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1.  MolProbity’s Ultimate Rotamer-Library Distributions for Model Validation 
Proteins  2016;84(9):1177-1189.
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 sidechains 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 sidechain conformations. Further, along with favored and outlier, an allowed category (0.3% to 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 Mol-Probity 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 sidechain with backbone criteria.
PMCID: PMC4983197  PMID: 27018641
sidechain rotamer library; rare sidechain conformations; structural bioinformatics; structure validation; Phenix; high-quality dataset; protein conformation
2.  A joint effort over a period of time: factors affecting use of urate-lowering therapy for long-term treatment of gout 
Although international guidelines encourage urate lowering therapy (ULT) for people who have more than two attacks of gout, only 30 % of patients are prescribed it and only 40 % of those adhere to the treatment. The aim was to explore reasons for this through an exploration of patient experience and understanding of ULT treatment for gout.
A qualitative study was conducted throughout the United Kingdom. Narrative and semi-structured video-recorded interviews and thematic analysis were used.
Participants talked about their views and experiences of treatment, and the factors that affected their use of ULT. The analysis revealed five main themes: 1) knowledge and understanding of gout and its treatment; 2) resistance to taking medication; 3) uncertainty about when to start ULT; 4) experiences of using ULT; and 5) desire for information and monitoring.
Patients’ understanding and experiences of gout and ULT are complex and it is important for clinicians to be aware of these when working with patients. It is also important for clinicians to know that patients’ perceptions and behaviour are not fixed, but can change over time, with changes to their condition, with dialogue and increased understanding. Patients want this interaction with their clinicians, through “a joint effort over a period of time”.
PMCID: PMC4895958  PMID: 27267878
Gout; Urate lowering therapy (ULT); Patient perceptions; Qualitative; Primary care
3.  The acceptability to patients of PhysioDirect telephone assessment and advice services; a qualitative interview study 
In response to long waiting lists and problems with access to primary care physiotherapy, several Primary Care Trusts (PCTs) (now Clinical Commissioning Groups CCGs) developed physiotherapy-led telephone assessment and treatment services. The Medical Research Council (MRC) funded PhysioDirect trial was a randomised control trial (RCT) in four PCTs, with a total of 2252 patients that compared this approach with usual physiotherapy care. This nested qualitative study aimed to explore the acceptability of the PhysioDirect telephone assessment and advice service to patients with musculoskeletal conditions.
We conducted 57 semi-structured interviews with adults from 4 PCTs who were referred from general practice to physiotherapy with musculoskeletal conditions and were participating in the PhysioDirect trial. The Framework method was used to analyse the qualitative data.
The PhysioDirect service was largely viewed as acceptable although some saw it as a first step to subsequent face-to-face physiotherapy. Most participants found accessing the PhysioDirect service straightforward and smooth, and they valued the faster access to physiotherapy advice offered by the telephone service. Participants generally viewed both the PhysioDirect service and the physiotherapists providing the service as helpful. Participants’ preferences and priorities for treatment defined the acceptable features of PhysioDirect but the acceptable features were traded off against less acceptable features. Some participants felt that the PhysioDirect service was impersonal and impaired the development of a good relationship with their physiotherapist, which made the service feel remote and less valuable.
The PhysioDirect service was broadly acceptable to participants since it provided faster access to physiotherapy advice for their musculoskeletal conditions. Participants felt that it is best placed as one method of accessing physiotherapy services, in addition to, rather than as a replacement for, more traditional face-to-face physiotherapy assessment and treatment.
PMCID: PMC4810506  PMID: 27020840
Physiotherapy; Service delivery; Patient experience; Interview; Qualitative study
4.  “Why me? I don’t fit the mould … I am a freak of nature”: a qualitative study of women’s experience of gout 
BMC Women's Health  2015;15:122.
