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1.
Acta Crystallogr D Struct Biol ; 75(Pt 10): 861-877, 2019 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-31588918

RESUMO

Diffraction (X-ray, neutron and electron) and electron cryo-microscopy are powerful methods to determine three-dimensional macromolecular structures, which are required to understand biological processes and to develop new therapeutics against diseases. The overall structure-solution workflow is similar for these techniques, but nuances exist because the properties of the reduced experimental data are different. Software tools for structure determination should therefore be tailored for each method. Phenix is a comprehensive software package for macromolecular structure determination that handles data from any of these techniques. Tasks performed with Phenix include data-quality assessment, map improvement, model building, the validation/rebuilding/refinement cycle and deposition. Each tool caters to the type of experimental data. The design of Phenix emphasizes the automation of procedures, where possible, to minimize repetitive and time-consuming manual tasks, while default parameters are chosen to encourage best practice. A graphical user interface provides access to many command-line features of Phenix and streamlines the transition between programs, project tracking and re-running of previous tasks.

2.
J Infect Dis ; 2019 Sep 24.
Artigo em Inglês | MEDLINE | ID: mdl-31549165

RESUMO

BACKGROUND: Annual influenza immunization is recommended for people with chronic obstructive pulmonary disease (COPD) by all major COPD clinical practice guidelines. We sought to determine the seasonal influenza vaccine effectiveness (VE) against laboratory-confirmed influenza-associated hospitalizations among older adults with COPD. METHODS: We conducted a test-negative study of influenza VE in community-dwelling older adults with COPD in Ontario, Canada using health administrative data and respiratory specimens collected from patients tested for influenza during the 2010-11 to 2015-16 influenza seasons. Influenza vaccination was ascertained from physician and pharmacist billing claims. Multivariable logistic regression was used to estimate the adjusted odds ratio of influenza vaccination in people with, compared to those without, laboratory-confirmed influenza. RESULTS: Receipt of seasonal influenza vaccine was associated with an adjusted 22% (95% confidence interval [CI], 15%-27%) reduction in laboratory-confirmed influenza-associated hospitalization. Adjustment for potential misclassification of vaccination status increased this to 43% (95% CI, 35%-52%). Vaccine effectiveness was not found to vary by patient- or influenza-related variables. CONCLUSIONS: During the studied influenza seasons, influenza vaccination was at least modestly effective in reducing laboratory-confirmed influenza-associated hospitalizations in people with COPD. The imperfect effectiveness emphasizes the need for better influenza vaccines and other preventive strategies.

3.
J Clin Oncol ; 37(30): 2795-2804, 2019 Oct 20.
Artigo em Inglês | MEDLINE | ID: mdl-31465264

RESUMO

PURPOSE: Seasonal influenza vaccination is recommended for patients with cancer despite concerns of disease or treatment-associated immunosuppression. The objective of this study was to evaluate vaccine effectiveness (VE) against laboratory-confirmed influenza for patients with cancer. PATIENTS AND METHODS: We conducted an observational test-negative design study of previously diagnosed patients with cancer 18 years of age and older who underwent influenza testing during the 2010-2011 to 2015-2016 influenza seasons in Ontario, Canada. We linked individual-level cancer registry, respiratory virus testing, and health administrative data to identify the study population and outcomes. Vaccination status was determined from physician and pharmacist billing claims. We used multivariable logistic regression to estimate VE, adjusting for age, sex, rurality, income quintile, cancer characteristics, chemotherapy exposure, comorbidities, previous health care use, influenza season, and calendar time. RESULTS: We identified 26,463 patients with cancer who underwent influenza testing, with 4,320 test-positive cases (16%) and 11,783 (45%) vaccinated. Mean age was 70 years, 52% were male, mean time since diagnosis was 6 years, 69% had solid tumor malignancies, and 23% received active chemotherapy. VE against laboratory-confirmed influenza was 21% (95% CI, 15% to 26%), and VE against laboratory-confirmed influenza hospitalization was 20% (95% CI, 13% to 26%). For patients with solid tumor malignancies, VE was 25% (95% CI, 18% to 31%), compared with 8% (95% CI, -5% to 19%) for patients with hematologic malignancies (P = .015). Active chemotherapy usage did not significantly affect VE, especially among patients with solid tumor cancer. CONCLUSION: Our results support recommendations for influenza vaccination for patients with cancer. VE was decreased for patients with hematologic malignancies, and there was no significant difference in VE among patients with solid tumor cancer receiving active chemotherapy. Strategies to optimize influenza prevention among patients with cancer are warranted.

