Subassemblies and Asymmetry in Assembly of Herpes Simplex Virus Procapsid

The herpes simplex virus 1 (HSV-1) capsid is a massive particle (~200 MDa; 1,250-Å diameter) with T=16 icosahedral symmetry. It initially assembles as a procapsid with ~4,000 protein subunits of 11 different kinds. The procapsid undergoes major changes in structure and composition as it matures, a process driven by proteolysis and expulsion of the internal scaffolding protein. Assembly also relies on an external scaffolding protein, the triplex, an α2β heterotrimer that coordinates neighboring capsomers in the procapsid and becomes a stabilizing clamp in the mature capsid. To investigate the mechanisms that regulate its assembly, we developed a novel isolation procedure for the metastable procapsid and collected a large set of cryo-electron microscopy data. In addition to procapsids, these preparations contain maturation intermediates, which were distinguished by classifying the images and calculating a three-dimensional reconstruction for each class. Appraisal of the procapsid structure led to a new model for assembly; in it, the protomer (assembly unit) consists of one triplex, surrounded by three major capsid protein (MCP) subunits. The model exploits the triplexes’ departure from 3-fold symmetry to explain the highly skewed MCP hexamers, the triplex orientations at each 3-fold site, and the T=16 architecture. These observations also yielded new insights into maturation

FSC-Q: a CryoEM map-to-atomic model quality validation based on the local Fourier shell correlation

In recent years, advances in cryoEM have dramatically increased the resolution of reconstructions and, with it, the number of solved atomic models. It is widely accepted that the quality of cryoEM maps varies locally; therefore, the evaluation of the maps-derived structural models must be done locally as well. In this article, a method for the local analysis of the map-to-model fit is presented. The algorithm uses a comparison of two local resolution maps. The first is the local FSC (Fourier shell correlation) between the full map and the model, while the second is calculated between the half maps normally used in typical single particle analysis workflows. We call the quality measure “FSC-Q”, and it is a quantitative estimation of how much of the model is supported by the signal content of the map. Furthermore, we show that FSC-Q may be helpful to detect overfitting. It can be used to complement other methods, such as the Q-score method that estimates the resolvability of atomsWe thank Prof. David Veesler for providing us the half maps of the spike glycoprotein of SARS-CoV-2. The authors would like to acknowledge financial support from: the Comunidad de Madrid through grant CAM (S2017/BMD-3817), the Spanish National Research Council (PIE/COVID-19 number 202020E079), the Spanish Ministry of Economy and Competitiveness through grants SEV 2017-0712, PID2019-104757RB-I00/AEI/10.13039/501100011033, the Instituto de Salud Carlos III through grant PT17/0009/0010 (ISCIII-GEFI/ERDF-). Instruct-ULTRA (Grant 731005), an EU H2020 project to further develop the services of Instruct-ERIC. UE H2020 grant HighResCells (ERC-2018-SyG, Proposal: 810057). This work was supported by the Intramural Research Program of the National Institute for Arthritis, musculoskeletal, and Skin Diseases, NIH. The authors acknowledge the support and the use of resources of Instruct, a Landmark ESFRI projec

Recent advances in electron cryotomography and their application to imaging purified protein complexes, isolated organelles, and small cells

A new 300kV, FEG, helium-cooled, energy-filtered "G2 Polara" electron cryoTEM was recently installed at Caltech. Using this instrument, we have now extensively tested the potential advantages of liquid helium cooling and the inclusion of two orthogonal tilt-series in the context of electron cryotomography. These results and the application of the technology to several specific biological problems are reported

Pain, quality of life, self perception of health and depression in patients with fibromyalgia, submited to hydrocinesiotherapy