Gout is more common in men, and is often perceived by both patients and health practitioners to be a disorder of men, but its prevalence in women is increasing. Little is known about women’s experience of gout and the impact it has on their lives. It is important for practitioners to be aware of these areas, given the increasing numbers of women with gout they are likely to see in the future. This study aimed to explore women’s experiences of gout.
A qualitative research design was used. Semi-structured interviews were conducted with 43 people, of whom 14 were women. Interviews were video and/or tape recorded and transcribed verbatim. Data from the interviews was first grouped into broad categories, followed by a more detailed thematic analysis and interpretation.
Participants’ ages ranged from 32 to 82. Nine participants were retired and five were in fulltime work. Four themes emerged: (1) experience of onset, help seeking and diagnosis (2) understanding and finding information about gout, (3) impact on identity, and (4) impact on roles and relationships.
The diagnostic process for women with gout can be uncertain due to lack of awareness of gout in women (by health care professionals and women themselves). Women do not have a good understanding of the condition and find it difficult to find information that feels relevant to them. Gout has a major impact on women’s identity and on their roles and relationships. These findings are of importance to health care professionals dealing with women with potential gout and those with an existing diagnosis.
PMCID: PMC4693432  PMID: 26710971
Gout; Inflammatory arthritis; Qualitative research; Patient experience
5.  Multiscale conformational heterogeneity in the protein-binding domains of staphylococcal protein A: Possible determinant of functional plasticity§ 
Structure (London, England : 1993)  2014;22(10):1467-1477.
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 sidechain conformational networks and contacting protein interfaces. This represents the kind of structural plasticity that could enable SpA to bind multiple partners.
PMCID: PMC4191857  PMID: 25295398
6.  Mapping patients’ experiences from initial symptoms to gout diagnosis: a qualitative exploration 
BMJ Open  2015;5(9):e008323.
To explore patients’ experiences from initial symptoms to receiving a diagnosis of gout.
Data from in-depth semistructured interviews were used to construct themes to describe key features of patients’ experiences of gout diagnosis.
Participants and setting
A maximum variation sample of 43 UK patients with gout (29 men; 14 women; age range 32–87 years) were recruited from general practices, rheumatology clinics, gout support groups and through online advertising.
Severe joint pain, combined with no obvious signs of physical trauma or knowledge of injury, caused confusion for patients attempting to interpret their symptoms. Reasons for delayed consultation included self-diagnosis and/or self-medication, reluctance to seek medical attention, and financial/work pressures. Factors potentially contributing to delayed diagnosis after consultation included reported misdiagnosis, attacks in joints other than the first metatarsophalangeal joint, and female gender. The limitations in using serum uric acid (SUA) levels for diagnostic purposes were not always communicated effectively to patients, and led to uncertainty and lack of confidence in the accuracy of the diagnosis. Resistance to the diagnosis occurred in response to patients’ beliefs about the causes of gout and characteristics of the people likely to be affected. Diagnosis prompted actions, such as changes in diet, and evidence was found of self-monitoring of SUA levels.
This study is the first to report data specifically about patients’ pathways to initial consultation and subsequent experiences of gout diagnosis. A more targeted approach to information provision at diagnosis would improve patients’ experiences.
PMCID: PMC4577947  PMID: 26369796
PRIMARY CARE; QUALITATIVE RESEARCH; Gout; Patient experiences; Diagnosis
7.  “You want to get on with the rest of your life”: a qualitative study of health-related quality of life in gout 
Clinical Rheumatology  2015;35:1197-1205.