4.
Vaccine ; 37(31): 4392-4400, 2019 Jul 18.
Artigo em Inglês | MEDLINE | ID: mdl-31221563

RESUMO

BACKGROUND: Linking data on laboratory specimens collected during clinical practice with health administrative data permits highly powered vaccine effectiveness (VE) studies to be conducted at relatively low cost, but bias from using convenience samples is a concern. We evaluated the validity of using such data for estimating VE. METHODS: We created the Flu and Other Respiratory Viruses Research (FOREVER) Cohort by linking individual-level data on respiratory virus laboratory tests, hospitalizations, emergency department visits, and physician services. For community-dwelling adults aged > 65 years, we assessed the presence and magnitude of information and selection biases, generated VE estimates under various conditions, and compared our VE estimates with those from other studies. RESULTS: We included 65,648 unique testing episodes obtained from 54,434 individuals during the 2010-11 to 2015-16 influenza seasons. To examine information bias, we found the proportion testing positive for influenza for patients with unknown interval from illness onset to specimen collection was more similar to patients for whom illness onset date was ≤ 7 days before specimen collection than to patients for whom illness onset was > 7 days before specimen collection. To assess the presence of selection bias, we found the likelihood of influenza testing was comparable between vaccinated and unvaccinated individuals, although the adjusted odds ratios were significantly greater than 1 for some healthcare settings and during some influenza seasons. Over 6 seasons, VE estimates ranged between 36% (95%CI, 27-44%) in 2010-11 and 5% (95%CI, -2, 11%) in 2014-15. VE estimates were similar under a range of conditions, but were consistently higher when accounting for misclassification of vaccination status through a quantitative sensitivity analysis. VE estimates from the FOREVER Cohort were comparable to those from other studies. CONCLUSIONS: Routinely collected laboratory and health administrative data contained in the FOREVER Cohort can be used to estimate influenza VE in community-dwelling older adults.

5.
Pediatr Infect Dis J ; 38(4): 362-369, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30882725

RESUMO

BACKGROUND: Respiratory illnesses are a major contributor to pediatric hospitalizations, with influenza and respiratory syncytial virus (RSV) causing substantial morbidity and cost each season. We compared the characteristics and outcomes of children 0-59 months of age who were hospitalized with laboratory-confirmed influenza or RSV between 2009 and 2014 in Ontario, Canada. METHODS: We included hospitalized children who were tested for influenza A, influenza B and RSV and were positive for a single virus. We characterized individuals by their demographics and healthcare utilization patterns and compared their hospital outcomes, in-hospital cost and postdischarge healthcare use by virus type and by presence of underlying comorbidities. RESULTS: We identified and analyzed 7659 hospitalizations during which a specimen tested positive for influenza or RSV. Children with RSV were the youngest whereas children with influenza B were the oldest [median ages 6 months (interquartile range: 2-17 months) and 25 months (interquartile range: 10-45 months), respectively]. Complex chronic conditions were more prevalent among children with all influenza (sub)types than RSV (31%-34% versus 20%). In-hospital outcomes were similar by virus type, but in children with comorbidities, postdischarge outcomes varied. We observed no differences in in-hospital cost between viruses or by presence of comorbidities [overall median cost: $4150 Canadian dollars (interquartile range: $3710-$4948)]. CONCLUSIONS: Influenza and RSV account for large numbers of pediatric hospitalizations. RSV and influenza were similar in terms of severity and cost in hospitalized children. Influenza vaccination should be promoted in pregnant women and young children, and a vaccine against RSV would mitigate the high burden of RSV.

