. Among other factors, the lack of maternal milk use in the diet offered to these babies during hospitalization favors weaning. Common reasons for the lack of use of human milk is the anxiety of the health team regarding the rate of weight gain, although the use of an infant formula in the present study did not show nutritional advantages compared to maternal milk at the time of discharge. Another reason is the long time between birth and the transition to oral feeding, with the need for much stimulation and involvement of the health team to maintain lactation during the period of hospitalization of premature babies (29,30).
|
study
| 63.8 |
In the present study, the anthropometric measurement was made by the nursing team, being subject to error, which was a limitation. In addition, the retrospective nature of the study was another limitation, since the proportion of each type of milk offered during hospitalization for the included patients was not known. It is important to note that even patients who had a complete transition to artificial feeding also received human milk for some period during hospitalization. Therefore, we could not be sure that the group that was exclusively breastfed at the time of discharge received a greater proportion of human milk during hospitalization than the other groups.
|
study
| 100.0 |
The nutritional status of very low birth weight premature infants at discharge from the hospital was not influenced by the type of feeding offered during hospitalization. In view of the countless nutritional and immunological advantages of breast milk regarding neurodevelopment, we suggest that premature babies be exclusively fed maternal milk within neonatal units whenever possible.
|
other
| 99.06 |
Mental illness is an important public health priority . There is a wealth of research evidence suggesting that regular physical activity is beneficial for individuals with mental health disorders yet an additional lifestyle factor that may be important is sedentary behavior. Sedentary behavior can be defined as any waking activity characterised by an energy expenditure ≤ 1.5 metabolic equivalents and a sitting or reclining posture . It is now well established that sedentary behavior is independent of an individual’s physical activity levels in their associations with health . Epidemiological studies have consistently shown that spending excessive time engaged in sedentary behaviors may have a negative impact on several physical health outcomes yet the research examining the relationship between sedentary behavior and mental health outcomes (e.g., depression, anxiety, quality of life) is somewhat sparse with limitations in the assessment and classification of sedentary behavior.
|
review
| 99.9 |
It has been suggested that adults spend between 6–10 hours sitting each day . Recent data has highlighted that for the majority of working adults, time spent sitting during the working day is more likely to contribute to overall time spent sitting than the time spent sitting during leisure time . Examining sitting time during the working week compared to at weekends could provide additional insight into the links with mental health and well-being, particularly in employees. It could be suggested that there may be different effects of weekday and weekend day sitting due to the lack of volition individuals have over their sitting behaviours within the workplace, in comparison to their leisure time. Current proposed mechanisms as to why engaging in prolonged periods of sedentary behavior could lead to poorer mental health include: i) the social withdrawal hypothesis ii) the time displacement hypothesis and iii) the involvement of inflammatory markers . The majority of cross-sectional evidence suggests that watching TV is associated with poorer mental health yet the evidence for other sedentary behaviors, for example computer use, is mixed and it has been argued that the association with mental health is often dependent on the purpose and content of the computer use . Longitudinal evidence has shown that engaging in more TV watching and computer use at baseline predicts a greater risk of depression at follow-up and that depressive symptoms at baseline are predictive of more TV viewing at follow-up . A recent meta-analysis examining sedentary behavior and the risk of depression across 13 cross-sectional and 11 longitudinal studies suggested that different types of sedentary activities may have differing associations with mental health.
|
review
| 99.8 |
It could also be suggested that sedentary behavior influences different types of mental illness in varying degrees, yet the majority of studies focus on depression. In a recent systematic review, Teychenne, Costigan and Parker suggested that a positive relationship may exist between overall sitting time and anxiety yet there was inconsistent evidence for other types of sedentary behavior (e.g., TV viewing, computer use) and the links with the risk of anxiety. As highlighted in this review and previous research, an important identifiable limitation in the research to date is the reliance on self-report items to assess sedentary behavior, often only measuring one type of sedentary behavior, usually TV viewing, which is not considered an accurate measure of total sedentary time. The aim of the study was to examine objectively measured sedentary behavior and mental health and quality of life across both week and weekend days in adults.
|
review
| 99.9 |
Participants provided informed consent, completed a demographic questionnaire and stature and mass were also assessed. Participants were then asked to wear an activPAL on their left thigh for 24 hours/day for seven consecutive days. Participants were given a wear diary, where they were asked to record what time they woke up and when they went to sleep and to record if the activPAL was removed and the reasons for removal. After one week, participants returned the activPAL and the wear diary to the researchers, and completed the Hospital Anxiety and Depression Scale (HADS) and the Short Form-12 questionnaire (SF-12). Sedentary time was calculated as monitor recorded sitting or lying time minus wear diary recorded sleep time. Wear diary sleep time was verified against monitor recorded activity.
|
study
| 100.0 |
The HADS is a 14-item self-report questionnaire used to assess anxiety and depression, which requires participants to recall how they have felt in the previous week. Scores are summed where a score of 0–7 signifies no presence of clinical symptoms; 8–10 indicates mild symptoms; 11–14 indicates moderate symptoms and a score of 15–21 indicates severe symptoms.
|
other
| 99.9 |
The SF-12 is 12-item self-report questionnaire used to assess aspects of overall health-related quality of life. There are eight subscales relating to physical and mental health (general health; physical functioning; emotional role functioning; physical role functioning; bodily pain; mental health; vitality and social functioning). A higher % score on the SF-12 subscales represents a higher level of physical and mental health.
|
other
| 99.9 |
Sedentary behavior was measured using an activPAL mini, an inclinometer-based activity monitor which can directly identify periods of sitting/lying, standing and stepping. The activPAL has been shown to be 32.4% more accurate than actigraphs with 99.1% accuracy for sitting, standing, and slow walking . A minimum of three days of data were required to be included in analysis (including at least one weekend day), which is in accordance with similar studies [24, 25].
|
study
| 100.0 |
Average total daily time spent in sedentary, standing and stepping activity, in addition to total daily step counts and sit to stand transitions, were computed from the activPAL software for 1) weekdays and 2) weekend days. Paired t-tests were used to examine differences in total daily time spent in sedentary, standing and stepping activity, in addition to total daily step counts and sit to stand transitions between weekdays and weekend days. Participants were grouped according to time spent sitting based on the overall group mean. In relation to weekday sedentary behavior (n = 42), 10 participants were in group 1; 14 participants were in group 2 and 18 participants were in group 3. In relation to weekend sedentary behavior (n = 39), 13 participants were in group 1; 12 participants were in group 2 and 14 participants were in group 3. Differences between sitting (Group 1 = <8hrs/day, Group 2 = 8–10 hrs/day, Group 3 = >10hrs/day) and components of the HADS and SF12 were examined using an ANCOVA with a measure of physical activity (step count) included as a covariate. Partial eta-squared (ηp2) effect sizes were used to evaluate the strength of association for between group differences. Values of 0.01–0.03, 0.06–0.09 and >0.14 indicate a small, medium and large effect, respectively .
|
study
| 100.0 |
The mean hours spent in sedentary, standing and stepping activity, in addition to total daily step counts and sit to stand transitions during weekdays and weekend days, are reported in Table 1. Paired t-tests revealed that average sedentary time on weekdays was higher (p ≤ 0.05) than weekend days. Time spent standing was higher (p ≤ 0.05) at weekends than weekdays. Time spent stepping was similar between weekdays and weekend, however step count was lower (p ≤ 0.05) on weekend days. The number of sit to stand transitions was higher (p ≤ 0.05) during weekdays than on weekend days.
|
study
| 100.0 |
The mean (SD) values for anxiety and depression (HADS) and the eight subscales of the SF-12 are shown in Table 2. There was a main effect for weekday sitting time on anxiety (F(1, 41) = 3.05, p = 0.040, ƞ2 = 0.18), depression (F(1, 41) = 2.91, p = 0.047, ƞ2 = 0.16), mental health (F(1, 41) = 3.50, p = 0.025, ƞ2 = 0.17) and vitality (F(1, 41) = 3.70, p = 0.020, ƞ2 = 0.22). Planned contrasts identified individuals in group 1 had lower anxiety and depression (Fig 1) and higher mental health and vitality scores (Fig 2) than individuals in groups 2 or 3 (p ≤ 0.05). No differences were found between individuals in group 2 and group 3 (p ≤ 0.05). No main effects were found for weekend sitting (p ≥ 0.05) across any of the mental health outcome variables.
|
study
| 100.0 |
The aim was to examine objectively measured sedentary behavior and mental health and quality of life across both week and weekend days in adults. There were significant differences in sedentary behavior across weekdays compared to weekends. Participants spent more time sitting and less time standing during the week compared to the weekend yet this was accompanied by a significantly greater step count. The increase in time spent sitting during weekdays could be attributed to occupation-related behavior within the sample as all of the participants worked at least 30 hours during the week in variable occupations. Our findings contradict those reported in the Health Survey England where adults self-reported spending 4.8 hours being sedentary on weekdays and 5.3 hours at weekends. Yet it should be noted that the higher values of sedentary behavior reported in the current study could be due to the use of an objective method to assess sedentary behavior more accurately. Our findings are similar to those reported in a study by Thorp et al. . They assessed sedentary behavior objectively, comparing work days and non-work days, and found that on work days, participants spent 10.7 hours in sedentary activities yet 8.6 hours in sedentary activities on non-work days. These findings, along with those in the current study, provide support that the workplace could be a key setting for sedentary behavior and should be targeted for appropriate public health intervention strategies.
|
study
| 99.94 |
Understanding the association between varying levels of sedentary behavior on mental health outcomes was also examined. Findings indicated that those participants who engaged in less than 8 hours of sedentary behavior per day on weekdays had significantly lower levels of anxiety and depression compared with those who engaged in greater than 8 hours per day. Findings also indicated a large effect of sitting less than 8 hours per day on lower levels of anxiety and depression. These findings support conclusions drawn from systematic reviews and a meta-analysis examining the links between anxiety, depression and sedentary behavior. In support of the current findings, data from the meta-analysis indicated there was a significant association between sedentary behavior and risk of depression yet only three of the twenty studies included assessed sedentary behavior using an objective measure. Our findings add to the limited research examining the association between anxiety and sedentary behavior, contributing to only one known study that used an objective measure of sedentary behavior.
|
review
| 99.8 |
In relation to aspects of health-related quality of life, findings indicated that those participants who engaged in less than 8 hours of sedentary behavior per day on weekdays had significantly higher levels of vitality and mental health compared with those who engaged in greater than 8 hours per day. Findings also indicated a large effect of sitting less than 8 hours per day on higher levels of vitality and mental health. There is limited research examining the links between aspects of health-related quality of life and sedentary behavior yet our findings support a prospective cohort study conducted with older adults . They found significant increases in levels of vitality across decreased sitting time yet the measure of sedentary behavior was self-report and focused on sitting time during leisure activities as opposed to the waking day as in the current study. Understanding how and why sedentary behavior could influence mental well-being (e.g., depression, anxiety and vitality) is an area of research that is lacking and needs further attention.
|
study
| 99.94 |
A strength of the current study is the use of an objective measure to assess sedentary behavior across both weekdays and weekends and relate to a range of mental health outcomes in an adult population. Our findings highlighted that there were weekday and weekend differences in sitting time and that those participants who spent less than 8 hours per day sitting, were less depressed, anxious and had higher levels of vitality than those who spent over 8 hours sitting. Yet it is important to note that due to the cross-sectional nature of the study, it could be argued that the participants who were more depressed, anxious and had lower levels of vitality could be more prone to spending greater amounts of time sitting. Furthermore, this association was only evident during the weekdays. Contextual information regarding the type of sedentary activities participated in during measured sitting time and where sitting behavior occurred was not obtained. This is an identifiable limitation of the study, along with the cross-sectional nature of the data, making it difficult to infer cause and effect. This would have allowed us to further explore the workplace as a potential important context for sedentary behavior and mental health. Whilst our findings suggest a large positive effect for sitting less than 8 hours per day on improved mental well-being, we acknowledge a requirement for further research with larger groups of participants in order to translate our findings into meaningful recommendations. Our data indicates that reducing sedentary behavior during the working day could be important for improving employees’ mental health and quality of life. Therefore, encouraging workplace intervention studies to include mental health and quality of life outcome measures is warranted to explore the resultant effect of reducing sedentary behavior during the working day.