OBJECTIVES: The aim of this study was to analyze the effects of treatment by hydrotherapy on quality of life, perception of pain and the severity of depression in a group of patients with fibromyalgia. MATERIALS AND METHODS: We evaluated 64 females divided into two groups: hydrocinesiotherapy (n = 33, 58.2 ± 10.6 years) and control group (n = 31 with 59.6 ± 9.4 years) with clinical diagnosis of fibromyalgia. Individuals were assessed by Visual Analog Scale of Pain (VAS), the Fibromyalgia Impact Questionnaire (FIQ) and the Beck Depression Inventory. Participants underwent a treatment in a hydrotherapy pool heated to 33ºC over a period of 15 weeks, two sessions per week of 45 minutes, a total of 30 sessions. The exercises were underwater: cardiovascular conditioning, strength training, mobility, coordination, balance and still, stretching exercises and muscle relaxation. The ANOVA 2×2 and Kruskall-Wallis was used for statistical analysis RESULTS: There were statistically significant improvements in the perception of pain intensity (Δ% = -28.2%, p < 0, 01), quality of life (Δ% = -32.4%, p < 0, 05) and depression symptoms (Δ% = -35.4%, p < 0, 05) in favor of the Hydrotherapy group compared to the control group. CONCLUSIONS: The study suggests that hydrocinesiotherapy was effective as an alternative therapy for fibromyalgia, however further studies are recommended to test the associations between the variables and intervention programs and using the water activities, and the modifiability of the parameters of physical and mental health when these individuals undergo programs of short, medium and long duration.OBJETIVOS: Analisar os efeitos do tratamento hidrocinesioterapêutico na qualidade de vida, percepção de dor e gravidade de episódios depressivos em um grupo de pacientes com fibromialgia. MATERIAIS E MÉTODOS: Foram avaliados 64 indivíduos do sexo feminino, separados em dois grupos: hidrocinesioterapia (n = 33; 58,2 ± 10,6 anos) e grupo controle (n = 31; 59,6 ± 9,4 anos), com diagnóstico de fibromialgia. Os indivíduos foram avaliados através da Escala Analógica Visual de Dor (EVA), o Fibromyalgia Impact Questionnaire (FIQ), e o Inventário de Beck. Os participantes foram submetidos a um tratamento hidrocinesioterápico numa piscina aquecida a 33ºC com duas sessões de 45 minutos por semana, ao longo 15 semanas, num total de 30 sessões. Os exercícios subaquáticos foram: de condicionamento cardiovascular, de força, de mobilidade, de coordenação, de equilíbrio, de alongamento e de relaxamento muscular. Utilizou-se a ANOVA 2×2 e Kruskal-Wallis para análise estatística. RESULTADOS: Foram observadas melhorias estatisticamente significativas na percepção da intensidade da dor (Δ% = -28,2%, p < 0,01), na qualidade de vida (Δ% = -32,4%, p < 0,05) e nos sintomas de depressão (Δ% = -35,4%, p < 0,05) favoráveis ao grupo hidrocinesioterapia comparado ao grupo controle. CONCLUSÕES: O estudo sugere que a hidrocinesioterapia mostrou-se eficaz como terapia alternativa da fibromialgia. No entanto, recomenda novos estudos que testem as associações existentes entre as variáveis analisadas e os programas de intervenção, utilizando as atividades aquáticas, bem como a modificabilidade dos parâmetros de saúde física e psíquica quando estes indivíduos são submetidos a programas de curta, média e longa duração.Faculdade Católica Rainha do SertãoUniversidade de Coimbra Faculdade de Ciências do Desporto e Educação FísicaCentro Universitário da Fundação Educacional GuaxupéUniversidade Federal do ParanáFederal do Estado do Rio de JaneiroUniversidade Federal de São Paulo (UNIFESP)UNIFESPSciEL

Global patient outcomes after elective surgery: prospective cohort study in 27 low-, middle- and high-income countries.