6.
J Struct Biol ; 204(2): 301-312, 2018 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-30107233

RESUMO

We find that the overall quite good methods used in the CryoEM Model Challenge could still benefit greatly from several strategies for improving local conformations. Our assessments primarily use validation criteria from the MolProbity web service. Those criteria include MolProbity's all-atom contact analysis, updated versions of standard conformational validations for protein and RNA, plus two recent additions: first, flags for cis-nonPro and twisted peptides, and second, the CaBLAM system for diagnosing secondary structure, validating Cα backbone, and validating adjacent peptide CO orientations in the context of the Cα trace. In general, automated ab initio building of starting models is quite good at backbone connectivity but often fails at local conformation or sequence register, especially at poorer than 3.5 Šresolution. However, we show that even if criteria (such as Ramachandran or rotamer) are explicitly restrained to improve refinement behavior and overall validation scores, automated optimization of a deposited structure seldom corrects specific misfittings that start in the wrong local minimum, but just hides them. Therefore, local problems should be identified, and as many as possible corrected, before starting refinement. Secondary structures are confusing at 3-4 Šbut can be better recognized at 6-8 Å. In future model challenges, specific steps being tested (such as segmentation) and the required documentation (such as PDB code of starting model) should each be explicitly defined, so competing methods on a given task can be meaningfully compared. Individual local examples are presented here, to understand what local mistakes and corrections look like in 3D, how they probably arise, and what possible improvements to methodology might help avoid them. At these resolutions, both structural biologists and end-users need meaningful estimates of local uncertainty, perhaps through explicit ensembles. Fitting problems can best be diagnosed by validation that spans multiple residues; CaBLAM is such a multi-residue tool, and its effectiveness is demonstrated.

7.
Pediatr Infect Dis J ; 2018 Aug 27.
Artigo em Inglês | MEDLINE | ID: mdl-30153230

RESUMO

BACKGROUND: Respiratory illnesses are a major contributor to pediatric hospitalizations, with influenza and respiratory syncytial virus (RSV) causing substantial morbidity and cost each season. We compared the characteristics and outcomes of children 0-59 months of age who were hospitalized with laboratory-confirmed influenza or RSV between 2009 and 2014 in Ontario, Canada. METHODS: We included hospitalized children who were tested for influenza A, influenza B, and RSV, and were positive for a single virus. We characterized individuals by their demographics and healthcare utilization patterns, and compared their hospital outcomes, in-hospital cost, and post-discharge healthcare use by virus type and by presence of underlying comorbidities. RESULTS: We identified and analyzed 7,659 hospitalizations during which a specimen tested positive for influenza or RSV. Children with RSV were the youngest whereas children with influenza B were the oldest (median ages 6 months [interquartile range {IQR}: 2-17 months] and 25 months [IQR: 10-45 months], respectively). Complex chronic conditions were more prevalent among children with all influenza (sub)types than RSV (31%-34% vs 20%). In-hospital outcomes were similar by virus type, but in children with comorbidities, post-discharge outcomes varied. We observed no differences in in-hospital cost between viruses or by presence of comorbidities (overall median cost: $4,150 CAD [IQR: $3,710-$4,948]). CONCLUSION: Influenza and RSV account for large numbers of pediatric hospitalizations. RSV and influenza were similar in terms of severity and cost in hospitalized children. Influenza vaccination should be promoted in pregnant women and young children, and a vaccine against RSV would mitigate the high burden of RSV.