The objective of the study is to examine the impact of gout and its treatments on health-related quality of life (HRQOL) using focus group interviews. From the baseline phase of a cohort study of HRQOL in gout, 17 participants (15 males, mean age 71 years) with varying attack frequency and treatment with and without allopurinol participated in one of four focus group interviews. All interviews were audio-recorded and transcribed verbatim. Data was analysed thematically. Physical and psychosocial HRQOL in gout was affected by characteristics of acute gout (particularly the unpredictable nature of attacks, location of joint involved in an attack, pain and modifications in lifestyle), lack of understanding of gout by others (association with unhealthy lifestyle, symptoms ridiculed as non-severe and non-serious) as well as participants (not considered a disease) and the lack of information provided by physicians (about causes and pharmacological as well as non-pharmacological treatments of gout). Participants emphasised the impact of acute attacks of gout and prioritised dietary modifications and treatment of acute attacks over long-term urate-lowering therapy. Characteristics of acute gout, lack of understanding and information about gout and its treatments perpetuate poor HRQOL. HRQOL (maintenance of usual diet and reduced frequency of attacks) was associated with urate-lowering treatment. Better patient, public and practitioner education about gout being a chronic condition associated with co-morbidities and poor HRQOL may improve understanding and long-term treatment of gout.
PMCID: PMC4844632  PMID: 26245722
Focus group interviews; Gout; Health-related quality of life; Primary care; Qualitative study; Thematic analysis
8.  Foot problems in people with gout in primary care: baseline findings from a prospective cohort study 
Foot problems are common in people with gout yet the prevalence of current foot problems in people with gout and the burden they present to healthcare systems is not known. This cross-sectional study aimed to determine the prevalence and associations of hallux valgus, foot pain and disability in people with gout, and to assess the frequency with which foot problems lead to consultation with healthcare professionals.
Adults registered with 20 general practices who had consulted their GP about gout or been prescribed allopurinol or colchicine in the preceding two years were mailed a questionnaire. Prevalence of hallux valgus, foot pain in the last month, and disabling foot pain in the mailed population were ascertained using validated instruments and estimated by inverse-weighted logistic regression. Associations with socio-demographic, comorbid and gout-specific factors were examined using logistic regression. Participants were asked if they had seen health care professionals for foot problems within the preceding 12 months.
One thousand one hundred eighty-four questionnaires were received (response 66 %). Prevalence of hallux valgus was 36.3 %, foot pain in the last month 22.3 % and disabling foot pain 14.5 %. Hallux valgus associated with age (adjusted OR 1.47 per 10-year increase, 95 % CI 1.26, 1.72) and female gender (2.03; 1.31, 3.15). Foot pain in the last month associated with age (1.24; 1.00, 1.55), obesity (BMI 30.0–34.9 2.67; 1.32, 5.38; BMI ≥ 35.0 3.16; 1.44, 6.93), mild depression (2.04; 1.09, 3.81) and polyarticular gout attacks (1.86; 1.18, 2.95). Disabling foot pain associated with age (1.42; 1.08, 1.87), obesity (BMI 30.0–34.9 3.73; 1.54, 9.09; BMI ≥ 35.0 4.36; 1.64, 11.64), depression (mild 2.63; 1.25, 5.53; moderate 3.53; 1.11, 11.26) and ischaemic heart disease (2.45; 1.32, 4.53). In the previous 12 months, 495 (42.8 %) reported consulting their GP about their feet and 281 (23.7 %) a podiatrist/chiropodist.
Foot problems are common in people with gout and frequently lead to healthcare consultation. Hallux valgus has similar associations to those seen in the general population, whereas foot pain associates with obesity and gout characteristics, and disabling foot pain with obesity and comorbidity. Patient assessment should consider foot problems and offer specific treatment where relevant.
PMCID: PMC4512156  PMID: 26207143
Foot; Gout; Pain; Hallux valgus; Epidemiology
9.  New insights into Hoogsteen base pairs in DNA duplexes from a structure-based survey 
Nucleic Acids Research  2015;43(7):3420-3433.
