AlphaFold predictions are valuable hypotheses and accelerate but do not replace experimental structure determination.

Artificial intelligence-based protein structure prediction methods such as AlphaFold have revolutionized structural biology. The accuracies of these predictions vary, however, and they do not take into account ligands, covalent modifications or other environmental factors. Here, we evaluate how well AlphaFold predictions can be expected to describe the structure of a protein by comparing predictions directly with experimental crystallographic maps. In many cases, AlphaFold predictions matched experimental maps remarkably closely. In other cases, even very high-confidence predictions differed from experimental maps on a global scale through distortion and domain orientation, and on a local scale in backbone and side-chain conformation. We suggest considering AlphaFold predictions as exceptionally useful hypotheses. We further suggest that it is important to consider the confidence in prediction when interpreting AlphaFold predictions and to carry out experimental structure determination to verify structural details, particularly those that involve interactions not included in the prediction.

Outcomes of the EMDataResource cryo-EM Ligand Modeling Challenge.

The EMDataResource Ligand Model Challenge aimed to assess the reliability and reproducibility of modeling ligands bound to protein and protein-nucleic acid complexes in cryogenic electron microscopy (cryo-EM) maps determined at near-atomic (1.9-2.5 Å) resolution. Three published maps were selected as targets: Escherichia coli beta-galactosidase with inhibitor, SARS-CoV-2 virus RNA-dependent RNA polymerase with covalently bound nucleotide analog and SARS-CoV-2 virus ion channel ORF3a with bound lipid. Sixty-one models were submitted from 17 independent research groups, each with supporting workflow details. The quality of submitted ligand models and surrounding atoms were analyzed by visual inspection and quantification of local map quality, model-to-map fit, geometry, energetics and contact scores. A composite rather than a single score was needed to assess macromolecule+ligand model quality. These observations lead us to recommend best practices for assessing cryo-EM structures of liganded macromolecules reported at near-atomic resolution.

Improved AlphaFold modeling with implicit experimental information.

Machine-learning prediction algorithms such as AlphaFold and RoseTTAFold can create remarkably accurate protein models, but these models usually have some regions that are predicted with low confidence or poor accuracy. We hypothesized that by implicitly including new experimental information such as a density map, a greater portion of a model could be predicted accurately, and that this might synergistically improve parts of the model that were not fully addressed by either machine learning or experiment alone. An iterative procedure was developed in which AlphaFold models are automatically rebuilt on the basis of experimental density maps and the rebuilt models are used as templates in new AlphaFold predictions. We show that including experimental information improves prediction beyond the improvement obtained with simple rebuilding guided by the experimental data. This procedure for AlphaFold modeling with density has been incorporated into an automated procedure for interpretation of crystallographic and electron cryo-microscopy maps.

Cryo-EM model validation recommendations based on outcomes of the 2019 EMDataResource challenge.

This paper describes outcomes of the 2019 Cryo-EM Model Challenge. The goals were to (1) assess the quality of models that can be produced from cryogenic electron microscopy (cryo-EM) maps using current modeling software, (2) evaluate reproducibility of modeling results from different software developers and users and (3) compare performance of current metrics used for model evaluation, particularly Fit-to-Map metrics, with focus on near-atomic resolution. Our findings demonstrate the relatively high accuracy and reproducibility of cryo-EM models derived by 13 participating teams from four benchmark maps, including three forming a resolution series (1.8 to 3.1 Å). The results permit specific recommendations to be made about validating near-atomic cryo-EM structures both in the context of individual experiments and structure data archives such as the Protein Data Bank. We recommend the adoption of multiple scoring parameters to provide full and objective annotation and assessment of the model, reflective of the observed cryo-EM map density.

OECD harmonised template 201: Structuring and reporting mechanistic information to foster the integration of new approach methodologies for hazard and risk assessment of chemicals.