8.
Acta Crystallogr D Struct Biol ; 74(Pt 2): 132-142, 2018 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-29533239

RESUMO

Traditionally, validation was considered to be a final gatekeeping function, but refinement is smoother and results are better if model validation actively guides corrections throughout structure solution. This shifts emphasis from global to local measures: primarily geometry, conformations and sterics. A fit into the wrong local minimum conformation usually produces outliers in multiple measures. Moving to the right local minimum should be prioritized, rather than small shifts across arbitrary borderlines. Steric criteria work best with all explicit H atoms. `Backrub' motions should be used for side chains and `P-perp' diagnostics to correct ribose puckers. A `water' may actually be an ion, a relic of misfitting or an unmodeled alternate. Beware of wishful thinking in modeling ligands. At high resolution, internally consistent alternate conformations should be modeled and geometry in poor density should not be downweighted. At low resolution, CaBLAM should be used to diagnose protein secondary structure and ERRASER to correct RNA backbone. All atoms should not be forced inside density, beware of sequence misalignment, and very rare conformations such as cis-non-Pro peptides should be avoided. Automation continues to improve, but the crystallographer still must look at each outlier, in the context of density, and correct most of them. For the valid few with unambiguous density and something that is holding them in place, a functional reason should be sought. The expectation is a few outliers, not zero.

9.
N Engl J Med ; 378(4): 345-353, 2018 Jan 25.
Artigo em Inglês | MEDLINE | ID: mdl-29365305

RESUMO

BACKGROUND: Acute myocardial infarction can be triggered by acute respiratory infections. Previous studies have suggested an association between influenza and acute myocardial infarction, but those studies used nonspecific measures of influenza infection or study designs that were susceptible to bias. We evaluated the association between laboratory-confirmed influenza infection and acute myocardial infarction. METHODS: We used the self-controlled case-series design to evaluate the association between laboratory-confirmed influenza infection and hospitalization for acute myocardial infarction. We used various high-specificity laboratory methods to confirm influenza infection in respiratory specimens, and we ascertained hospitalization for acute myocardial infarction from administrative data. We defined the "risk interval" as the first 7 days after respiratory specimen collection and the "control interval" as 1 year before and 1 year after the risk interval. RESULTS: We identified 364 hospitalizations for acute myocardial infarction that occurred within 1 year before and 1 year after a positive test result for influenza. Of these, 20 (20.0 admissions per week) occurred during the risk interval and 344 (3.3 admissions per week) occurred during the control interval. The incidence ratio of an admission for acute myocardial infarction during the risk interval as compared with the control interval was 6.05 (95% confidence interval [CI], 3.86 to 9.50). No increased incidence was observed after day 7. Incidence ratios for acute myocardial infarction within 7 days after detection of influenza B, influenza A, respiratory syncytial virus, and other viruses were 10.11 (95% CI, 4.37 to 23.38), 5.17 (95% CI, 3.02 to 8.84), 3.51 (95% CI, 1.11 to 11.12), and 2.77 (95% CI, 1.23 to 6.24), respectively. CONCLUSIONS: We found a significant association between respiratory infections, especially influenza, and acute myocardial infarction. (Funded by the Canadian Institutes of Health Research and others.).


Assuntos
Hospitalização/estatística & dados numéricos , Influenza Humana/complicações , Infarto do Miocárdio/etiologia , Idoso , Idoso de 80 Anos ou mais , Feminino , Humanos , Incidência , Vírus da Influenza A/isolamento & purificação , Vírus da Influenza B/isolamento & purificação , Influenza Humana/diagnóstico , Masculino , Pessoa de Meia-Idade , Infarto do Miocárdio/epidemiologia , Ontário/epidemiologia , Risco
10.
Protein Sci ; 27(1): 293-315, 2018 01.
Artigo em Inglês | MEDLINE | ID: mdl-29067766