Hoogsteen (HG) base pairs (bps) provide an alternative pairing geometry to Watson–Crick (WC) bps and can play unique functional roles in duplex DNA. Here, we use structural features unique to HG bps (syn purine base, HG hydrogen bonds and constricted C1′–C1′ distance across the bp) to search for HG bps in X-ray structures of DNA duplexes in the Protein Data Bank. The survey identifies 106 A•T and 34 G•C HG bps in DNA duplexes, many of which are undocumented in the literature. It also uncovers HG-like bps with syn purines lacking HG hydrogen bonds or constricted C1′–C1′ distances that are analogous to conformations that have been proposed to populate the WC-to-HG transition pathway. The survey reveals HG preferences similar to those observed for transient HG bps in solution by nuclear magnetic resonance, including stronger preferences for A•T versus G•C bps, TA versus GG steps, and also suggests enrichment at terminal ends with a preference for 5′-purine. HG bps induce small local perturbations in neighboring bps and, surprisingly, a small but significant degree of DNA bending (∼14°) directed toward the major groove. The survey provides insights into the preferences and structural consequences of HG bps in duplex DNA.
PMCID: PMC4402545  PMID: 25813047
10.  New tools provide a second look at HDV ribozyme structure, dynamics and cleavage 
Nucleic Acids Research  2014;42(20):12833-12846.
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.
PMCID: PMC4227795  PMID: 25326328
11.  Modelling the continental-scale spread of Schmallenberg virus in Europe: Approaches and challenges 
Preventive Veterinary Medicine  2014;116(4):404-411.
Following its emergence in northern Europe in 2011 Schmallenberg virus (SBV), a vector-borne disease transmitted by the bites of Culicoides midges, has spread across much of the continent. Here we develop simple models to describe the spread of SBV at a continental scale and, more specifically, within and between NUTS2 regions in Europe. The model for the transmission of SBV between regions suggests that vector dispersal is the principle mechanism for transmission, even at the continental scale. The within-region model indicates that there is substantial heterogeneity amongst regions in the force of infection for cattle and sheep farms. Moreover, there is considerable under-ascertainment of SBV-affected holdings, though the level of under-ascertainment varies between regions. We contrast the relatively simple approach adopted in this study with the more complex continental-scale micro-simulation models which have been developed for pandemic influenza and discuss the strengths, weaknesses and data requirements of both approaches.
PMCID: PMC4204989  PMID: 24630403
Epidemiology; Modelling; SBV; Bayesian methods; Under-ascertainment
12.  Crystallographic Model Validation: from Diagnosis to Healing 
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.
PMCID: PMC3805755  PMID: 24064406
13.  Recommendations of the wwPDB NMR Validation Task Force 
Structure (London, England : 1993)  2013;21(9):10.1016/j.str.2013.07.021.
As methods for analysis of biomolecular structure and dynamics using nuclear magnetic resonance spectroscopy (NMR) continue to advance, the resulting 3D structures, chemical shifts, and other NMR data are broadly impacting biology, chemistry, and medicine. Structure model assessment is a critical area of NMR methods development, and is an essential component of the process of making these structures accessible and useful to the wider scientific community. For these reasons, the Worldwide Protein Data Bank (wwPDB) has convened an NMR Validation Task Force (NMR-VTF) to work with the wwPDB partners in developing metrics and policies for biomolecular NMR data harvesting, structure representation, and structure quality assessment. This paper summarizes the recommendations of the NMR-VTF, and lays the groundwork for future work in developing standards and metrics for biomolecular NMR structure quality assessment.
PMCID: PMC3884077  PMID: 24010715
14.  Extending the authority for sickness certification beyond the medical profession: the importance of ‘boundary work’ 
BMC Family Practice  2014;15:100.
The study aimed to explore the views of general practitioners (GPs), nurses and physiotherapists towards extending the role of sickness certification beyond the medical profession in primary care.
Fifteen GPs, seven nurses and six physiotherapists were selected to achieve varied respondent characteristics including sex, geographical location, service duration and post-graduate specialist training. Constant-comparative qualitative analysis of data from 28 semi-structured telephone interviews was undertaken.