|
study
| 99.94 |
In recent years, total thyroidectomy has become increasingly popular in the treatment of bilateral multinodular non-toxic goiter (MNG), replacing subtotal thyroidectomy in many high-volume endocrine surgery units worldwide [1, 2]. Nevertheless, the major benefits of total thyroidectomy understood as the risk of recurrent goiter reduced to almost zero and abolished need for revision thyroid surgery in the future should be balanced against the risk of postoperative morbidity. Thus, total thyroidectomy for benign thyroid disease continues to remain controversial, as there are many conflicting data published in the literature regarding the risk of hypoparathyroidism and recurrent laryngeal nerve injury stratified to indications for surgery, extent of thyroid resection and surgical volume [2–5]. Cirocchi et al. published recently a Cochrane systematic review focused on total and near-total thyroidectomy versus subtotal thyroidectomy for MNG in adults and identified only four randomized controlled trials (RCT) in the field. Despite the fact that goiter recurrence was found to be reduced following total thyroidectomy (TT), the effects on other major endpoints, such as the need for revision surgery for goiter recurrence, prevalence of adverse events, and thyroid cancer incidence, remained unclear. Hence, new RCTs with a long-term follow-up and with additional focus on data such as surgical experience, surgical volume, and more attention to surgical technique were found to be needed . To fulfill this gap in evidence with more data, it was decided at our institution to continue follow-up of all consenting patients previously included into RCT for 5 years . Thus, the aim of this study was to validate in a 10-year follow-up the outcomes of RCT run at our institution and published in 2010 comparing results of various thyroid resection modes hitherto assessed within 5 years following surgery . The hypothesis explored at the present study was that the prevalence of recurrent goiter and need for revision thyroidectomy would increase with time of follow-up and that the cumulative risk of postoperative and post-revision morbidity of more limited thyroid resection modes would overweight the initial risk of total thyroidectomy.
|
study
| 99.94 |
Patients referred to the Department of Endocrine Surgery, Third Department of General Surgery, Jagiellonian University Medical College in Krakow, for first-time thyroid surgery between January 2000 and December 2003 were registered. Eligible patients with MNG were assessed for the study. The study was approved by the institutional review board.
|
study
| 99.94 |
The exclusion criteria included: MNG involving the posterior aspect/s of thyroid lobe/s, suspicion of thyroid cancer, previous thyroid surgery, thyroiditis, subclinical or clinically overt hypothyroidism or hyperthyroidism, pregnancy or lactation, age < 18 years or > 65 years, ASA 4 grade (American Society of Anesthesiology), and inability to comply with the follow-up protocol.
|
other
| 96.75 |
The randomization sequence was generated by a computer. Sequencing was based on permuted blocks of two and three to balance the number of patients in the treatment groups. The patients were allocated randomly to one of the three treatment groups in a 1:1:1 ratio. Information on the type of intervention remained in consecutively numbered and sealed envelopes that were stored in the operating theater. An envelope containing the allocation was added to the patient’s file in the operating room. The envelope was opened, and the surgeon performed the assigned intervention. All the participants were blinded to treatment assignment for the duration of the study.
|
other
| 99.9 |
Operations in both groups were performed under general anesthesia. Two anesthesiologists involved in the study followed a strict protocol including premedication with IV midazolam and anesthesia induction with fentanyl, thiopental, and pancuronium at the body mass-dependent dose. After the endotracheal intubation, all the patients were put on mechanical ventilation (sevoflurane and oxygen mixture).
|
other
| 52.25 |
All the operations were performed by one of the three experienced endocrine surgeons involved in the study. Each of them performed approximately one-third of the operations in each study arm. In the TT group, the operation consisted of extracapsular total thyroidectomy, and in the DO group, the operation consisted of unilateral extracapsular total thyroidectomy and contralateral subtotal thyroid lobe resection (leaving a thyroid stump of approximately 2 g of normal remnant tissue), whereas in the ST group, the operation consisted of bilateral subtotal thyroidectomy (leaving thyroid stumps of approximately 2 g of normal remnant tissue each on both sides of the neck). In each group, efforts were made to identify and remove the entire pyramidal thyroid lobe. In each patient, the recurrent laryngeal nerves were exposed and the branches of the superior and inferior thyroid arteries were divided close to the thyroid capsule (peripheral ligation). Intraoperative nerve monitoring (IONM) was not used for initial surgery in this study. However, IONM was utilized for 15 of 20 (75%) revision operations for recurrent goiter in this study, depending on individual surgical preferences. A lateral approach was routinely used for all reoperative cases. The surgical technique of reoperation for recurrent goiter was described in detail in our previous publication . The parathyroid glands were meticulously dissected from the thyroid gland, and effort was made to identify all the four parathyroid glands and preserve as many as possible “in situ.” Any inadvertently removed parathyroid gland found on inspection to be lying on the thyroid capsule, any gland that was anatomically impossible to be preserved, as well as any devascularized gland were electively reimplanted into the sternocleidomastoid muscle using the standard technique of parathyroid autotransplantation as described by Wells et al. .
|
study
| 99.94 |
High-resolution Doppler ultrasound of the neck with both 7.5- and 12-MHz linear array transducers (Logiq 7; GE, Solingen, Germany) was performed during an outpatient visit prior to admission by a single endocrine surgeon (MB) experienced in thyroid ultrasound imaging. Thyroid volumes were calculated according to the spherical ellipsoid formula: volume = π/6 × anteroposterior diameter (cm) × width (cm) × length (cm). Fine-needle aspiration (FNA) was performed in all the patients prior to enrollment. Preoperative evaluation included serum free T3, free T4, thyroid-stimulating hormone (TSH) concentrations (respectively by commercial radioimmunoassay kits and ultrasensitive method) and serum thyroid peroxidase antibodies (TPOAb) levels.
|
study
| 99.94 |
All the patients underwent ultrasonographic, cytological, and biochemical follow-up for 60 months postoperatively. However, for all the consenting patients, the follow-up period was extended to 120 months postoperatively. All the patients were evaluated at 3, 6, 9, and 12 months postoperatively during the first year, and every 12 months for the following years. Biochemical evaluation consisted of determining serum TSH concentrations. Thyroid ultrasonography was performed by the same operator (MB) using the same equipment as in preoperative evaluation (Logiq 7; GE, Solingen, Germany). All the patients in this study, irrespectively of the individual group assignment, received postoperative levothyroxine treatment. The levothyroxine dose was adjusted to serum TSH concentrations to keep it within the lowest two-thirds of the reference range (0.3–2.5 mU/L). This therapeutic strategy was focused on avoiding the risk of mild thyrotoxicosis and limiting the excessive TSH stimulation of the thyroid remnants (in the DO and BST groups).
|
study
| 100.0 |
The following criteria were used to define recurrence of nodular lesions within the remnant thyroid tissue (the same as previously reported in the Miccoli study): presence of hypoechoic or hyperechoic nodular pattern at least 5 mm in diameter, identification of perinodular hypoechogenic or hyperechogenic halo, and presence of an anechoic lesion with a reinforced posterior wall . FNA was electively performed during the follow-up period in all the cases of identified thyroid lesions larger than 1 cm in diameter within the remnant thyroid tissue. The following indications for reoperation were used: presence of a 3 cm, or larger nodule, result of FNA suggestive of an increased risk for malignancy, and presence of compressive symptoms. Indirect laryngoscopy by an ENT specialist was mandatory before surgery and before discharge. In patients with RLN paresis, an additional examination was scheduled at 2 weeks and 1, 2, 4, 6, and 12 months after surgery, or until the vocal cord function recovered. Vocal cord paresis for more than 12 months after the operation was regarded as permanent palsy.
|
study
| 99.7 |
The patients were monitored for postoperative biochemical hypocalcemia at 12, 24, 48, and 72 h postoperatively (during hospitalization and after discharge on morning outpatient visits), with hypocalcemia being defined as a total serum calcium level lower than 2.0 mmol/L, in either asymptomatic or symptomatic patients. Persistent hypocalcemia for more than 6 months after the operation was regarded as permanent hypoparathyroidism.
|
study
| 93.75 |
The sample size was estimated based on the principle of detecting a 5% difference in the prevalence of recurrent goiter with a 90% probability at p < 0.05. The univariate relation between patient characteristics and the development of goiter recurrence and the need for reoperation were examined. The statistical significance of categorical variables was evaluated by the χ 2 test and F test, whereas the Student’s t-test was used to evaluate continuous variables. Ten-year recurrence-free survival was calculated using the Kaplan–Meier method, with the log rank test for comparison between study groups. All the data were entered onto a dedicated spreadsheet (Microsoft Excel 2010; Microsoft Corporation, San Jose, CA, USA) by a medical assistant and then analyzed by a statistician (MedCalc, version 16, Belgium). p < 0.05 was considered to indicate significance.
|
study
| 100.0 |
Three thousands one hundred and thirty-three patients were referred to the Department of Endocrine Surgery, Third Chair of General Surgery, Jagiellonian University College of Medicine in Krakow, Poland, for first-time thyroid surgery between January 2000 and December 2003. Of this group, 694 patients were eligible for this study, while 94 patients refused to participate. Finally, 600 patients who signed the informed consent were randomized to three groups equal in size (n = 200): TT, DO, and BST. Thirty patients were lost to follow-up at 5 years, leaving 570 who were followed for at least 60 months: 191 in the TT group, 189 in the DO group, and 190 in the BST group. Further 44 patients were lost to follow-up at 10 years, leaving 526 who were followed for at least 120 months: 177 in the TT group, 174 in the DO group, and 175 in the BST group (Fig. 1). Demographic characteristics of patients in this study are shown in Table 1. In this study, indications for initial surgery for MNG were: presence of compressive symptoms in a patient with a thyroid nodule/s of 3 cm or larger (n = 412), indeterminate result of FNA suggestive of an increased risk for malignancy (n = 91), discrepancy between the suspicious ultrasound phenotype of the dominant thyroid lesion/s, and the FNA result suggestive for non-diagnostic or benign lesion (n = 45), particularly in cases of a cold thyroid scanning, multiple bilateral thyroid lesions not responding to TSH suppressive treatment with l-thyroxine (n = 49), and on patient’s request for cosmetic indications (n = 3). All indications were equally distributed between the three study arms.Fig. 1Flow diagram of the study Table 1Demographic characteristics of 526 patients who completed the 10-year follow-upTT n = 177DO n = 174BST n = 175Sex ratioa (M:F)15:16218:15615:160Mean ageb (years)45.9 ± 13.947.0 ± 15.347.9 ± 15.2Preoperative TSHb (mIU/L)1.87 ± 0.841.82 ± 0.811.80 ± 0.91Preoperative thyroid volume (assessed by ultrasound)a (mL)75.8 ± 38.477.8 ± 39.578.8 ± 39.9 TT total thyroidectomy, DO Dunhill operation, BST bilateral subtotal thyroidectomy a χ 2 test b t-test; statistically nonsignificant differences for all values
|
study
| 99.94 |
Recurrent goiter was found at 10 years in 1 (0.6%) TT versus 15 (8.6%) DO versus 39 (22.4%) BST patients (p < 0.001 for TT vs. DO or BST and for DO vs. BST), and revision thyroidectomy was necessary in 1 (0.6%) TT versus 5 (2.8%) DO versus 14 (8.0%) BST subjects (p < 0.001 for TT vs. BST and p = 0.019 for DO vs. BST). Detailed data are shown in Table 2. Recurrence-free survival (RFS) for the cohort of 600 patients treated for multinodular non-toxic goiter by total thyroidectomy (TT), Dunhill operation (DO), and bilateral subtotal thyroidectomy (BST) is shown in Fig. 2. TT significantly decreased the risk of recurrence when compared to BST: HR 0.795 (0.643–0.982), p < 0.001 at 10 years (log rank test).Table 2Prevalence of recurrent goiter and need for revision thyroidectomy for 526 patients who completed the 10-year follow-upTTPTT versus DODOPDO versus BSTBSTPTT versus BSTLost to follow-up at 10 years23 (11.5)0.29626 (13.0)0.32925 (12.5)0.343Recurrent goiter [no (%)]1 (0.6) <0.001 15 (8.6) <0.001 39 (22.3) <0.001 Need for revision thyroidectomy [no (%)]1 (0.6)0.0705 (2.9) 0.019 14 (8.0) <0.001 Bold values are statistically significant (p < 0.05) TT total thyroidectomy, DO Dunhill operation, BST bilateral subtotal thyroidectomy χ 2 test for all Fig. 2Recurrence free survival (RFS) for the cohort of 600 patients treated for multinodular non-toxic goiter by total thyroidectomy (TT), Dunhill operation (DO), and bilateral subtotal thyroidectomy (BST). TT significantly decreased the risk of recurrence when compared to BST: HR 0.795 (0.643–0.982), p < 0.001 at 10 years (log rank test)
|
study
| 99.94 |
Recurrence free survival (RFS) for the cohort of 600 patients treated for multinodular non-toxic goiter by total thyroidectomy (TT), Dunhill operation (DO), and bilateral subtotal thyroidectomy (BST). TT significantly decreased the risk of recurrence when compared to BST: HR 0.795 (0.643–0.982), p < 0.001 at 10 years (log rank test)
|
study
| 99.94 |
Cumulative permanent postoperative hypoparathyroidism after initial surgery and revision thyroidectomy was present in 1 (0.6%) TT versus 2 (1.1%) DO versus 5 (2.9%) BST patients (nonsignificant differences), whereas the cumulative permanent recurrent laryngeal nerve injury was found in 4 (1.1%) TT versus 5 (1.4%) DO versus 5 (1.4%) BST nerves at risk (nonsignificant differences). Detailed data are shown in Table 3. The primary outcomes were twice as inferior at 10 years when compared to 5-year results for DO and BST, but not for TT . No significant differences were observed between the three surgeons involved in this study and the outcomes of surgery they performed.Table 3Complications after initial versus revision thyroidectomy and cumulative risk of morbidity among 526 patients who completed the 10-year follow-upTTPTT versus DODOPDO versus BSTBSTPTT versus BST Risk of initial thyroid surgery (n = 600) a n 200200200Hypoparathyroidism [no (%)] Total22 (11.5) 0.007 8 (4.2)0.2414 (2.1) <0.001 Transient21 (11.0) 0.012 8 (4.2)0.2414 (2.1) <0.001 Permanent1 (0.5)0.3160 (0)1.0000 (0)0.316Recurrent laryngeal nerve injury [no (%)] Total25 (6.5)0.35219 (5.0)0.08810 (2.6) 0.009 Temporary21 (5.5)0.39916 (4.2)0.0978 (2.1) 0.014 Permanent4 (1.0)0.7043 (0.8)0.6532 (0.5)0.412Hemorrhage [no (%)]0 (0)0.3161 (0.5)0.5622 (1.0)0.156 Risk of revision thyroidectomy (n = 20) b n 1514Hypoparathyroidism [no (%)] Total0 (0)1.0003 (60.0)1.00010 (71.4)0.333 Transient0 (0)1.0001 (10.0)1.0005 (35.7)1.000 Permanent0 (0)1.0002 (40.0)1.0005 (35.7)1.000Recurrent laryngeal nerve injury [no (%)] Total0 (0)1.0003 (60.0)0.4115 (17.8)1.000 Temporary0 (0)1.0001 (20.0)1.0002 (7.1)1.000 Permanent0 (0)1.0002 (40.0)0.5923 (10.7)1.000Hemorrhage [no (%)]0 (0)1.0000 (0)1.0001 (7.1)1.000 Cumulative risk of initial and revision thyroidectomy (n = 526) a Permanent hypoparathyroidism [no (%)]1 (0.6)0.5552 (1.1)0.2525 (2.9)0.097Permanent recurrent laryngeal nerve injury [no (%)]4 (1.1)0.7175 (1.4)1.0005 (1.4)0.724Hemorrhage [no (%)]1 (0.6)1.0001 (0.6)0.3173 (1.7)0.309Bold values are statistically significant (p < 0.05) TT total thyroidectomy, DO Dunhill operation, BST bilateral subtotal thyroidectomy calculation for nerves at risk, not for patients a χ 2 test b F test
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| 99.94 |
Total thyroidectomy was proposed as the definitive treatment for MNG in order to reduce the risk of goiter recurrence to almost zero [11, 12]. The evidence regarding the balance between the effectiveness and safety of TT compared with more limited thyroid resection modes, such as DO or BST for MNG, is conflicting and no consensus has been reached [3, 6, 13].