RESUMO

This paper describes the current update on macromolecular model validation services that are provided at the MolProbity website, emphasizing changes and additions since the previous review in 2010. There have been many infrastructure improvements, including rewrite of previous Java utilities to now use existing or newly written Python utilities in the open-source CCTBX portion of the Phenix software system. This improves long-term maintainability and enhances the thorough integration of MolProbity-style validation within Phenix. There is now a complete MolProbity mirror site at http://molprobity.manchester.ac.uk. GitHub serves our open-source code, reference datasets, and the resulting multi-dimensional distributions that define most validation criteria. Coordinate output after Asn/Gln/His "flip" correction is now more idealized, since the post-refinement step has apparently often been skipped in the past. Two distinct sets of heavy-atom-to-hydrogen distances and accompanying van der Waals radii have been researched and improved in accuracy, one for the electron-cloud-center positions suitable for X-ray crystallography and one for nuclear positions. New validations include messages at input about problem-causing format irregularities, updates of Ramachandran and rotamer criteria from the million quality-filtered residues in a new reference dataset, the CaBLAM Cα-CO virtual-angle analysis of backbone and secondary structure for cryoEM or low-resolution X-ray, and flagging of the very rare cis-nonProline and twisted peptides which have recently been greatly overused. Due to wide application of MolProbity validation and corrections by the research community, in Phenix, and at the worldwide Protein Data Bank, newly deposited structures have continued to improve greatly as measured by MolProbity's unique all-atom clashscore.


Assuntos
Bases de Dados de Proteínas , Modelos Moleculares , Linguagens de Programação , Proteínas/química , Proteínas/genética
11.
Acta Crystallogr D Struct Biol ; 73(Pt 10): 852-859, 2017 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-28994414

RESUMO

Hoogsteen base pairs are seen in DNA crystal structures, but only rarely. This study tests whether Hoogsteens or other syn purines are either under-modeled or over-modeled, which are known problems for rare conformations. Candidate purines needing a syn/anti 180° flip were identified by diagnostic patterns of difference electron-density peaks. Manual inspection narrowed 105 flip candidates to 20 convincing cases, all at ≤2.7 Šresolution. Rebuilding and refinement confirmed that 14 of these were authentic purine flips. Seven examples are modeled as Watson-Crick base pairs but should be Hoogsteens (commonest at duplex termini), and three had the opposite issue. Syn/anti flips were also needed for some single-stranded purines. Five of the 20 convincing cases arose from an unmodeled alternate duplex running in the opposite direction. These are in semi-palindromic DNA sequences bound by a homodimeric protein and show flipped-purine-like difference peaks at residues where the palindrome is imperfect. This study documents types of incorrect modeling which are worth avoiding. However, the primary conclusions are that such mistakes are infrequent, the bias towards fitting anti purines is very slight, and the occurrence rate of Hoogsteen base pairs in DNA crystal structures remains unchanged from earlier estimates at ∼0.3%.


Assuntos
DNA/química , Purinas/química , Pareamento de Bases , Ligações de Hidrogênio , Modelos Moleculares , Conformação de Ácido Nucleico , Software
12.
J Mol Biol ; 429(9): 1321-1335, 2017 May 05.
Artigo em Inglês | MEDLINE | ID: mdl-28336403

RESUMO

Vicinal disulfides between sequence-adjacent cysteine residues are very rare and rather startling structural features which play a variety of functional roles. Typically discussed as an isolated curiosity, they have never received a general treatment covering both cis and trans forms. Enabled by the growing database of high-resolution structures, required deposition of diffraction data, and improved methods for discriminating reliable from dubious cases, we identify and describe distinct protein families with reliably genuine examples of cis or trans vicinal disulfides and discuss their conformations, conservation, and functions. No cis-trans interconversions and only one case of catalytic redox function are seen. Some vicinal disulfides are essential to large, functionally coupled motions, whereas most form the centers of tightly packed internal regions. Their most widespread biological role is providing a rigid hydrophobic contact surface under the undecorated side of a sugar or multiring ligand, contributing an important aspect of binding specificity.