The majority of respondents supported the extended role concept; however members of each professional group also rejected the notion. Respondents employed four different legitimacy claims to justify their views and define their occupational boundaries in relation to sickness certification practice. Condition-specific legitimacy, the ability to adopt a holistic approach to sickness certification, system efficiency and control-related arguments were used to different degrees by each occupation. Practical suggestions for the extension of the sickness certification role beyond the medical profession are underpinned by the sociological theory of professional identity.
Extending the authority to certify sickness absence beyond the medical profession is not simply a matter of addressing practical and organisational obstacles. There is also a need to consider the impact on, and preferences of, the specific occupations and their respective boundary claims. This paper explores the implications of extending the sick certification role beyond general practice. We conclude that the main policy challenge of such a move is to a) persuade GPs to relinquish this role (or to share it with other professions), and b) to understand the ‘boundary work’ involved.
PMCID: PMC4030269  PMID: 24884678
Professional boundaries; Sick certification; Qualitative methods; Sociology of professions; Primary care
15.  Automated identification of elemental ions in macromolecular crystal structures 
The solvent-picking procedure in phenix.refine has been extended and combined with Phaser anomalous substructure completion and analysis of coordination geometry to identify and place elemental ions.
Many macromolecular model-building and refinement programs can automatically place solvent atoms in electron density at moderate-to-high resolution. This process frequently builds water molecules in place of elemental ions, the identification of which must be performed manually. The solvent-picking algorithms in phenix.refine have been extended to build common ions based on an analysis of the chemical environment as well as physical properties such as occupancy, B factor and anomalous scattering. The method is most effective for heavier elements such as calcium and zinc, for which a majority of sites can be placed with few false positives in a diverse test set of structures. At atomic resolution, it is observed that it can also be possible to identify tightly bound sodium and magnesium ions. A number of challenges that contribute to the difficulty of completely automating the process of structure completion are discussed.
PMCID: PMC3975891  PMID: 24699654
refinement; ions; PHENIX
16.  Studying and Polishing the PDB's Macromolecules 
Biopolymers  2012;99(3):170-182.
Macromolecular crystal structures are among the best of scientific data, providing detailed insight into these complex and biologically important molecules with a relatively low level of error and subjectivity. However, there are two notable problems with getting the most information from them. The first is that the models are not perfect: there is still opportunity for improving them, and users need to evaluate whether the local reliability in a structure is up to answering their question of interest. The second is that protein and nucleic acid molecules are highly complex and individual, inherently handed and 3-dimensional, and the cooperative and subtle interactions that govern their detailed structure and function are not intuitively evident. Thus there is a real need for graphical representations and descriptive classifications that enable molecular 3D literacy. We have spent our career working to understand these elegant molecules ourselves, and building tools to help us and others determine and understand them better. The Protein Data Bank (PDB) has of course been vital and central to this undertaking. Here we combine some history of our involvement as depositors, illustrators, evaluators, and end-users of PDB structures with commentary on how best to study and draw scientific inferences from them.
PMCID: PMC3535681  PMID: 23023928
17.  Exploring patient priorities among long-term conditions in multimorbidity: A qualitative secondary analysis 
SAGE Open Medicine  2013;1:2050312113503955.
A lack of agreement between health-care providers and patient priorities can impact the health-care provider–patient relationship, treatment concordance and potentially health outcomes. Evidence suggests that people living with multiple morbidities do prioritise among their long-term conditions. However, the evidence revealing the underlying reasons behind this prioritisation remains limited. Given the potential implications for day-to-day self-management activity and ultimately patient outcomes, this study aims to explore how and why people with multimorbidity prioritise some long-term conditions over others and what the potential implications may be for self-management activity, and in turn, suggest how such information may help clinicians negotiate the management of multimorbidity patients.
A secondary analysis of qualitative data was conducted utilising four existing data sets collated from the three research centres involved. Purposive sampling provided a sample of 41 participants who had multimorbidity. The research team collectively coded and analysed the data thematically.