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review
| 99.9 |
In the present study, the prevalence of recurrent goiter and need for future revision thyroidectomy during 10-year follow-up were significantly reduced after TT as compared to DO and BST (Table 2). Prevalence of goiter recurrence tended to increase with time of follow-up (Fig. 2), and goiter recurrence noted at 5 years (8.2%) was almost doubled at 10 years (15.5%) following the initial surgery being more limited than TT (p = 0.002) . In addition, the need for revision thyroidectomy for goiter recurrence in procedures more limited than TT was more than sixfold higher during 10-year follow-up (5.4%) as compared to values noted during 5-year follow-up (0.8%), whereas it remained almost abolished following TT (0.6%) during the entire study period (p < 0.001) .
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| 100.0 |
On the other hand, the risk of temporary, but not permanent RLN injury, as well as transient, but not permanent hypoparathyroidism, was significantly higher following initial TT as compared to less than total thyroid resections. However, the risk of initial TT did not overweight the cumulative risk of postoperative and post-revision permanent morbidity of more limited thyroid resection modes (Table 3).
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| 99.94 |
Much of the debate regarding the extent of surgical resection in MNG was inspired by the previously reported higher prevalence of permanent morbidity following TT [14–16]. However, more recent data indicate the safety of TT for benign thyroid disease if surgery is performed by high-volume surgeons [2, 5, 7, 11, 12, 17–20], which is also supported by the outcomes of the present study. Patients after procedures more limited than TT are at an increased risk of goiter recurrence and need lifelong surveillance . Subclinical ultrasound-detectable lesions within the thyroid stumps following subtotal thyroidectomy are much more frequent than previously thought, approaching 50% of patients undergoing lifelong follow-up . Some of these ultrasound findings were even the reasons for surgical malpractice claims . However, clinically overt recurrent MNG occurs in up to one-third of patients with ultrasound-detectable lesions, with a peak incidence between 10 and 20 years following the initial procedures more limited than TT . The longer the follow-up period, the higher the prevalence of recurrent goiter. Despite the fact that indications for reoperation for goiter recurrence are rare, the redo surgery may be necessary in approximately 5% of patients following initial subtotal thyroidectomy . Similar observations were made in the present study. However, Delbridge et al. reported much higher values and showed that subtotal thyroidectomy for MNG resulted in reoperation for recurrent disease in up to 20% of patients, reaching a top incidence 13 years following the primary surgery. According to the 2015 ATA management guidelines, surgery may be considered for growing nodules that are benign after repeat FNA if they are large (> 4 cm), causing compressive or structural symptoms, or the indications may be based upon clinical concerns (Recommendation 27 A) . Moreover, patients with indeterminate nodules who have bilateral nodular disease, those with significant medical comorbidities, or those who prefer to undergo bilateral thyroidectomy to avoid the possibility of requiring a future surgery on the contralateral lobe, may undergo total or near-total thyroidectomy, assuming completion thyroidectomy would be recommended if the indeterminate nodule proved malignant following lobectomy (Recommendation 20 B) . In addition, indications for completion thyroidectomy can be expected in one-third of patients with incidentally diagnosed thyroid cancer following BST . As a result, at least one in ten patients following a procedure more limited than TT may require revision thyroidectomy in the future. It has been repeatedly shown that the risk of repeat thyroid surgery is up to 20-fold higher as compared to the risk of initial TT [3, 4, 23]. Identification and preservation of vital anatomic structures, such as recurrent laryngeal nerves and parathyroid glands, may be compromised during dissection of the scar tissues, leading to a markedly increased prevalence of permanent morbidity following thyroid reoperations. In the present study, the risk of initial TT did not overweight the cumulative risk of postoperative and post-revision permanent morbidity of more limited thyroid resection modes. This evidence is in favor of initial TT for treatment of benign MNG. Nevertheless, recurrent goiter was described even after TT, the condition that is extremely rare (0.3%), and was usually caused by an inadequate resection of embryologic remnant thyroid tissue along the thyrothymic ligament or pyramidal tract . Hence, thyroidectomy with much attention paid to identify and remove the entire thyroid gland along its embryologic descent should effectively eliminate benign goiter recurrence following TT .
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| 80.8 |
The major strength of this study is that it is an RCT with a long-term follow-up of 10 years that allowed for an objective estimate of the treatment effects of utilizing different thyroid resection modes for patients with MNG. On the other hand, the study protocol has several limitations that should be taken into consideration. First, patients included into this study were recruited from the southern part of the Polish territory that historically has been classified as an iodine-deficient area and endemic goiter area according to the International Council for Control of Iodine Deficiency (ICCIDD) criteria. Hence, primary outcomes of this study may not be universally translated into other cohorts of patients with MNG, particularly living in iodine-sufficient areas of the world. However, we believe that in iodine-deficient areas both iodine prophylaxis and levothyroxine treatment are needed for efficient long-term prevention of goiter recurrence following other than total thyroidectomy . In addition, all the operations in this study were performed by high-volume thyroid surgeons performing more than 200 thyroid operations annually each. Hence, the prevalence of surgical morbidity reported in this study cohort may not be achievable for low-volume thyroid surgeons. Adam et al. reported recently that 81% of all thyroid surgeries in the USA were undertaken by low-volume surgeons and 51% of surgeons performed only 1 case per year. Thus, more limited thyroid resections for benign thyroid disease may still be a safe and effective alternative for low-volume surgeons, whereas TT should be reserved for high-volume thyroid surgeons. Last but not least, the novel surgical adjuncts having a potential for improving functional outcomes of thyroidectomy, such as IONM of the laryngeal nerves or near-infrared fluorescence technique for parathyroid imaging, were not utilized in this study for initial surgery. However, one can expect that the rising popularity of these adjuncts in the future can make safe TT at experienced hands even safer [25, 26]. Encouraging data were recently published by Wojtczak et al. who found that experience with IONM led to an increase in RLN identification, a decrease of RLN injury, and an increase in the safe utilization of TT in non-monitored thyroid operations. Thus, if surgery is chosen as definitive treatment for MNG and the permanent complication rate can be kept below 2%, TT should be recommended, as it prevents recurrent nodular goiter and eliminates the risk of reoperation in the future.
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| 91.5 |
Total thyroidectomy performed by a high-volume thyroid surgeon can be regarded as the procedure of choice for patients with MNG, as it entails a significantly lower prevalence of recurrent goiter and need for revision thyroidectomy than other more limited thyroid resections. In addition, the risk of initial TT does not overweight the cumulative risk of postoperative and post-revision permanent morbidity of more limited thyroid resection modalities.
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other
| 99.9 |
Gram-positive bacteria, including the common wound-infecting Staphylococcus aureus, the virulent food-borne pathogen Listeria monocytogenes, and Streptococcus pneumoniae that causes pneumonia, utilize cholesterol-dependent cytolysins (CDCs) to attack and kill mammalian and human cells (Gilbert, 2005; Tweten, 2005). The bacteria produce and release CDCs as water-soluble monomers that attach to cholesterol-containing cell membranes, where they assemble into large, 200–500 Å cytolytic pores (Olofsson et al., 1993). X-ray structures of pneumolysin (PLY) from S. pneumoniae (Lawrence et al., 2015; Marshall et al., 2015; van Pee et al., 2016) show that the soluble toxin monomers are roughly rod-shaped and consist of four domains (D1-D4). Sequence comparison suggests that all CDCs have the same domain structure and therefore insert into target membranes in essentially the same way (Tweten et al., 2001). Although numerous biochemical studies have addressed the arrangement of monomers in the CDC prepore or pore complex (Hotze and Tweten, 2012; Tilley et al., 2005) and how they might penetrate the lipid bilayer (Shatursky et al., 1999), the detailed structure of the pore complex, and hence the exact mechanism of membrane insertion is unknown. The size heterogeneity of CDC prepores and pores has so far precluded structure determination at high resolution. We determined the structure of the ring-shaped ~2.2 MDa PLY pore complex by cryoEM. Together with our 2.4 Å x-ray structure of soluble PLY (van Pee et al., 2016) and a map of the prepore complex obtained by electron cryo-tomography, we can now describe the entire process of membrane attachment, prepore and pore formation in near-atomic detail. Given the high degree of sequence conservation amongst CDCs, it is likely that the same mechanism of membrane insertion holds for related bacterial toxins.