Assuntos
Dissulfetos/química , Dissulfetos/metabolismo , Glicosilação , Proteínas/química , Proteínas/metabolismo , Biologia Computacional , Modelos Moleculares , Conformação Proteica
13.
Infect Control Hosp Epidemiol ; 38(1): 61-67, 2017 01.
Artigo em Inglês | MEDLINE | ID: mdl-27821194

RESUMO

OBJECTIVE To measure transmission frequencies and risk factors for household acquisition of community-associated and healthcare-associated (HA-) methicillin-resistant Staphylococcus aureus (MRSA). DESIGN Prospective cohort study from October 4, 2008, through December 3, 2012. SETTING Seven acute care hospitals in or near Toronto, Canada. PARTICIPANTS Total of 99 MRSA-colonized or MRSA-infected case patients and 183 household contacts. METHODS Baseline interviews were conducted, and surveillance cultures were collected monthly for 3 months from household members, pets, and 8 prespecified high-use environmental locations. Isolates underwent pulsed-field gel electrophoresis and staphylococcal cassette chromosome mec typing. RESULTS Overall, of 183 household contacts 89 (49%) were MRSA colonized, with 56 (31%) detected at baseline. MRSA transmission from index case to contacts negative at baseline occurred in 27 (40%) of 68 followed-up households. Strains were identical within households. The transmission risk for HA-MRSA was 39% compared with 40% (P=.95) for community-associated MRSA. HA-MRSA index cases were more likely to be older and not practice infection control measures (P=.002-.03). Household acquisition risk factors included requiring assistance and sharing bath towels (P=.001-.03). Environmental contamination was identified in 78 (79%) of 99 households and was more common in HA-MRSA households. CONCLUSION Household transmission of community-associated and HA-MRSA strains was common and the difference in transmission risk was not statistically significant. Infect Control Hosp Epidemiol 2016;1-7.


Assuntos
Portador Sadio/diagnóstico , Infecções Comunitárias Adquiridas/transmissão , Infecção Hospitalar/transmissão , Características da Família , Staphylococcus aureus Resistente à Meticilina/isolamento & purificação , Infecções Estafilocócicas/diagnóstico , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Técnicas de Tipagem Bacteriana , Canadá , Criança , Pré-Escolar , Eletroforese em Gel de Campo Pulsado , Microbiologia Ambiental , Feminino , Humanos , Lactente , Recém-Nascido , Masculino , Pessoa de Meia-Idade , Estudos Prospectivos , Fatores de Risco , Infecções Estafilocócicas/transmissão , Adulto Jovem
14.
Proteins ; 84(9): 1177-89, 2016 09.
Artigo em Inglês | MEDLINE | ID: mdl-27018641

RESUMO

Here we describe the updated MolProbity rotamer-library distributions derived from an order-of-magnitude larger and more stringently quality-filtered dataset of about 8000 (vs. 500) protein chains, and we explain the resulting changes and improvements to model validation as seen by users. To include only side-chains with satisfactory justification for their given conformation, we added residue-specific filters for electron-density value and model-to-density fit. The combined new protocol retains a million residues of data, while cleaning up false-positive noise in the multi- χ datapoint distributions. It enables unambiguous characterization of conformational clusters nearly 1000-fold less frequent than the most common ones. We describe examples of local interactions that favor these rare conformations, including the role of authentic covalent bond-angle deviations in enabling presumably strained side-chain conformations. Further, along with favored and outlier, an allowed category (0.3-2.0% occurrence in reference data) has been added, analogous to Ramachandran validation categories. The new rotamer distributions are used for current rotamer validation in MolProbity and PHENIX, and for rotamer choice in PHENIX model-building and refinement. The multi-dimensional χ distributions and Top8000 reference dataset are freely available on GitHub. These rotamers are termed "ultimate" because data sampling and quality are now fully adequate for this task, and also because we believe the future of conformational validation should integrate side-chain with backbone criteria. Proteins 2016; 84:1177-1189. © 2016 Wiley Periodicals, Inc.