All participants, except two, identified one ‘main’ priority long-term condition. Current priorities were arrived at by participants making comparisons between their long-term conditions, specifically by trading off the various attributes, impacts and perceived consequences of their individual long-term conditions. Two main themes emerged as to why participants identified a particular main long-term condition: (a) proximate issues surrounding barriers to functional health and (b) prioritisation of long-term conditions perceived to have a particular future risk.
The recent focus on multimorbidity within the medical literature reflects its prevalence. It is therefore important to understand the complexities of the multimorbidity illness experience. We have added to the limited literature on condition prioritisation by revealing some novel understandings of the process of condition prioritisation which can feed into patient–provider consultations in order to allow better communication and treatment planning as well as, ultimately, optimise patient outcomes.
PMCID: PMC4687770  PMID: 26770680
Multimorbidity; primary care; priorities; self-management; risk
18.  Health-related quality of life in gout: a systematic review 
Rheumatology (Oxford, England)  2013;52(11):2031-2040.
Objectives. To identify the instruments that have been used to measure health-related quality of life (HRQOL) in gout and assess their clinimetric properties, determine the distribution of HRQOL in gout and identify factors associated with poor HRQOL.
Methods. Medline, CINAHL, EMBASE and PsycINFO were searched from inception to October 2012. Search terms pertained to gout, health or functional status, clinimetric properties and HRQOL. Study data extraction and quality assessment were performed by two independent reviewers.
Results. From 474 identified studies, 22 met the inclusion criteria. Health Assessment Questionnaire Disability Index (HAQ-DI) and Short Form 36 (SF-36) were most frequently used and highest rated due to robust construct and concurrent validity, despite high floor and ceiling effects. The Gout Impact Scale had good content validity. Gout had a greater impact on physical HRQOL compared to other domains. Both gout-specific features (attack frequency and intensity, intercritical pain and number of joints involved) and comorbid disease were associated with poor HRQOL. Evidence for objective features such as tophi and serum uric acid was less robust. Limitations of existing studies include cross-sectional design, recruitment from specialist clinic settings and frequent use of generic instruments.
Conclusion. Most studies have used the generic HAQ-DI and SF-36. Gout-specific characteristics and comorbidities contribute to poor HRQOL. There is a need for a cohort study in primary care (where most patients with gout are treated) to determine which factors predict changes in HRQOL over time. This will enable those at risk of deterioration to be identified and better targeted for treatment.
PMCID: PMC3798715  PMID: 23934311
gout; health-related quality of life; clinimetrics
19.  Scientific Benchmarks for Guiding Macromolecular Energy Function Improvement 
Methods in enzymology  2013;523:109-143.
Accurate energy functions are critical to macromolecular modeling and design. We describe new tools for identifying inaccuracies in energy functions and guiding their improvement, and illustrate the application of these tools to improvement of the Rosetta energy function. The feature analysis tool identifies discrepancies between structures deposited in the PDB and low energy structures generated by Rosetta; these likely arise from inaccuracies in the energy function. The optE tool optimizes the weights on the different components of the energy function by maximizing the recapitulation of a wide range of experimental observations. We use the tools to examine three proposed modifications to the Rosetta energy function: improving the unfolded state energy model (reference energies), using bicubic spline interpolation to generate knowledge based torisonal potentials, and incorporating the recently developed Dunbrack 2010 rotamer library (Shapovalov and Dunbrack, 2011).