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| 99.94 |
Ring-shaped pore complexes of wildtype PLY (PLYWT) forming on unilamellar, cholesterol-containing liposomes were solubilized with detergent. The size and homogeneity of solubilized PLY pores is detergent-dependent, as indicated by negative-stain and cryoEM (Figure 1—figure supplement 1). Pore diameters varied from 310 to 500 Å in DDM or 350 to 400 Å in Cymal-6. Replacing the detergent Cymal-6 by amphipol A8-35 (Althoff et al., 2011; Lu et al., 2014) resulted in a stable population that was sufficiently homogenous for single-particle cryoEM. 2D class averages indicated a roughly even distribution of views (Figure 1—figure supplement 2A). Pore complexes were rings of 42 subunits with an aggregate molecular mass of 2.2 MDa. In total, 6461 ring images were combined to generate a 3D map with 42-fold symmetry at 4.5 Å resolution (Figure 1; Figure 1—figure supplements 2B and 3,). The final map of the pore complex has an outer diameter of 400 Å and a total height of 110 Å, of which ~80 Å protrudes from the membrane surface (Figure 1).10.7554/eLife.23644.002Figure 1.Overall structure of the PLY pore complex.(A) Single-particle cryoEM map of PLY at 4.5 Å resolution with fitted model. The four PLY domains (D1–D4) are red (D1), yellow (D2), green/cyan (D3) and blue (D4). Inset: refolded β-hairpins (HP1 and HP2) fitted to the map. Cyan β-strands have refolded from helix bundles in D3 of the soluble form. (B) Cross-section with overall dimensions of the pore complex. The grey bar indicates the position of the lipid bilayer. Inset: side view of membrane-inserted monomer with toroid density of disordered amphipol (broken line).DOI: http://dx.doi.org/10.7554/eLife.23644.00210.7554/eLife.23644.003Figure 1—figure supplement 1.Negative stain and cryoEM of PLY solubilized in DDM, Cymal-6 and Amphipol.PLY rings solubilized with DDM vary in size and tend to orient on the specimen support or the air/water interface. Rings solubilized with Cymal-6 are more homogenous in size but tend to stack in pairs. Exchange of detergent against Amphipol A8-35 results in uniform, randomly oriented, single PLY rings. Scale bar: 50 nm.DOI: http://dx.doi.org/10.7554/eLife.23644.00310.7554/eLife.23644.004Figure 1—figure supplement 2.Image processing of PLY rings.(A) Sample micrograph of amphipol-solubilized PLY rings picked for single-particle processing (red boxes). Scale bar: 50 nm. Class averages after 2D classification are shown on the right. (B) FSC curve for phase-randomized, masked map (red curve) indicates 4.5 Å resolution by the FSC0.143 criterion (red line). The sharp peak at 4.8 Å, reflecting the repeat distance between the 168 β-strands in the β-barrel, and a steep drop beyond that, indicating a resolution of 4.5 Å at the 0.143 theshold for the masked FSC, and of 4.6 Å at the 0.3 threshold for the map-vs-model FSC. The two values are in excellent agreement.DOI: http://dx.doi.org/10.7554/eLife.23644.00410.7554/eLife.23644.005Figure 1—figure supplement 3.Local resolution estimate of the PLY monomers and of the complete pore complex.(A) Local resolution map of one PLY monomer from the final density map of the pore complex. The monomer is rotated in 90° steps around the y-axis. (B) Local resolution map of the PLY pore complex.DOI: http://dx.doi.org/10.7554/eLife.23644.005
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| 100.0 |
(A) Single-particle cryoEM map of PLY at 4.5 Å resolution with fitted model. The four PLY domains (D1–D4) are red (D1), yellow (D2), green/cyan (D3) and blue (D4). Inset: refolded β-hairpins (HP1 and HP2) fitted to the map. Cyan β-strands have refolded from helix bundles in D3 of the soluble form. (B) Cross-section with overall dimensions of the pore complex. The grey bar indicates the position of the lipid bilayer. Inset: side view of membrane-inserted monomer with toroid density of disordered amphipol (broken line).
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| 100.0 |
10.7554/eLife.23644.003Figure 1—figure supplement 1.Negative stain and cryoEM of PLY solubilized in DDM, Cymal-6 and Amphipol.PLY rings solubilized with DDM vary in size and tend to orient on the specimen support or the air/water interface. Rings solubilized with Cymal-6 are more homogenous in size but tend to stack in pairs. Exchange of detergent against Amphipol A8-35 results in uniform, randomly oriented, single PLY rings. Scale bar: 50 nm.DOI: http://dx.doi.org/10.7554/eLife.23644.003
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| 99.94 |
PLY rings solubilized with DDM vary in size and tend to orient on the specimen support or the air/water interface. Rings solubilized with Cymal-6 are more homogenous in size but tend to stack in pairs. Exchange of detergent against Amphipol A8-35 results in uniform, randomly oriented, single PLY rings. Scale bar: 50 nm.
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other
| 77.3 |
10.7554/eLife.23644.004Figure 1—figure supplement 2.Image processing of PLY rings.(A) Sample micrograph of amphipol-solubilized PLY rings picked for single-particle processing (red boxes). Scale bar: 50 nm. Class averages after 2D classification are shown on the right. (B) FSC curve for phase-randomized, masked map (red curve) indicates 4.5 Å resolution by the FSC0.143 criterion (red line). The sharp peak at 4.8 Å, reflecting the repeat distance between the 168 β-strands in the β-barrel, and a steep drop beyond that, indicating a resolution of 4.5 Å at the 0.143 theshold for the masked FSC, and of 4.6 Å at the 0.3 threshold for the map-vs-model FSC. The two values are in excellent agreement.DOI: http://dx.doi.org/10.7554/eLife.23644.004
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| 100.0 |
(A) Sample micrograph of amphipol-solubilized PLY rings picked for single-particle processing (red boxes). Scale bar: 50 nm. Class averages after 2D classification are shown on the right. (B) FSC curve for phase-randomized, masked map (red curve) indicates 4.5 Å resolution by the FSC0.143 criterion (red line). The sharp peak at 4.8 Å, reflecting the repeat distance between the 168 β-strands in the β-barrel, and a steep drop beyond that, indicating a resolution of 4.5 Å at the 0.143 theshold for the masked FSC, and of 4.6 Å at the 0.3 threshold for the map-vs-model FSC. The two values are in excellent agreement.
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| 100.0 |
10.7554/eLife.23644.005Figure 1—figure supplement 3.Local resolution estimate of the PLY monomers and of the complete pore complex.(A) Local resolution map of one PLY monomer from the final density map of the pore complex. The monomer is rotated in 90° steps around the y-axis. (B) Local resolution map of the PLY pore complex.DOI: http://dx.doi.org/10.7554/eLife.23644.005
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| 99.94 |
Where possible, individual domains of the PLY x-ray structure (van Pee et al., 2016) were moved manually into the 4.5 Å cryoEM map as rigid bodies. Rebuilding of domains that refolded upon membrane insertion and readjustment of secondary structure elements and sidechains within domains yielded an atomic model of the pore complex (Figure 1). Comparison to the soluble form (Figure 2A,B) revealed a complete reorganization of the toxin upon membrane insertion. Of the four protein domains D1-D4 in the x-ray structure, D1 and D4 fitted the map of the pore complex with minimal modifications (Figure 2—figure supplements 1–3, Video 1). In the membrane-inserted form, the loop linking D1 to D2 refolds into a helix (α3a) at the interface between the rearranged domains D1, D2 and D3 (Figures 2A,E, 3 and 4; Figure 2—figure supplement 4). In D4, the highly conserved undecapeptide loop (Figure 3) that renders PLY cholesterol-specific (Soltani et al., 2007b) was shifted by up to ~9 Å into the cryoEM map (Figure 2—figure supplements 2 and 3, Video 1). The undecapeptide loops of adjacent protomers in the pore complex are located in one plane on the outer membrane surface of the target cell, where they interact closely with the lipid head groups (Figures 1B and 2A,C). In the linear rows of soluble PLY monomers that are found in the PLY crystal structures (Lawrence et al., 2015; Marshall et al., 2015; van Pee et al., 2016), the distance between the loops of neighbouring monomers is ~14 Å. In the pore complex this distance decreases to 4–5 Å, which enables an interaction of the loop that connects β-strands 18 and 19 (β18/19) in D4 with the β22/23 loop in D4 of the next monomer along the ring (Figure 2—figure supplements 2 and 3, Video 1). The close proximity of loop β18/19 that contains Asp403, Thr405, and His407 of one monomer to loop β18/19 and the uncedapeptide containing Trp433 of the adjacent monomer suggests a critical role of these D4 loops not only in receptor recognition, but also in oligomer formation.10.7554/eLife.23644.006Figure 2.Soluble and membrane-inserted PLY monomer.One subunit of membrane-inserted form of PLY in the cryoEM structure (A) and x-ray structure of soluble PLY (van Pee et al., 2016) (B) seen from the side (left) and from the pore center (right). Both helix bundles (HB1 and HB2, cyan) in the PLY monomer refold to form two long, membrane-spanning β-hairpins (HP1 and HP2). The upper end of the β-hairpins is sandwiched between a helix-turn-helix motif (HTH, green) on the inside and helix α3a (red) of the next monomer on the outside of the pore. One of the helices is a refolded β-strand of D3 (green). Helix α3a is the refolded linker that connects D2 to D1 in the soluble monomer. (C) D4 contains the conserved undecapetide (green sticks) that confers cholesterol specificity to PLY. The peptide includes three resolved Trp sidechains, one of which interacts with Thr405 in the adjacent monomer. (D) Cross section through a segment of the 168-strand β−barrel with resolved bulky sidechains. The two β-hairpins of one monomer are labelled. (E) Helix α3a interacts with HP1 and D1 of the neighbouring monomer, stabilizing the pore complex.DOI: http://dx.doi.org/10.7554/eLife.23644.00610.7554/eLife.23644.007Figure 2—figure supplement 1.Stereo view of domain 1 presented as ribbon-and-stick model.Selected large sidechains are shown as sticks. The map is contoured at 7.0 and 9.0 σ.DOI: http://dx.doi.org/10.7554/eLife.23644.00710.7554/eLife.23644.008Figure 2—figure supplement 2.Superposition of domain 4 of the PLY monomer in the crystal structure (blue) and in the pore complex (green), viewed from the pore center.Residues of the undecapeptide and selected residues in other loops are shown as sticks. The map is contoured at 6.5 and 7.5 σ.DOI: http://dx.doi.org/10.7554/eLife.23644.00810.7554/eLife.23644.009Figure 2—figure supplement 3.Stereo view of domain 4 in the pore complex with potential inter- and intramolecular interactions of loops viewed from two sides.The undecapeptide is green and selected residues are shown as sticks. The closest distance between the loops from different monomers is 4–5 Å. The map is contoured at 6.0 and 7.5 σ.DOI: http://dx.doi.org/10.7554/eLife.23644.00910.7554/eLife.23644.010Figure 2—figure supplement 4.Stereo view of domain 3 with the upper part of the β-barrel and the new helix-turn-helix motif (HTH) that forms the α-barrel inside the pore.The new helix α3a (red) from the neighboring monomer is on the right. Hydrophobic residues at the interface between the β-sheet and HTH and intermolecular interactions to the next helix α3a are shown as stick models. The map is contoured at 6.5 and 8 σ.DOI: http://dx.doi.org/10.7554/eLife.23644.01010.7554/eLife.23644.011Figure 2—figure supplement 5.Stereo view of domain 2 seen along the ring axis.The main chain is presented as cartoon and the side chains as sticks. The map is contoured at 7.0 and 9.0 σ.DOI: http://dx.doi.org/10.7554/eLife.23644.01110.7554/eLife.23644.012Figure 3.Sequence alignment of selected cholesterol-dependent cytolysins.Elements of secondary structure are shown for the crystal structure of soluble PLY (above) and for the cyroEM structure of the pore complex (below). Colors of the secondary structure elements and residue numbers correspond to the PLY crystal structure (Figure 2B; pdb code 5aod). Asterisks indicate conserved residues.DOI: http://dx.doi.org/10.7554/eLife.23644.01210.7554/eLife.23644.013Figure 4.Intermolecular interactions in the 4.5 Å cryoEM map of the PLY pore complex.Slices parallel (A–C) and perpendicular (D–F) to the membrane reveal interactions of secondary structure elements between adjacent monomers in the pore complex. Section planes are indicated on top. Note that the scale of sections D to F is 50% larger to show molecular detail more clearly. The β5-α4-β6 region in domain D1 and the long membrane-parallel helix α5 (red box in A and D) alternate around the top of the pore complex. On the outside, the 168-strand β-barrel is flanked by helix α3a (orange box in B and D) and by the α-barrel of the helix-turn-helix motifs (green box in B and F) on the inside. The 4-strand β-sheets of domain D4 (blue box in C and E) are offset against D4 of the next monomer, forming a ring of 8-strand β-sheets. The 85 Å-long β-strands forming the 168-strand β-barrel are clearly resolved (cyan box in C and E).DOI: http://dx.doi.org/10.7554/eLife.23644.013Video 1.Superposition of domain 4 of the PLY monomer from the crystal structure (blue) on domain 4 of the PLY monomer in the pore complex (green) rotated around an axis perpendicular to the membrane.Residues of the undecapeptide and selected residues in other loops are shown as sticks.DOI: http://dx.doi.org/10.7554/eLife.23644.01410.7554/eLife.23644.014
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| 100.0 |
One subunit of membrane-inserted form of PLY in the cryoEM structure (A) and x-ray structure of soluble PLY (van Pee et al., 2016) (B) seen from the side (left) and from the pore center (right). Both helix bundles (HB1 and HB2, cyan) in the PLY monomer refold to form two long, membrane-spanning β-hairpins (HP1 and HP2). The upper end of the β-hairpins is sandwiched between a helix-turn-helix motif (HTH, green) on the inside and helix α3a (red) of the next monomer on the outside of the pore. One of the helices is a refolded β-strand of D3 (green). Helix α3a is the refolded linker that connects D2 to D1 in the soluble monomer. (C) D4 contains the conserved undecapetide (green sticks) that confers cholesterol specificity to PLY. The peptide includes three resolved Trp sidechains, one of which interacts with Thr405 in the adjacent monomer. (D) Cross section through a segment of the 168-strand β−barrel with resolved bulky sidechains. The two β-hairpins of one monomer are labelled. (E) Helix α3a interacts with HP1 and D1 of the neighbouring monomer, stabilizing the pore complex.