In the European Union, the Chemicals Strategy for Sustainability (CSS) highlights the need to enhance the identification and assessment of substances of concern while reducing animal testing, thus fostering the development and use of New Approach Methodologies (NAMs) such as in silico, in vitro and in chemico. In the United States, the Tox21 strategy aims at shifting toxicological assessments away from traditional animal studies towards target-specific, mechanism-based and biological observations mainly obtained by using NAMs. Many other jurisdictions around the world are also increasing the use of NAMs. Hence, the provision of dedicated non-animal toxicological data and reporting formats as a basis for chemical risk assessment is necessary. Harmonising data reporting is crucial when aiming at re-using and sharing data for chemical risk assessment across jurisdictions. The OECD has developed a series of OECD Harmonised Templates (OHT), which are standard data formats designed for reporting information used for the risk assessment of chemicals relevant to their intrinsic properties, including effects on human health (e.g., toxicokinetics, skin sensitisation, repeated dose toxicity) and the environment (e.g., toxicity to test species and wildlife, biodegradation in soil, metabolism of residues in crops). The objective of this paper is to demonstrate the applicability of the OHT standard format for reporting information under various chemical risk assessment regimes, and to provide users with practical guidance on the use of OHT 201, in particular to report test results on intermediate effects and mechanistic information.

VectorNet: collaborative mapping of arthropod disease vectors in Europe and surrounding areas since 2010.

BackgroundArthropod vectors such as ticks, mosquitoes, sandflies and biting midges are of public and veterinary health significance because of the pathogens they can transmit. Understanding their distributions is a key means of assessing risk. VectorNet maps their distribution in the EU and surrounding areas.AimWe aim to describe the methodology underlying VectorNet maps, encourage standardisation and evaluate output.Methods: Vector distribution and surveillance activity data have been collected since 2010 from a combination of literature searches, field-survey data by entomologist volunteers via a network facilitated for each participating country and expert validation. Data were collated by VectorNet members and extensively validated during data entry and mapping processes.ResultsAs of 2021, the VectorNet archive consisted of ca 475,000 records relating to > 330 species. Maps for 42 species are routinely produced online at subnational administrative unit resolution. On VectorNet maps, there are relatively few areas where surveillance has been recorded but there are no distribution data. Comparison with other continental databases, namely the Global Biodiversity Information Facility and VectorBase show that VectorNet has 5-10 times as many records overall, although three species are better represented in the other databases. In addition, VectorNet maps show where species are absent. VectorNet's impact as assessed by citations (ca 60 per year) and web statistics (58,000 views) is substantial and its maps are widely used as reference material by professionals and the public.ConclusionVectorNet maps are the pre-eminent source of rigorously validated arthropod vector maps for Europe and its surrounding areas.

A Reflection on the Tensions of Acting in Dual Roles of Doctoral Researcher and Practitioner when Evaluating a Lifestyle Intervention for Breast Cancer Patients.

This reflection was completed as part of a doctoral project to develop and trial a lifestyle intervention for people following the completion of their treatment for breast cancer. In this study the graduate student acted in the dual roles of nutrition practitioner and researcher. This article uses the experience, reflection, action (ERA) cycle of reflection to consider some of the tensions faced due to the divergent priorities and requirements of these two roles. One challenge occurred during study recruitment when a few potential participants did not meet the inclusion criteria for the study but still wished to attend the intervention sessions. It was also a challenge to mitigate the risks of distress of potentially vulnerable participants during group intervention sessions. In both instances there was a potential conflict between the needs of patients and research requirements. This reflection concluded that the obligations of both roles should be adhered to where possible, but if in doubt, the needs of the participants were paramount.

Seeing the PDB.

Ever since the first structures of proteins were determined in the 1960s, structural biologists have required methods to visualize biomolecular structures, both as an essential tool for their research and also to promote 3D comprehension of structural results by a wide audience of researchers, students, and the general public. In this review to celebrate the 50th anniversary of the Protein Data Bank, we present our own experiences in developing and applying methods of visualization and analysis to the ever-expanding archive of protein and nucleic acid structures in the worldwide Protein Data Bank. Across that timespan, Jane and David Richardson have concentrated on the organization inside and between the macromolecules, with ribbons to show the overall backbone "fold" and contact dots to show how the all-atom details fit together locally. David Goodsell has explored surface-based representations to present and explore biological subjects that range from molecules to cells. This review concludes with some ideas about the current challenges being addressed by the field of biomolecular visualization.