Assuntos
Algoritmos , Elétrons , Biblioteca de Peptídeos , Proteínas/química , Aminoácidos/química , Bases de Dados de Proteínas , Conjuntos de Dados como Assunto , Conformação Proteica , Proteínas/classificação , Distribuições Estatísticas , Termodinâmica
15.
Methods Enzymol ; 558: 181-212, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26068742

RESUMO

With increasing recognition of the roles RNA molecules and RNA/protein complexes play in an unexpected variety of biological processes, understanding of RNA structure-function relationships is of high current importance. To make clean biological interpretations from three-dimensional structures, it is imperative to have high-quality, accurate RNA crystal structures available, and the community has thoroughly embraced that goal. However, due to the many degrees of freedom inherent in RNA structure (especially for the backbone), it is a significant challenge to succeed in building accurate experimental models for RNA structures. This chapter describes the tools and techniques our research group and our collaborators have developed over the years to help RNA structural biologists both evaluate and achieve better accuracy. Expert analysis of large, high-resolution, quality-conscious RNA datasets provides the fundamental information that enables automated methods for robust and efficient error diagnosis in validating RNA structures at all resolutions. The even more crucial goal of correcting the diagnosed outliers has steadily developed toward highly effective, computationally based techniques. Automation enables solving complex issues in large RNA structures, but cannot circumvent the need for thoughtful examination of local details, and so we also provide some guidance for interpreting and acting on the results of current structure validation for RNA.


Assuntos
Biologia Computacional/estatística & dados numéricos , RNA/química , Software , Biologia Computacional/métodos , Cristalização , Cristalografia por Raios X , Interpretação Estatística de Dados , Humanos , Ligações de Hidrogênio , Modelos Moleculares , Conformação de Ácido Nucleico , Dobramento de RNA , Ribostamicina/química
16.
Structure ; 22(10): 1467-77, 2014 Oct 07.
Artigo em Inglês | MEDLINE | ID: mdl-25295398

RESUMO

The Staphylococcus aureus virulence factor staphylococcal protein A (SpA) is a major contributor to bacterial evasion of the host immune system, through high-affinity binding to host proteins such as antibodies. SpA includes five small three-helix-bundle domains (E-D-A-B-C) separated by conserved flexible linkers. Prior attempts to crystallize individual domains in the absence of a binding partner have apparently been unsuccessful. There have also been no previous structures of tandem domains. Here we report the high-resolution crystal structures of a single C domain, and of two B domains connected by the conserved linker. Both structures exhibit extensive multiscale conformational heterogeneity, which required novel modeling protocols. Comparison of domain structures shows that helix1 orientation is especially heterogeneous, coordinated with changes in side chain conformational networks and contacting protein interfaces. This represents the kind of structural plasticity that could enable SpA to bind multiple partners.


Assuntos
Proteína Estafilocócica A/química , Proteína Estafilocócica A/metabolismo , Staphylococcus aureus/química , Cristalografia por Raios X , Modelos Moleculares , Conformação Proteica , Estrutura Terciária de Proteína
17.
Nucleic Acids Res ; 42(20): 12833-46, 2014 Nov 10.
Artigo em Inglês | MEDLINE | ID: mdl-25326328