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| 100.0 |
10.7554/eLife.23644.008Figure 2—figure supplement 2.Superposition of domain 4 of the PLY monomer in the crystal structure (blue) and in the pore complex (green), viewed from the pore center.Residues of the undecapeptide and selected residues in other loops are shown as sticks. The map is contoured at 6.5 and 7.5 σ.DOI: http://dx.doi.org/10.7554/eLife.23644.008
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| 99.9 |
10.7554/eLife.23644.009Figure 2—figure supplement 3.Stereo view of domain 4 in the pore complex with potential inter- and intramolecular interactions of loops viewed from two sides.The undecapeptide is green and selected residues are shown as sticks. The closest distance between the loops from different monomers is 4–5 Å. The map is contoured at 6.0 and 7.5 σ.DOI: http://dx.doi.org/10.7554/eLife.23644.009
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| 99.94 |
10.7554/eLife.23644.010Figure 2—figure supplement 4.Stereo view of domain 3 with the upper part of the β-barrel and the new helix-turn-helix motif (HTH) that forms the α-barrel inside the pore.The new helix α3a (red) from the neighboring monomer is on the right. Hydrophobic residues at the interface between the β-sheet and HTH and intermolecular interactions to the next helix α3a are shown as stick models. The map is contoured at 6.5 and 8 σ.DOI: http://dx.doi.org/10.7554/eLife.23644.010
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| 99.94 |
Elements of secondary structure are shown for the crystal structure of soluble PLY (above) and for the cyroEM structure of the pore complex (below). Colors of the secondary structure elements and residue numbers correspond to the PLY crystal structure (Figure 2B; pdb code 5aod). Asterisks indicate conserved residues.
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| 99.9 |
Slices parallel (A–C) and perpendicular (D–F) to the membrane reveal interactions of secondary structure elements between adjacent monomers in the pore complex. Section planes are indicated on top. Note that the scale of sections D to F is 50% larger to show molecular detail more clearly. The β5-α4-β6 region in domain D1 and the long membrane-parallel helix α5 (red box in A and D) alternate around the top of the pore complex. On the outside, the 168-strand β-barrel is flanked by helix α3a (orange box in B and D) and by the α-barrel of the helix-turn-helix motifs (green box in B and F) on the inside. The 4-strand β-sheets of domain D4 (blue box in C and E) are offset against D4 of the next monomer, forming a ring of 8-strand β-sheets. The 85 Å-long β-strands forming the 168-strand β-barrel are clearly resolved (cyan box in C and E).
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| 100.0 |
Domains 2 and 3 undergo massive rearrangement and refolding (Figure 2A,B) in the membrane-inserted form. The elongated two-stranded β-sheet of D2 rotates from its vertical position in the x-ray structure by 90° around a short glycine linker to an orientation parallel to the membrane plane in the pore complex. D2 connects D1 to D4, and its rotation results in a roughly linear translation of D1 by 35 Å towards the membrane surface and by 30 Å towards the pore centre. Otherwise, D2 required only minor adjustments of secondary structure for an optimal fit to the cryoEM map (Figures 1B and 2A, Figure 2—figure supplement 5).
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By contrast, the structure of D3 changes entirely upon membrane insertion. In the soluble toxin, the central, five-stranded β-sheet in D3 is flanked by two bundles of short α-helices (Figure 2B). In the pore complex, both bundles refold into four 85 Å β-strands, which form two parallel β-hairpins that insert into and traverse the lipid bilayer (Figure 2A). The β-hairpins of neighbouring subunits coalesce into one extensive, 168-strand β-barrel with an inner diameter of 260 Å (Figure 1B). The new β-strands can be traced unambiguously, because they are straight, apparently rigid and continuous with four β-strands in the D3 x-ray structure that are preserved in the membrane form. The two new, long β-hairpins are inclined by 20° relative to the membrane normal, in good agreement with predictions for perfringolysin O on the basis of cysteine crosslinking experiments (Sato et al., 2013). The chain trace in the refolded domain is confirmed by the positions of bulky densities for large sidechains in the β-barrel (Figure 2D; Figure 2—figure supplement 4). While five of the six helices in bundles HB1 and HB2 refold into β-strands in the pore complex, one remains intact. Conversely, the loop and fifth β-strand in the central β-sheet of D3 refold into a helix (Figure 2A,B). Together, the two α-helices form a helix-turn-helix (HTH) motif (Figure 2—figure supplement 4). In the ring, the HTH motifs of the 42 subunits line up in one plane on the exoplasmic side of the molecule and form an α-barrel inside the β-barrel (Figures 1B and 4), restricting the pore diameter locally to 250 Å (Figure 1B).
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The outside of the membrane-inserted part of the β-barrel is almost entirely hydrophobic (Figure 5A,E) and covered by the toroid density of disordered amphipol that replaces the membrane lipid (Figure 1B), as in the cryoEM structures of amphipol-solubilized membrane proteins (Althoff et al., 2011; Liao et al., 2013). The inside pore surface is highly polar, with three aspartates and two glutamates forming a 15 by 9 Å patch of negative charge, flanked above and below by positive charges (Figure 5B,F,G). Apart from the hydrogen bonds between the 168 β-strands in the barrel and the interactions of helix α3a with D1 and β-hairpin one (Figure 2E, Figure 2—figure supplement 4), three other factors contribute to pore stability: (1) the surface and charge complementarity of the membrane-inserted monomers (Figure 5C); (2) the alternating positive and negative charges of helices α13a and α13b in the α-barrel (Figure 5D); and (3) ionic interactions between charged sidechains in adjacent β-strands of the pore barrel (Figure 5G). In particular, Asp168 and Glu170 in β-strand β7 are in a good position for forming a salt bridge with Lys271 in β-strand β10 of the next-door monomer (Figure 5G).10.7554/eLife.23644.015Figure 5.Charge distribution in the PLY pore complex.(A) Positive (blue) and negative charges (red) are evenly distributed on the polar outer surface of the pore complex. The membrane-inserted region is marked by a band of neutral hydrophobic residues (arrows). (B) The inner surface of the pore complex is highly charged. (C) Contact surfaces of adjacent PLY monomers in the pore complex. (D) Charge complementarity of the two helices in the helix-turn-helix motif forming the internal α-barrel. Positive charges are shown in blue and negative charges in red. (E) The membrane-inserted region of the β-barrel is hydrophobic on the outside and negatively charged on the inside (F). The inset (G) shows map density for the salt bridge between Asp168 of one PLY monomer with Lys271 in the next monomer along the ring. Glutamates and aspartates forming the negatively charged patches on the inner surface of the β-barrel are drawn as sticks. The map is contoured at 5.0 and 6.0 σ.DOI: http://dx.doi.org/10.7554/eLife.23644.015
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(A) Positive (blue) and negative charges (red) are evenly distributed on the polar outer surface of the pore complex. The membrane-inserted region is marked by a band of neutral hydrophobic residues (arrows). (B) The inner surface of the pore complex is highly charged. (C) Contact surfaces of adjacent PLY monomers in the pore complex. (D) Charge complementarity of the two helices in the helix-turn-helix motif forming the internal α-barrel. Positive charges are shown in blue and negative charges in red. (E) The membrane-inserted region of the β-barrel is hydrophobic on the outside and negatively charged on the inside (F). The inset (G) shows map density for the salt bridge between Asp168 of one PLY monomer with Lys271 in the next monomer along the ring. Glutamates and aspartates forming the negatively charged patches on the inner surface of the β-barrel are drawn as sticks. The map is contoured at 5.0 and 6.0 σ.
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To investigate the structures of the PLY prepore and pore complex in native membranes prior to detergent solubilisation, we incubated cholesterol-containing liposomes with purified PLY and generated 3D volumes by electron cryo-tomography (cryoET). The liposomes were studded with numerous ring-shaped complexes of 290 to 360 Å outer diameter (Figure 6A). Assemblies were identified as PLY pores or prepores by the absence or presence of a lipid bilayer within the ring (Figure 7A). Pore and prepore complexes were classified independently and processed by subtomogram averaging (Figure 6B). The subtomogram average map of the prepore complex at a resolution of 22 Å (Figure 6C) had an outer diameter of 320 Å and accommodated 34 subunits (Figure 6D). Of the available PLY crystal structures (Lawrence et al., 2015; Marshall et al., 2015; van Pee et al., 2016), the model 5cr6 (Marshall et al., 2015) fitted the map best (Figure 6—figure supplement 1), indicating that in this structure the soluble toxin is in the pre-pore state. The subtomogram average map of the pore complex at 27 Å resolution (Figure 6C) indicated a slightly larger diameter of around 350 Å, but likewise consisted of 34 subunits (Figure 6E). The cross-section profile resembled that of the high-resolution cryoEM map of the pore complex closely (Figure 1), indicating that solubilisation with detergent or amphipol does not change the structure of the membrane form significantly. The model of membrane-inserted PLY fitted the subtomogram average of the pore complex without significant adjustment, including the 20° inclination of the long β-hairpins relative to the membrane normal.10.7554/eLife.23644.016Figure 6.CryoET of PLY prepores and pores.(A) PLY assembles into prepores and pores upon incubation with cholesterol-containing liposomes. Scale bar, 50 nm. (B) Sections through subtomogram average volumes of the prepore (top) and pore complex (below). Left panel: sections perpendicular to the membrane; right panel: sections parallel to the membrane. Colors indicate section planes. (C) Fourier shell correlation for subtomogram averages indicate 22 Å resolution for the prepore and 27 Å for the pore at FSC0.5 or 20 Å and 21 Å resolution at at FSC0.3. Oblique view (D) and cross section (E) of PLY prepore (left) and pore (right). Both maps accommodate 34 PLY monomers. The prepore map was fitted with the crystal structure of the soluble PLY monomer (Marshall et al., 2015), and the pore map with the cryoEM structure of the pore monomer (Figure 2A). PLY domains are red (D1), yellow (D2), green/cyan (D3) and blue (D4). The lipid bilayer is continuous in the prepore complex, but absent in the pore complex.DOI: http://dx.doi.org/10.7554/eLife.23644.01610.7554/eLife.23644.017Figure 6—figure supplement 1.CryoET map of the PLY prepore with rigid-body fitted x-ray structures of the water-soluble toxin that forms rows in the 3D crystals.PLY monomers of 5AOD (green) are straight and protrude by 10 Å at the top of the map. Monomers of 5CR6 (red) are bent at the hinge between domain 2 and 4 (blue arrow) and match the map volume closely, indicating that this structure represents the pre-pore form of PLY.DOI: http://dx.doi.org/10.7554/eLife.23644.01710.7554/eLife.23644.018Figure 7.Membrane binding and hemolytic activity of PLY .(A) Wildtype PLY (PLYWT) and PLYD168A form rings on cholesterol-containing liposomes. PLYWT lyses the majority of liposomes, while PLYD168Aleaves them mostly intact. Lipid-filled rings with a narrow rim (blue arrows) are prepores, while rings with a wider rim that do not contain lipid (red arrows) are pores. Slits (green arrows) and arcs (yellow arrows) are observed occasionally, but mostly PLY forms complete rings. Mutant PLYD168A prepores detach easily from the liposomes due to reduced binding affinity, and then break into fragments. Curves indicate the hemolytic activity of PLYWT, PLYD168A, and PLYΔ146/147. PLYΔ146/147 is inactive, in line with the inability of this mutant to form oligomers on cholesterol-containing liposomes. Scale bar: 50 nm.DOI: http://dx.doi.org/10.7554/eLife.23644.018
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(A) PLY assembles into prepores and pores upon incubation with cholesterol-containing liposomes. Scale bar, 50 nm. (B) Sections through subtomogram average volumes of the prepore (top) and pore complex (below). Left panel: sections perpendicular to the membrane; right panel: sections parallel to the membrane. Colors indicate section planes. (C) Fourier shell correlation for subtomogram averages indicate 22 Å resolution for the prepore and 27 Å for the pore at FSC0.5 or 20 Å and 21 Å resolution at at FSC0.3. Oblique view (D) and cross section (E) of PLY prepore (left) and pore (right). Both maps accommodate 34 PLY monomers. The prepore map was fitted with the crystal structure of the soluble PLY monomer (Marshall et al., 2015), and the pore map with the cryoEM structure of the pore monomer (Figure 2A). PLY domains are red (D1), yellow (D2), green/cyan (D3) and blue (D4). The lipid bilayer is continuous in the prepore complex, but absent in the pore complex.