PMCID: PMC3724755  PMID: 23422428
Rosetta; energy function; scientific benchmarking; parameter estimation; decoy discrimination
20.  Doing Molecular Biophysics: Finding, Naming, and Picturing Signal Within Complexity 
A macromolecular structure, as measured data or as a list of coordinates or even on-screen as a full atomic model, is an extremely complex and confusing object. The underlying rules of how it folds, moves, and interacts as a biological entity are even less evident or intuitive to the human mind. To do science on such molecules, or to relate them usefully to higher levels of biology, we need to start with a natural history that names their features in meaningful ways and with multiple representations (visual or algebraic) that show some aspect of their organizing principles. The two of us have jointly enjoyed a highly varied and engrossing career in biophysical research over nearly 50 years. Our frequent changes of emphasis are tied together by two threads: first, by finding the right names, visualizations, and methods to help both ourselves and others to better understand the 3D structures of protein and RNA molecules, and second, by redefining the boundary between signal and noise for complex data, in both directions—sometimes identifying and promoting real signal up out of what seemed just noise, and sometimes demoting apparent signal into noise or systematic error. Here we relate parts of our scientific and personal lives, including ups and downs, influences, anecdotes, and guiding principles such as the title theme.
PMCID: PMC3695750  PMID: 23451888
scientific biography; structural biology; molecular graphics; ribbon drawings; structure validation; all-atom contacts
21.  OSPREY: Protein Design with Ensembles, Flexibility, and Provable Algorithms 
Methods in enzymology  2013;523:87-107.
We have developed a suite of protein redesign algorithms that improves realistic in silico modeling of proteins. These algorithms are based on three characteristics that make them unique: (1) improved flexibility of the protein backbone, protein side chains, and ligand to accurately capture the conformational changes that are induced by mutations to the protein sequence; (2) modeling of proteins and ligands as ensembles of low-energy structures to better approximate binding affinity; and (3) a globally-optimal protein design search, guaranteeing that the computational predictions are optimal with respect to the input model. Here, we illustrate the importance of these three characteristics. We then describe OSPREY, a protein redesign suite that implements our protein design algorithms. OSPREY has been used prospectively, with experimental validation, in several biomedically-relevant settings. We show in detail how OSPREY has been used to predict resistance mutations and explain why improved flexibility, ensembles, and provability are essential for this application.
PMCID: PMC3692370  PMID: 23422427
protein design; OSPREY; Dead-end elimination; protein ensembles; protein flexibility; K*; minDEE
22.  Prospective observational cohort study of Health Related Quality of Life (HRQOL), chronic foot problems and their determinants in gout: a research protocol 
Gout is the commonest inflammatory arthritis affecting around 1.4% of adults in Europe. It is predominantly managed in primary care and classically affects the joints of the foot, particularly the first metatarsophalangeal joint. Gout related factors (including disease characteristics and treatment) as well as comorbid chronic disease are associated with poor Health Related Quality of Life (HRQOL) yet to date there is limited evidence concerning gout in a community setting. Existing epidemiological studies are limited by their cross-sectional design, selection of secondary care patients with atypical disease and the use of generic tools to measure HRQOL. This 3 year primary care-based prospective observational cohort study will describe the spectrum of HRQOL in community dwelling patients with gout, associated factors, predictors of poor outcome, and prevalence and incidence of foot problems in gout patients.
Adults aged ≥ 18 years diagnosed with gout or prescribed colchicine or allopurinol in the preceding 2 years will be identified through Read codes and mailed a series of self-completion postal questionnaires over a 3-year period. Consenting participants will have their general practice medical records reviewed.
This is the first prospective cohort study of HRQOL in patients with gout in primary care in the UK. The combination of survey data and medical record review will allow an in-depth understanding of factors that are associated with and lead to poor HRQOL and foot problems in gout. Identification of these factors will improve the management of this prevalent, yet under-treated, condition in primary care.
PMCID: PMC3517766  PMID: 23148573
Gout; HRQOL; Foot; Patient experience; Prospective cohort; Primary care
23.  The Phenix Software for Automated Determination of Macromolecular Structures 
Methods (San Diego, Calif.)  2011;55(1):94-106.