Improving SARS-CoV-2 structures: Peer review by early coordinate release.

This work builds upon the record-breaking speed and generous immediate release of new experimental three-dimensional structures of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) proteins and complexes, which are crucial to downstream vaccine and drug development. We have surveyed those structures to catch the occasional errors that could be significant for those important uses and for which we were able to provide demonstrably higher-accuracy corrections. This process relied on new validation and correction methods such as CaBLAM and ISOLDE, which are not yet in routine use. We found such important and correctable problems in seven early SARS-CoV-2 structures. Two of the structures were soon superseded by new higher-resolution data, confirming our proposed changes. For the other five, we emailed the depositors a documented and illustrated report and encouraged them to make the model corrections themselves and use the new option at the worldwide Protein Data Bank for depositors to re-version their coordinates without changing the Protein Data Bank code. This quickly and easily makes the better-accuracy coordinates available to anyone who examines or downloads their structure, even before formal publication. The changes have involved sequence misalignments, incorrect RNA conformations near a bound inhibitor, incorrect metal ligands, and cis-trans or peptide flips that prevent good contact at interaction sites. These improvements have propagated into nearly all related structures done afterward. This process constitutes a new form of highly rigorous peer review, which is actually faster and more strict than standard publication review because it has access to coordinates and maps; journal peer review would also be strengthened by such access.

Assessment of detailed conformations suggests strategies for improving cryoEM models: Helix at lower resolution, ensembles, pre-refinement fixups, and validation at multi-residue length scale.

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.

Constructing atomic structural models into cryo-EM densities using molecular dynamics - Pros and cons.

Accurate structure determination from electron density maps at 3-5 Šresolution necessitates a balance between extensive global and local sampling of atomistic models, yet with the stereochemical correctness of backbone and sidechain geometries. Molecular Dynamics Flexible Fitting (MDFF), particularly through a resolution-exchange scheme, ReMDFF, provides a robust way of achieving this balance for hybrid structure determination. Employing two high-resolution density maps, namely that of ß-galactosidase at 3.2 Šand TRPV1 at 3.4 Å, we showcase the quality of ReMDFF-generated models, comparing them against ones submitted by independent research groups for the 2015-2016 Cryo-EM Model Challenge. This comparison offers a clear evaluation of ReMDFF's strengths and shortcomings, and those of data-guided real-space refinements in general. ReMDFF results scored highly on the various metric for judging the quality-of-fit and quality-of-model. However, some systematic discrepancies are also noted employing a Molprobity analysis, that are reproducible across multiple competition entries. A space of key refinement parameters is explored within ReMDFF to observe their impact within the final model. Choice of force field parameters and initial model seem to have the most significant impact on ReMDFF model-quality. To this end, very recently developed CHARMM36m force field parameters provide now more refined ReMDFF models than the ones originally submitted to the Cryo-EM challenge. Finally, a set of good-practices is prescribed for the community to benefit from the MDFF developments.

New insights into Hoogsteen base pairs in DNA duplexes from a structure-based survey.

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.

Molprobity's ultimate rotamer-library distributions for model validation.

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.

New tools provide a second look at HDV ribozyme structure, dynamics and cleavage.

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.

The acceptability to patients of PhysioDirect telephone assessment and advice services; a qualitative interview study.

BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSION: 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.

A joint effort over a period of time: factors affecting use of urate-lowering therapy for long-term treatment of gout.

BACKGROUND: 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. METHODS: A qualitative study was conducted throughout the United Kingdom. Narrative and semi-structured video-recorded interviews and thematic analysis were used. RESULTS: 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. CONCLUSION: 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".

"Why me? I don't fit the mould I am a freak of nature": a qualitative study of women's experience of gout.

BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

Biophysical highlights from 54 years of macromolecular crystallography.

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.

Automated identification of elemental ions in macromolecular crystal structures.

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.