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10.7554/eLife.23644.017Figure 6—figure supplement 1.CryoET map of the PLY prepore with rigid-body fitted x-ray structures of the water-soluble toxin that forms rows in the 3D crystals.PLY monomers of 5AOD (green) are straight and protrude by 10 Å at the top of the map. Monomers of 5CR6 (red) are bent at the hinge between domain 2 and 4 (blue arrow) and match the map volume closely, indicating that this structure represents the pre-pore form of PLY.DOI: http://dx.doi.org/10.7554/eLife.23644.017
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PLY monomers of 5AOD (green) are straight and protrude by 10 Å at the top of the map. Monomers of 5CR6 (red) are bent at the hinge between domain 2 and 4 (blue arrow) and match the map volume closely, indicating that this structure represents the pre-pore form of PLY.
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(A) Wildtype PLY (PLYWT) and PLYD168A form rings on cholesterol-containing liposomes. PLYWT lyses the majority of liposomes, while PLYD168Aleaves them mostly intact. Lipid-filled rings with a narrow rim (blue arrows) are prepores, while rings with a wider rim that do not contain lipid (red arrows) are pores. Slits (green arrows) and arcs (yellow arrows) are observed occasionally, but mostly PLY forms complete rings. Mutant PLYD168A prepores detach easily from the liposomes due to reduced binding affinity, and then break into fragments. Curves indicate the hemolytic activity of PLYWT, PLYD168A, and PLYΔ146/147. PLYΔ146/147 is inactive, in line with the inability of this mutant to form oligomers on cholesterol-containing liposomes. Scale bar: 50 nm.
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Atomic force microscopy (AFM) had revealed two different forms of PLY prepores in planar bilayers, rising 110 or 80 Å above the membrane surface (van Pee et al., 2016). The low form has the same height as the pore complex, but the lipid bilayer is still intact. We propose that in this lower prepore, D1, D2 and D4 have moved to their positions in the pore assembly, while D3 has not yet refolded and the long, membrane-spanning β-hairpins have not yet formed. We assign the tall form to an early prepore state, which then rearranges into the lower, late prepore state. The cross-section profile (Figure 6B,D) shows that the early prepore is essentially a ring-shaped side-by-side arrangement of soluble PLY monomers (Figure 6—figure supplement 1), similar to that in the crystal lattice (Lawrence et al., 2015; Marshall et al., 2015; van Pee et al., 2016). While the late prepore was observed for 13% of the rings on planar bilayers examined by AFM, it was not observed in tomographic volumes of PLY on liposomes, implying that it is an intermediate, transient state which inserts more readily into curved liposomes than into planar bilayers. Cross-sections of pore complexes in liposomes (Figure 6B,E) show that the lipid bilayer around the prepores and pores is curved, whereas in the AFM images it was flat (van Pee et al., 2016). Prepore stability and membrane insertion thus seem to be related to membrane curvature, such that pores form preferentially in lipid bilayers with a convex curvature.
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To understand the functional role of individual sidechains in oligomerization, prepore and pore formation, we mutated Asp168 that, as the structure suggests, might be involved in forming a salt bridge between adjacent subunits to alanine (PLYD168A). We also deleted Ala146 and Arg147 (PLYΔ146/147) in the loop that would clash with the last β-strand of the central β-sheet. The resulting mutants were characterized by negative-stain EM (Figure 7A), hemolytic activity assays (Figure 7B), and x-ray crystallography (Figure 8). X-ray data were collected to 2.45 and 2.5 Å resolution from crystals of PLYD168A and PLYΔ146/147 (Table 1). The structures were solved by molecular replacement with the x-ray structure of PLYWT (van Pee et al., 2016). As in PLYWT, the mutant monomers crystallized in rows. Superposition of both structures on PLYWT showed no significant overall differences, although a detailed comparison indicated small changes in domain D2, helix bundle HB2 and in two of the D4 loops (Figure 8). Around the mutated residue in PLYD168A differences were restricted to sidechain orientations, while the deletion of two residues in PLYΔ146/147 caused significant conformational changes in the loop connecting helix α5 to strand β7 of the central β-sheet. This deletion also caused a slight shift of helix α5 towards D3 (Figure 8).10.7554/eLife.23644.019Figure 8.Location of functionally important PLY residues.(A) Ala146 and Arg147 in the loop that induces refolding of the last β-strand in the central D3 β-sheet into helix α13a in the late prepore and pore complex. Deletion of both residues renders the toxin inactive. (B) In the pore complex, Asp168 near the end of one long trans-membrane β-hairpin (HP1) forms a salt bridge with Lys271 in the other trans-membrane β-hairpin (HP2) of the adjacent monomer. Replacing Asp168 by alanine inhibits membrane insertion. (C) α-carbon traces in the x-ray structures of PLYWT (pdb 5aod), PLYΔ146/147 (pdb 5aof), and PLYD168A (pdb 5aoe). Minor differences between wildtype and mutant structures are visible in the loop regions of D4 (blue arrows); D2 (yellow arrows) and HB2 (green arrows). In PLYΔ146/147, one loop connecting D1 to D3 is also slightly different (red arrows).DOI: http://dx.doi.org/10.7554/eLife.23644.01910.7554/eLife.23644.020Table 1.Data collection and refinement statistics.DOI: http://dx.doi.org/10.7554/eLife.23644.020PLYD168A (pdb-id 5aoe)PLYΔ146/147 (pdb-id 5aof)Data collectionBeamlinePXII @ Swiss Light Source x10saResolution (Å)40–2.5 (2.6–2.5)40–2.45 (2.55–2.45)Wavelength (Å)0.97860.978Space groupP21P 21 21 21Cell dimensionsa, b, c (Å)160.86 24.66 208.3524.73 163.5 207.8α, β, γ (°)90, 90.26, 9090, 90, 90Total reflections316531 (30235)137706 (13089)Unique reflections59222 (5685)29722 (2937)Multiplicity5.3 (5.3)4.6 (4.5)Completeness (%)99 (100)91 (94)Mean I / σI8.1 (1.4)9.1 (1.2)Wilson B-factor46.7545.05Rpim0.08 (0.593)0.07 (0.607)CC*0.998 (0.887)0.999 (0.792)RefinementReflections used in refinement59207 (5683)29718 (2936)Reflections in test set2962 (285)1486 (147)Rwork/Rfree (%)22.25/24.98 (32.9/33.7)20.87/23.44 (33.23/34.37)CC(work)/CC(free)0.946/0.934 (0.769/0.737)0.955/0.930 (0.626/0.567)Average B-Factor (Å2)72.564.3No. atoms in AU78784008Protein77243856Water154152r.m.s. deviations:Bond lengths (Å)0.0030.03Bond angles (°)0.720.69Ramachandran favored (%)9696Ramachandran allowed (%)3.63.1Ramachandran outliers (%)0.50.8Note: Values for the highest resolution shell are shown in parentheses.
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(A) Ala146 and Arg147 in the loop that induces refolding of the last β-strand in the central D3 β-sheet into helix α13a in the late prepore and pore complex. Deletion of both residues renders the toxin inactive. (B) In the pore complex, Asp168 near the end of one long trans-membrane β-hairpin (HP1) forms a salt bridge with Lys271 in the other trans-membrane β-hairpin (HP2) of the adjacent monomer. Replacing Asp168 by alanine inhibits membrane insertion. (C) α-carbon traces in the x-ray structures of PLYWT (pdb 5aod), PLYΔ146/147 (pdb 5aof), and PLYD168A (pdb 5aoe). Minor differences between wildtype and mutant structures are visible in the loop regions of D4 (blue arrows); D2 (yellow arrows) and HB2 (green arrows). In PLYΔ146/147, one loop connecting D1 to D3 is also slightly different (red arrows).
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Even though the structures of PLYD168A and PLYΔ146/147 were very similar to PLYWT, they displayed major differences in their membrane-binding and oligomerization behaviour. Like PLYWT, PLYD168A bound to cholesterol-containing liposomes and oligomerized into rings (Figure 7A). However, unlike PLYWT that lysed the liposomes within a short time, liposomes incubated with PLYD168A remained intact for hours. Frequently, the rings detached from the liposomes, indicating they were prepores that had not yet inserted into the membrane. This is also reflected by the hemolytic activity, which was reduced by 80% compared to PLYWT, highlighting the important role of Asp168 in ionic interactions between β-strands that stabilize the pore complex.
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PLYΔ146/147 was not able to bind to the membranes at all and had no detectable hemolytic activity (Figure 7B). Ionic interactions between the β-strands thus contribute significantly to pore stability, and any disruption of these interactions compromises pore formation. Both mutations pinpoint protein regions that are promising targets for drug development. Drugs that interfere with these interactions would render the toxin ineffective, and the bacteria that produce it non-pathogenic.
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With the ongoing resolution revolution in cryoEM (Kühlbrandt, 2014), structures of macromolecular assemblies can now be determined at high resolution without crystals. This is proving especially useful for membrane proteins and membrane protein complexes (Allegretti et al., 2015; Gu et al., 2016; Hahn et al., 2016; Letts et al., 2016; Liao et al., 2013), which tend to be unstable and flexible, and often do not crystallize. In the case of CDC pore complexes, structure determination represents a particular challenge, because they consist of variable numbers of 30 to 50 monomers (Dang et al., 2005; Shatursky et al., 1999; Shepard et al., 1998), and hence are intrinsically inhomogeneous. This fact has so far precluded structure determination of the membrane-attached or membrane-inserted form at high resolution. By a rigorous screen of the detergents used for solubilisation and purification of the pore complex from cholesterol-containing liposomes we were able to isolate a suitably homogenous population. Exchanging the detergent against amphipols appeared to stabilize rings of uniform size, as an important prerequisite for high-resolution single-particle cryoEM. The number of monomers in the amphipol-stabilized complexes was higher than in the prepores or the pores imaged by cryoET, even though the liposomes were prepared in the same way. This means that either the number of subunits in the ring varies from one liposome preparation to another, or that the rings rearrange into a more stable form upon detergent solubilisation, which we consider more likely. Interestingly, an earlier, low-resolution cryoEM structure of PLY pores and prepores in lipid bilayers deduced that there were 31 ± 3 subunits in the prepore and 38 or 44 in the pore complex (Tilley et al., 2005), in good agreement with our findings. Since the PLY pores in our subtomogram averages were smaller, consisting of 34 rather than 42 subunits, it seems that the rings can grow in the membrane by incorporating further subunits. Apparently, complexes of 42 monomers are more stable than both larger and smaller rings. The inherent variability in ring size restricts the number of particles that can be averaged in any one class. Moreover, the large ring-shaped assemblies are easily distorted and rarely, if ever, perfectly circular, which limits the accuracy to which they can be aligned by image processing. Both factors constrain the attainable resolution of the cryoEM reconstruction.