X-ray crystallography is a critical tool in the study of biological systems. It is able to provide information that has been a prerequisite to understanding the fundamentals of life. It is also a method that is central to the development of new therapeutics for human disease. Significant time and effort are required to determine and optimize many macromolecular structures because of the need for manual interpretation of complex numerical data, often using many different software packages, and the repeated use of interactive three-dimensional graphics. The Phenix software package has been developed to provide a comprehensive system for macromolecular crystallographic structure solution with an emphasis on automation. This has required the development of new algorithms that minimize or eliminate subjective input in favour of built-in expert-systems knowledge, the automation of procedures that are traditionally performed by hand, and the development of a computational framework that allows a tight integration between the algorithms. The application of automated methods is particularly appropriate in the field of structural proteomics, where high throughput is desired. Features in Phenix for the automation of experimental phasing with subsequent model building, molecular replacement, structure refinement and validation are described and examples given of running Phenix from both the command line and graphical user interface.
PMCID: PMC3193589  PMID: 21821126
Macromolecular Crystallography; Automation; Phenix; X-ray; Diffraction; Python
24.  The Role of Local Backrub Motions in Evolved and Designed Mutations 
PLoS Computational Biology  2012;8(8):e1002629.
Amino acid substitutions in protein structures often require subtle backbone adjustments that are difficult to model in atomic detail. An improved ability to predict realistic backbone changes in response to engineered mutations would be of great utility for the blossoming field of rational protein design. One model that has recently grown in acceptance is the backrub motion, a low-energy dipeptide rotation with single-peptide counter-rotations, that is coupled to dynamic two-state sidechain rotamer jumps, as evidenced by alternate conformations in very high-resolution crystal structures. It has been speculated that backrubs may facilitate sequence changes equally well as rotamer changes. However, backrub-induced shifts and experimental uncertainty are of similar magnitude for backbone atoms in even high-resolution structures, so comparison of wildtype-vs.-mutant crystal structure pairs is not sufficient to directly link backrubs to mutations. In this study, we use two alternative approaches that bypass this limitation. First, we use a quality-filtered structure database to aggregate many examples for precisely defined motifs with single amino acid differences, and find that the effectively amplified backbone differences closely resemble backrubs. Second, we directly apply a provably-accurate, backrub-enabled protein design algorithm to idealized versions of these motifs, and discover that the lowest-energy computed models match the average-coordinate experimental structures. These results support the hypothesis that backrubs participate in natural protein evolution and validate their continued use for design of synthetic proteins.
Author Summary
Protein design has the potential to generate useful molecules for medicine and chemistry, including sensors, drugs, and catalysts for arbitrary reactions. When protein design is carried out starting from an experimentally determined structure, as is often the case, one important aspect to consider is backbone flexibility, because in response to a mutation the backbone often must shift slightly to reconcile the new sidechain with its environment. In principle, one may model the backbone in many ways, but not all are physically realistic or experimentally validated. Here we study the "backrub" motion, which has been previously documented in atomic detail, but only for sidechain movements within single structures. By a twopronged approach involving both structural bioinformatics and computation with a principled design algorithm, we demonstrate that backrubs are sufficient to explain the backbone differences between mutation-related sets of very precisely defined motifs from the protein structure database. Our findings illustrate that backrubs are useful for describing evolutionary sequence change and, by extension, suggest that they are also appropriate for rational protein design calculations.
PMCID: PMC3410847  PMID: 22876172
25.  Graphical tools for macromolecular crystallography in PHENIX  
Journal of Applied Crystallography  2012;45(Pt 3):581-586.
The foundations and current features of a widely used graphical user interface for macromolecular crystallography are described.
A new Python-based graphical user interface for the PHENIX suite of crystallography software is described. This interface unifies the command-line programs and their graphical displays, simplifying the development of new interfaces and avoiding duplication of function. With careful design, graphical interfaces can be displayed automatically, instead of being manually constructed. The resulting package is easily maintained and extended as new programs are added or modified.
PMCID: PMC3359726  PMID: 22675231
macromolecular crystallography; graphical user interfaces; PHENIX

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