RESUMO

The hepatitis delta virus (HDV) ribozyme is a self-cleaving RNA enzyme essential for processing viral transcripts during rolling circle viral replication. The first crystal structure of the cleaved ribozyme was solved in 1998, followed by structures of uncleaved, mutant-inhibited and ion-complexed forms. Recently, methods have been developed that make the task of modeling RNA structure and dynamics significantly easier and more reliable. We have used ERRASER and PHENIX to rebuild and re-refine the cleaved and cis-acting C75U-inhibited structures of the HDV ribozyme. The results correct local conformations and identify alternates for RNA residues, many in functionally important regions, leading to improved R values and model validation statistics for both structures. We compare the rebuilt structures to a higher resolution, trans-acting deoxy-inhibited structure of the ribozyme, and conclude that although both inhibited structures are consistent with the currently accepted hammerhead-like mechanism of cleavage, they do not add direct structural evidence to the biochemical and modeling data. However, the rebuilt structures (PDBs: 4PR6, 4PRF) provide a more robust starting point for research on the dynamics and catalytic mechanism of the HDV ribozyme and demonstrate the power of new techniques to make significant improvements in RNA structures that impact biologically relevant conclusions.


Assuntos
Vírus Delta da Hepatite/enzimologia , RNA Catalítico/química , Pareamento de Bases , Modelos Moleculares , Conformação de Ácido Nucleico , Clivagem do RNA , RNA Catalítico/metabolismo , Ribonucleoproteína Nuclear Pequena U1/química , Ribonucleoproteína Nuclear Pequena U1/metabolismo
18.
Structure ; 22(8): 1184-1195, 2014 Aug 05.
Artigo em Inglês | MEDLINE | ID: mdl-25087509

RESUMO

Staphylococcal protein A (SpA) is a multidomain protein consisting of five globular IgG binding domains separated by a conserved six- to nine-residue flexible linker. We collected SAXS data on the N-terminal protein-binding half of SpA (SpA-N) and constructs consisting of one to five domain modules in order to determine statistical conformation of this important S. aureus virulence factor. We fit the SAXS data to a scattering function based on a new polymer physics model, which provides an analytical description of the SpA-N statistical conformation. We describe a protocol for systematically determining the appropriate level of modeling to fit a SAXS data set based on goodness of fit and whether the addition of parameters improves it. In the case of SpA-N, the analytical polymer physics description provides a depiction of the statistical conformation of a flexible protein that, while lacking atomistic detail, properly reflects the information content of the data.


Assuntos
Modelos Moleculares , Biologia Molecular/métodos , Proteína Estafilocócica A/química , Staphylococcus aureus/química , Sequência de Aminoácidos , Dados de Sequência Molecular , Conformação Proteica , Espalhamento a Baixo Ângulo
19.
Biophys J ; 106(3): 510-25, 2014 Feb 04.
Artigo em Inglês | MEDLINE | ID: mdl-24507592

RESUMO

The United Nations has declared 2014 the International Year of Crystallography, and in commemoration, this review features a selection of 54 notable macromolecular crystal structures that have illuminated the field of biophysics in the 54 years since the first excitement of the myoglobin and hemoglobin structures in 1960. Chronological by publication of the earliest solved structure, each illustrated entry briefly describes key concepts or methods new at the time and key later work leveraged by knowledge of the three-dimensional atomic structure.


Assuntos
Cristalografia por Raios X/métodos , Proteínas/química , Sequência de Aminoácidos , Animais , Humanos , Dados de Sequência Molecular , Conformação Proteica
20.
Curr Opin Struct Biol ; 23(5): 707-14, 2013 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-24064406

RESUMO

Model validation has evolved from a passive final gatekeeping step to an ongoing diagnosis and healing process that enables significant improvement of accuracy. A recent phase of active development was spurred by the worldwide Protein Data Bank requiring data deposition and establishing Validation Task Force committees, by strong growth in high-quality reference data, by new speed and ease of computations, and by an upswing of interest in large molecular machines and structural ensembles. Progress includes automated correction methods, concise and user-friendly validation reports for referees and on the PDB websites, extension of error correction to RNA and error diagnosis to ligands, carbohydrates, and membrane proteins, and a good start on better methods for low resolution and for multiple conformations.


Assuntos
Cristalografia por Raios X , Modelos Moleculares , Bases de Dados de Proteínas , Internet , Proteínas/química , Reprodutibilidade dos Testes
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