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Earlier cryoEM work, AFM and FRET studies have described the vertical height change of the prepore upon membrane insertion in terms of a collapse or unfolding of D2 (Czajkowsky et al., 2004; Ramachandran et al., 2005; Tilley et al., 2005). Our atomic model of the pore complex now shows that this change is due to a rigid-body rotation of D2, in which the structure of the domain remains intact. Time-lapse AFM of PLY and listeriolysin (LLO) on planar membranes indicated that the vertical height change is not directly linked to pore formation (Mulvihill et al., 2015; van Pee et al., 2016), in line with a mechanism that involves two different prepore states that we refer to as the early and late prepore. Our model of the late prepore monomer (Figure 9) is an intermediate between the x-ray structure of the soluble form and the cryoEM structure of the membrane-inserted form. The 5-stranded central β-sheet in the crystal structure of D3 fits into the cryoET map of the early prepore complex without any modification, but the late prepore model indicates a potential steric clash between the short β-strand in the central β-sheet of D3 and the adjacent monomer. The pivotal role of the loop that contains residues 145–147 connecting helix α5 of D1 to β7 of β-hairpin one is underlined by mutant PLYΔ146/147 that was completely inactive (Kirkham et al., 2006) and did not even bind to liposomes (Figure 7A). The detachment of PLYD168A rings indicates that the mutant does not insert into the membrane as easily as PLYWT to form pores. We have shown by time-lapse AFM that pore formation is irreversible (van Pee et al., 2016). Therefore the rings that become detached from the membrane are prepores, not pores.10.7554/eLife.23644.021Figure 9.Mechanism of membrane insertion and pore formation.Stepwise conformational changes of PLY toxin during pore formation shown for one monomer (above, side view) and for three neighbouring monomers (below, view from the pore centre). In the first step, soluble PLY monomers attach to the surface of cholesterol-containing cell membranes via the conserved D4 undecapeptide (Figure 2C) to form circular oligomers of the early prepore. A 90° rotation of D2 moves D1 and D3 towards the membrane in the late prepore. Helix bundles HB1 and HB2 are poised above the membrane surface to refold into 85 Å β-hairpins HP1 and HP2. In the final step of pore formation, both hairpins traverse the hydrophobic membrane core and assemble into a 168-strand, 260 Å β-barrel. Reorganization of the PLY monomer exposes numerous charges on the inside of the β-barrel (Figure 4) that would destabilize the lipid bilayer and repel membrane lipids, resulting in pore opening and cell lysis.DOI: http://dx.doi.org/10.7554/eLife.23644.021
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Stepwise conformational changes of PLY toxin during pore formation shown for one monomer (above, side view) and for three neighbouring monomers (below, view from the pore centre). In the first step, soluble PLY monomers attach to the surface of cholesterol-containing cell membranes via the conserved D4 undecapeptide (Figure 2C) to form circular oligomers of the early prepore. A 90° rotation of D2 moves D1 and D3 towards the membrane in the late prepore. Helix bundles HB1 and HB2 are poised above the membrane surface to refold into 85 Å β-hairpins HP1 and HP2. In the final step of pore formation, both hairpins traverse the hydrophobic membrane core and assemble into a 168-strand, 260 Å β-barrel. Reorganization of the PLY monomer exposes numerous charges on the inside of the β-barrel (Figure 4) that would destabilize the lipid bilayer and repel membrane lipids, resulting in pore opening and cell lysis.
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In earlier models of pore formation, the conserved domain D4 (Figure 3) was thought to be important for toxin targeting, receptor recognition and binding to cholesterol-containing membranes (Farrand et al., 2010; Ramachandran et al., 2002; Soltani et al., 2007a, 2007b), whereas oligomer formation was mainly attributed to intermolecular interactions via D1 and D3 (Lawrence et al., 2015; Ramachandran et al., 2002). It has however been shown that D4 of streptolysin, pyolysin and LLO can oligomerise by itself on cholesterol crystals (Harris et al., 2011; Weis and Palmer, 2001) or erythrocyte ghosts (Köster et al., 2014). Our cryoEM structure confirms that D4 can indeed play a role in oligomer formation through intra- and intermolecular interactions of its loops (Figure 2—figure supplements 2 and 3). Loop β22/23 and the conserved undecapeptide loop with Trp433 at its tip appear to interact with loop β18/19 of the neighbouring monomer, thereby stabilizing the pore complex by inter- and intramolecular interactions (Figure 2—figure supplements 2 and 3).
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The surface charge distribution on the toxin monomers is critical for CDC activity. Modification of surface charges affects ring formation, as we demonstrated by mutagenesis both for LLO (Köster et al., 2014) and PLY (van Pee et al., 2016). The alternating positive and negative charges of the HTH motif (Figure 5D) and the positive charges at the tips of the β-hairpins (Figure 5E,F) demonstrate the importance of charge complementarity for PLY ring formation. A similar HTH motif was found in the 8 Å cryoEM map of poly-C9 component of the human membrane attack complex (Dudkina et al., 2016). This HTH motif was already present in the soluble monomer of the C6 component (Aleshin et al., 2012) As in PLY, the HTH in the poly-C9 pore forms an α-barrel that determines the effective pore diameter.
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Together with the x-ray structure of the soluble form, our cryoEM structures of the pore and prepore explain the step-by-step process of membrane insertion and pore formation in near-atomic detail. Movies showing the transition from the soluble monomer to the pore complex via the intermediate stages of the early and late prepore illustrate the complete mechanism of pore formation (Videos 2–4). In the early prepore, PLY monomers assemble side by side into rings on the membrane surface. In the late prepore, the D3 helices are poised for re-folding immediately above membrane surface. Helix unfolding may be a stochastic yet cooperative event, such that the spontaneous transition of one monomer triggers the refolding of its neighbours in the ring, comparable to a zipper. As the 168-strand β-barrel assembles in the membrane, the charged patches on its inside surface would repel any trapped hydrophobic lipid. Most likely the lipid is pushed out of the nascent pore by successively inserted toxin monomers, rather than being ejected in the form of micelles or small vesicles, as has been proposed for suilysin (Leung et al., 2014). Upon pore formation, the membrane potential collapses, the cytoplasm leaks out and the cell dies.Video 2.Mechanism of pneumolysin pore formation.(1) Soluble PLY monomers attach to cholesterol-rich membranes by the cholesterol-binding undecapeptide (light green) of domain D4 (blue) and oligomerize into rings. For simplicity, only three ring subunits are shown. (2) Domain D2 (yellow) rotates by 90°, bringing domain D3 (green) with its two helix bundles (cyan) close to the membrane surface. (3) The helix bundles insert into the membrane and unfold into two trans-membrane 85 Å β-hairpins. β-hairpins of the 42 subunits in the ring merge into one large 168-strand β-barrel, which perforates the membrane.DOI: http://dx.doi.org/10.7554/eLife.23644.02210.7554/eLife.23644.022Video 3.Model of conformational changes in an entire ring of 42 PLY monomers from early prepore to late prepore to pore, seen from the side and from above.DOI: http://dx.doi.org/10.7554/eLife.23644.02310.7554/eLife.23644.023Video 4.Oblique view of PLY ring inserting into the membrane.DOI: http://dx.doi.org/10.7554/eLife.23644.02410.7554/eLife.23644.024
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(1) Soluble PLY monomers attach to cholesterol-rich membranes by the cholesterol-binding undecapeptide (light green) of domain D4 (blue) and oligomerize into rings. For simplicity, only three ring subunits are shown. (2) Domain D2 (yellow) rotates by 90°, bringing domain D3 (green) with its two helix bundles (cyan) close to the membrane surface. (3) The helix bundles insert into the membrane and unfold into two trans-membrane 85 Å β-hairpins. β-hairpins of the 42 subunits in the ring merge into one large 168-strand β-barrel, which perforates the membrane.
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The mechanism of membrane insertion by rearrangement of the conserved domains 1–4 is likely to be the same for all CDCs. Therefore, compounds that interfere with refolding and membrane insertion of soluble monomers would prevent infection by Streptococcus pneumonia and other CDC-producing Gram-positive bacteria that attack human cells with similar pore-forming toxins. The 4.5 Å cryoEM structure of the PLY pore complex thus paves the way towards the design of new drugs that inhibit pore formation as a promising approach towards combatting infections by dangerous and wide-spread Gram-positive pathogens.
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The gene coding for N-terminal His6-tagged PLY was inserted into the pET15b vector. E. coli BL21 (DE3) cells transformed with the expression plasmid were grown in TB medium containing 50 µg · ml−1 ampicillin. Protein expression was induced with 1 mM isopropyl-β-D-1-thiogalactopyranoside upon reaching an optical density of one at 600 nm. After 4 hr at 37°C the cells were pelleted, resuspended in Buffer A (50 mM Tris pH 7.0, 150 mM NaCl, 30 mM Imidazole, 5 mM β-mercaptoethanol) and disrupted with a Microfluidizer (M-110L, Microfluidics Corp., Newton, MA). PLY was purified on a HisTrap FF column, equilibrated with Buffer A. The protein was eluted in Buffer A containing 300 mM imidazole. Protein fractions were pooled and diluted in 50 mM Tris pH 7.0, 5 mM β−mercaptoethanol to a final NaCl concentration of 50 mM. The His6-tag was removed by overnight cleavage with thrombin at 4°C. The protein was further purified on a HiTrap Q FF ion-exchange column equilibrated in Buffer B (50 mM Tris-HCl pH 7.0, 50 mM NaCl, 5 mM β-mercaptoethanol). PLY was eluted in Buffer B containing 170 mM NaCl. Protein fractions were pooled, concentrated to 10 mg · ml−1 and stored at −80°C.
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Site-directed mutagenesis was performed with the QuikChange site-directed mutagenesis kit (Stratagene) according to the manufacturer’s instructions with the wildtype construct as a template. All constructs were verified by nucleotide sequencing. The hemolytic activity of PLY was determined by lysis of sheep red blood cells (SRBC) (Darji et al., 1995). Purified protein was serially diluted in hemolysis buffer (50 mM sodium phosphate pH 6.6, 150 mM NaCl, 5 mM DTT, 0.1% (v/v) BSA) in final volumes of 50 µl and incubated for 30 min at 37°C with 50 µl of a suspension of SRBC (I08 ml−1). Release of hemoglobin was monitored by recording the absorbance at 405 nm. The amount of toxin necessary to lyse 50% of erythrocytes was determined and expressed as percentage of the value for PLYWT. The absorbance upon incubation with 1% Triton-X100 was used as reference value for 100% lysis of erythrocytes. Three independent measurements were performed for each PLY mutant.
|
study
| 100.0 |
A lipid mixture containing 70 mol-% di-oleyl phosphatidyl choline (DOPC) and 30 mol-% cholesterol in chloroform was dried under a constant nitrogen stream. The lipid film was taken up in 50 mM Tris-HCl pH 7.0, 150 mM NaCl, 5 mM β-mercaptoethanol at a final concentration of 10 mg · ml−1 and stirred overnight at room temperature. After three freeze-thaw cycles (liquid nitrogen, 37°C), the suspension was passed through an extruder to obtain unilamellar ~200 nm liposomes. The extruded liposomes were flash-frozen in liquid nitrogen and stored at −20°C. The liposome suspension was incubated with PLY at a final lipid-to-protein ratio of 1:2 (wt/wt) at 37°C for 30 min. For the preparation of PLY pore complexes, the proteoliposomes were solubilized at a final concentration of 0.56% Cymal-6 at room temperature overnight. Amphipol A8-35 was added in fivefold molar excess and the suspension was incubated for 30 min at room temperature. Detergent was removed by dialysis against 50 mM Tris-HCl pH 7.0, 150 mM NaCl, 5 mM β-mercaptoethanol at room temperature for 72 hr.
|
study
| 100.0 |
PLY mutant pores were compared to wildtype protein formed on planar lipid layers or cholesterol-containing liposomes prepared as above. The liposome mixture was diluted to a final concentration of 0.5 mg · ml−1 and 25 µg · ml−1 PLY was added. Planar lipid bilayers and PLY were incubated in Teflon well plates. 25 µg · ml−1 PLY in reaction buffer (150 mM NaCl, 50 mM Tris-HCl pH 7.0, 5 mM β-mercaptoethanol) was transferred into a well and overlaid with a droplet of a 0.5 mg · ml−1 DOPC/cholesterol solution (1:1 molar ratio) in chloroform. Planar lipid layers formed upon chloroform evaporation. Wells were incubated for 30 min at 37°C, after which PLY-containing membranes were transferred to EM grids and stained with 1% (wt/vol) uranyl acetate. Negatively stained specimens were examined in an FEI Tecnai Spirit electron microscope at an acceleration voltage of 120 kV. Images were recorded on a 2 K Gatan CCD camera at a magnification of 25,000x–45,000x and ∼1.0–1.5 µm underfocus.
|
study
| 100.0 |
Amphipol-solubilized PLY pores were diluted to a final concentration of 1 mg · ml−1. A 3 µl aliquot was applied to freshly glow-discharged R2/2 holey carbon grids (Quantifoil Micro Tools, Jena, Germany), blotted for 3 to 4 s at 10°C and 100% humidity in a Vitrobot Mark IV (FEI). Preferential orientation of toxin rings on the air-water interface was overcome by carbon backing. Dose-fractionated 6.0 s movies of 30 frames with an electron dose of 1.02 e- · (Å2 · frame)−1 were recorded after coma-free alignment (Allegretti et al., 2014) with an FEI Polara electron microscope operating at 300 kV with 0.5–3.6 µm underfocus and a specimen pixel size of 1.4 Å on a K2 Summit direct electron detection camera (Gatan, Pleasanton, USA) operating in counting mode with an energy filter slit of 20 eV. Movie frames were corrected for beam-induced motion with Motioncorr (Li et al., 2013) and again with UNBLUR (Grant and Grigorieff, 2015), which also applied a dose-dependent filter. The contrast transfer function of each image was determined using CTFFIND3 (Mindell and Grigorieff, 2003). A total of 12308 particle images were hand-picked from 983 micrographs in RELION 1.3 or 1.4 (Scheres, 2012, 2015) and extracted into 360-pixel boxes. Initial 2D classification indicated that rings with 42-fold symmetry were most common, although rings with different symmetries were also present. For 3D classification in RELION, the pore structure of suilysin (EMD-2983) low-pass filtered to 50 Å was used as a reference. For the next processing step, the unsymmetrized low-resolution PLY map was used as the reference. Subsequent 3D classification yielded three classes, of which two had clear 42-fold symmetry. Rings that were distorted, damaged or of different size were sorted out at the 3D classification stage. A total of 6461 particles from 3D classes of 42-fold symmetry were auto-refined in RELION. A B-factor of −175 Å2 for map sharpening was determined using the modulation transfer function of the K2 Summit detector. The resolution of the processed data after B-factor sharpening was 4.5 Å (FSC0.143), with an estimated orientation accuracy of 0.75°. Local resolution was assessed with RELION 2.0 (Kimanius et al., 2016).
|
study
| 100.0 |
Preformed liposomes were incubated with PLY (1 mg · ml−1) at room temperature for 30 min to obtain prepores, or at 37°C for 30–180 min to obtain pores. For cryoET the liposomes were diluted 1:3 with buffer (150 mM NaCl, 50 mM Tris-HCl pH 7.0, 5 mM β-mercaptoethanol) and then 1:1 with 6 nm gold particles conjugated with protein A (Aurion) as fiducial markers. Samples of 3 µl were applied to glow-discharged Quantifoil EM grids (R2/2, Cu 300 mesh), excess liquid was blotted off with filter paper (Whatman #4) and samples were vitrified by plunge-freezing into liquid ethane (Dubochet et al., 1988). Single-axis tilt series were typically collected from −62° to +62° at 2° increments and 3–4 µm underfocus with a total electron dose of 60–80 e- · Å-2, on a Tecnai Polara electron microscope equipped with a field emisson gun operating at 300 kV (FEI, Hillsboro, OR), a post-column energy filter (GIF Quantum, Gatan) and a K2 summit direct electron detector (Gatan). Images were recorded in counting mode with a pixel size of 0.35 nm. Tilt series were CTF-corrected, binned 2 × 2 and aligned. Tomographic volumes were generated by weighted back projection in IMOD (Kremer et al., 1996).
|
study
| 100.0 |
For particle picking and initial rounds of sub-tomogram averaging (Frangakis and Hegerl, 2001), tomograms were filtered by nonlinear anisotropic diffusion to enhance contrast. Ring-shaped complexes were picked manually in 3dmod. A total of 752 prepore complexes and 2400 pore complexes were picked for averaging. Ring volumes were pre-aligned in one plane in 2 × 2 binned unfiltered tomograms. All prepores had roughly the same size and shape and were averaged in PEET (Nicastro et al., 2006). The average volume was used as an initial reference for symmetry determination by sub-tomogram averaging with IMOD (Kremer et al., 1996). Symmetries of 42-fold or lower were applied to each prepore volume on a trial-and-error basis. Volumes were aligned and averaged in an iterative process, which converged within about 15 iterations on an average volume with clear 34-fold symmetry. The best 70% of the prepore volumes contributed to the final average, in which 34 PLY monomers were resolved in the ring.
|
study
| 100.0 |
Pore complexes were separated into seven classes according to their shape and diameter by principal component analysis and clusterPCA in IMOD (Kremer et al., 1996). The best class with 844 pore complexes was used for further processing as described above. In the final pore complex average, individual subunits were clearly distinguished in one third of the ring, indicating some heterogeneity of ring size and symmetry (Figure 5B). The number of PLY monomers in the pore was determined as 34 from the angular distance between distinct subunits in the average volume and the ring diameter. As for the prepores, all averaging steps were performed in PEET (Nicastro et al., 2006). The resolution at FSC0.5 was 27 Å for the pore and 22 Å for the prepore (Figure 6c). At FSC0.3, the resolution of the pore and prepore complex would be 21 Å and 20 Å, respectively.
|
study
| 100.0 |
Model building was performed in COOT (Emsley and Cowtan, 2004) based on the x-ray structure of the water-soluble PLY monomer (pdb 5a0d; (van Pee et al., 2016). Initially, individual domains in the x-ray structure of soluble PLY were fitted manually into the cryoEM map as rigid bodies using COOT. Refolded or flexible protein regions were re-fitted manually, followed by geometry regularization in COOT. The complete backbone of PLY was traced in the pore complex map. Densities of bulky side chains were observed in well-ordered regions. The transmembrane β-hairpins, which form upon unfolding of HB1 and HB2 in domain 3 were readily fitted to the map density. The pore model with 42 subunits was generated in UCSF Chimera (Pettersen et al., 2004). Sub-tomogram average maps of the PLY prepore were fitted in UCSF Chimera with the x-ray structures of the PLY monomer (pdb 5cr6; [Marshall et al., 2015]). Sub-tomogram averages of the pore complex were fitted with the cryoEM structure of the membrane-inserted form. Figures were drawn with PyMol (Schrödinger, 2015). FSC curves of model versus map were calculated using the EMAN package (Tang et al., 2007).
|
study
| 100.0 |
Intitial crystallization trials were carried out with a protein concentration of 8 mg · ml−1 (PLYD168A) or 10 mg · ml−1 (PLYΔ146/147) after addition of glycerol to a final concentration of 10% in 96-well plates by vapour diffusion. 300 nl of protein solution were mixed with 300 nl of a commercially available crystallization solution (PGA Screen from Molecular Dimensions, JB Screen Classics I from Jena Bioscience, and Classics I Suite from Qiagen) in a pipetting robot (Mosquito, TTP Labtech). Hanging drops were incubated over 100 µl of reservoir solution at 18°C. Initial crystals hits were refined by varying the protein-to-reservoir ratio with a drop volume of 3 µl, incubated over 500 µl reservoirs at 18°C in 24-well plates. PLYD168A crystals grew after one day at 18°C in a 3 µl drop of 1.5 µl protein solution (8 mg · ml-1 in 10% glycerol) and 1.5 µl of reservoir solution (0.1 M sodium cacodylate pH 6.5, 1% PGA-LM, 0.2 M potassium bromide and 0.2 M potassium thiocyanate). PLYΔ146/147 crystals grew in a few days at 18°C in a 3 µl drop of 2.0 µl protein solution (10 mg · ml-1 in 10% glycerol) and 1.0 µl of reservoir solution (0.1 M imidazole pH 6.5 and 1.2 M sodium acetate). Crystals were transferred to reservoir solution as a cryo-protectant and flash-frozen in liquid nitrogen. Data were collected at beamline PXII (Paul Scherrer Institute, Villigen, Switzerland) under a constant stream of cold nitrogen gas (100 K). Data processing, integration and scaling was performed with the XDS package (Kabsch, 1993). Structures of the PLY mutants were solved by molecular replacement with PLYWT (pdb-id 4AOD) as a search model using PHASER (McCoy, 2007) in the CCP4 software package (Collaborative Computational Project, Number 4, 1994). The initial electron density map was improved by cycles of density modification, automatic model building in RESOLVE (Terwilliger, 2004) and refinement by REFMAC (Murshudov et al., 1997). The model was subjected to iterative rounds of rebuilding into 2Fo-Fc and Fo-Fc maps using COOT (Emsley and Cowtan, 2004) and refined with the phenix.refine subroutine in the PHENIX program suite (Zwart et al., 2008). Data collection, refinement, and model statistics are summarized in Table 1. Figures were generated with PyMOL (Schrödinger, 2015).
|
study
| 100.0 |
The cryoEM density map has been deposited in the Electron Microscopy Data Bank under the accession number EMD-4118. The structure coordinates have been deposited in the protein data bank under accession number 5LY6 (PLY pore complex), 5AOE (PLYD168A) and 5AOF (PLYΔ146/147)
|
other
| 99.9 |
In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.
|
other
| 99.94 |
Thank you for submitting your article "CryoEM structures of membrane pore and prepore complex reveal cytolytic mechanism of Pneumolysin" for consideration by eLife. Your article has been reviewed by three peer reviewers, one of whom, Sjors HW Scheres, is a member of our Board of Reviewing Editors, and the evaluation has been overseen by Richard Aldrich as the Senior Editor. The following individuals involved in review of your submission have agreed to reveal their identity: Christos G Savva (Reviewer #2).
|
other
| 99.94 |
The study by van Pee and co-workers describes the cryo-EM structures of the cholesterol dependent cytolysin (CDC) pneumolysin (PLY) in the membrane-inserted and pre-pore states by single particle and tomography respectively. Although the first crystal structure of a CDC (perfringolysin O) in the water soluble state was solved almost 20 years ago, a high-resolution structure of the membrane-inserted form has been elusive due to the inherent structural heterogeneity of these complexes with the best results so far limited to the resolution of ~10-15Å where domains can be approximately identified but the exact mechanics of the transition from soluble to membrane-inserted cannot be seen. Despite the recent developments of direct electron detectors and better software that have pushed the resolution limits of Cryo-EM, this class of pore forming toxins still poses difficulties due to the irregular size and shape of the pores. In this study authors have succeeded in preparing more homogeneous samples by systematically screening various detergents. The end result is the first near-atomic resolution structure of a CDC in the membrane-inserted state, which provides valuable information for the mechanics of pore formation including the large movements of domains 2 and 3, which have not been resolved previously by other studies. In general the manuscript is well written and is an important step for the field of pore forming proteins of the CDC/MACPF family. Therefore, the three reviewers were overall positive about publishing this work, although several important issues were raised that should be addressed in a revised version.
|
review
| 99.9 |
1) The overall resolution of the single-particle reconstruction is limited to 4.5A, whereas the entire paper discusses the resulting atomic model as if this were a atomic-resolution structure. Locally the resolution is probably even worse than 4.5A, as for example the β strands are not well separated in Figure 1A. Overall, too little information is given on the quality of the map and the corresponding atomic model. In fact, at 4.5A, the map alone will probably still leave considerable scope for registry or main chain tracing errors. Therefore, the authors should explicitly discuss these caveats where relevant in the main text. In addition, they should provide the following (supplementary?) information in their revision:
|
review
| 82.9 |
2) At the reported resolution the salt bridge between Asp168 and Lys271 should be interpreted carefully. A close up of the EM densities around these residues should be provided. Although the authors have mutated Asp168 and seen a 20% reduction in hemolytic activity this is not conclusive of a salt-bridge disruption. Did mutation of Lys271 also result in the same phenotype? In addition the mutation seems to affect membrane binding as the authors mention (Figure 7 legend) and the ring attachment from the membranes (subsection “Determinants of lytic activity”). Does this imply that pores are formed but are then released? Previous AFM studies by the authors (van Pee et. Al, Nano Letters, 2016) suggest the process is irreversible. Some clarity and caution interpreting this salt bridge would improve the manuscript.
|
review
| 99.6 |
3) Also related to the limited resolution: interactions of the Trp-rich loop are discussed (Results section) but the electron density map in that region looks poorly resolved. This is a very important part of a CDC and the motif defines the family so another panel is required to show the quality of the electron density map in that region. (A close up stereo showing side-chains would help). There is great interest in seeing what conformation this loop adopts in the prepore and pore states since it is thought to control prepore to pore conversion. In Results paragraph two, the authors state that the undecapeptide is interacting with Thr405. This sentence should be re-worded to provide more detail. What is the nature of this interaction and again is the resolution of this area sufficient to justify this comment? From Figure 2C the loop itself does not seem to be resolved even at the Cα backbone level.
|
other
| 97.94 |
4) A major finding of this work is that domain 2 collapses but does not lose its structure (Results section). Earlier EM studies suggested it does lose its structure but modeling and FRET studies suggested otherwise. More discussion of this controversy is required. Importantly, the electron density of this key region should be shown in a figure to satisfy the reader that the structure has been maintained. (Figure 2E is not convincing enough. A stereo with side-chains focused on D2 only would be easier to evaluate). Some new detailed insights are discussed; namely, the remodeled helix-turn-helix motif and helix alpha3a. Can stereo figures of the electron density with side-chains displayed of these regions be shown?
|
other
| 98.4 |
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