BACKGROUND: As global initiatives increase patient access to surgical treatments, there remains a need to understand the adverse effects of surgery and define appropriate levels of perioperative care. METHODS: We designed a prospective international 7-day cohort study of outcomes following elective adult inpatient surgery in 27 countries. The primary outcome was in-hospital complications. Secondary outcomes were death following a complication (failure to rescue) and death in hospital. Process measures were admission to critical care immediately after surgery or to treat a complication and duration of hospital stay. A single definition of critical care was used for all countries. RESULTS: A total of 474 hospitals in 19 high-, 7 middle- and 1 low-income country were included in the primary analysis. Data included 44 814 patients with a median hospital stay of 4 (range 2-7) days. A total of 7508 patients (16.8%) developed one or more postoperative complication and 207 died (0.5%). The overall mortality among patients who developed complications was 2.8%. Mortality following complications ranged from 2.4% for pulmonary embolism to 43.9% for cardiac arrest. A total of 4360 (9.7%) patients were admitted to a critical care unit as routine immediately after surgery, of whom 2198 (50.4%) developed a complication, with 105 (2.4%) deaths. A total of 1233 patients (16.4%) were admitted to a critical care unit to treat complications, with 119 (9.7%) deaths. Despite lower baseline risk, outcomes were similar in low- and middle-income compared with high-income countries. CONCLUSIONS: Poor patient outcomes are common after inpatient surgery. Global initiatives to increase access to surgical treatments should also address the need for safe perioperative care. STUDY REGISTRATION: ISRCTN5181700

Community recommendations on cryoEM data archiving and validation

In January 2020, a workshop was held at EMBL-EBI (Hinxton, UK) to discuss data requirements for the deposition and validation of cryoEM structures, with a focus on single-particle analysis. The meeting was attended by 47 experts in data processing, model building and refinement, validation, and archiving of such structures. This report describes the workshop’s motivation and history, the topics discussed, and the resulting consensus recommendations. Some challenges for future methods-development efforts in this area are also highlighted, as is the implementation to date of some of the recommendations.The workshop was supported by funding to PDBe and EMDB by the Wellcome Trust (grant No. 104948/Z/14/Z awarded to GJK, SV and AP) and by the European Molecular Biology Laboratory. Travel was supported by the PDBe, EMDB, RCSB PDB, PDBj, BMRB and EMDR. RCSB PDB is jointly funded by the National Science Foundation (grant No. DBI1832184); the US Department of Energy (grant No. DESC0019749); and the National Cancer Institute, National Institute of Allergy and Infectious Diseases, and National Institute of General Medical Sciences of the National Institutes of Health (grant No. R01GM133198). PDBj is funded by JST-NBDC and BMRB by the National Institute of General Medical Sciences (NIGMS) of the National Institutes of Health (NIH) (grant No. R24GM150793). EMDR was funded by the NIGMS of the NIH (grant No. R01GM079429).Peer reviewe

Validation of 3D EM Reconstructions: The Phantom in the Noise

Validation is a necessity to trust the structures solved by electron microscopy by single particle techniques. The impressive achievements in single particle reconstruction fuel its expansion beyond a small community of image processing experts. This poses the risk of inappropriate data processing with dubious results. Nowhere is it more clearly illustrated than in the recovery of a reference density map from pure noise aligned to that map—a phantom in the noise. Appropriate use of existing validating methods such as resolution-limited alignment and the processing of independent data sets (“gold standard”) avoid this pitfall. However, these methods can be undermined by biases introduced in various subtle ways. How can we test that a map is a coherent structure present in the images selected from the micrographs? In stead of viewing the phantom emerging from noise as a cautionary tale, it should be used as a defining baseline. Any map is always recoverable from noise images, provided a sufficient number of images are aligned and used in reconstruction. However, with smaller numbers of images, the expected coherence in the real particle images should yield better reconstructions than equivalent numbers of noise or background images, even without masking or imposing resolution limits as potential biases. The validation test proposed is therefore a simple alignment of a limited number of micrograph and noise images against the final reconstruction as reference, demonstrating that the micrograph images yield a better reconstruction. I examine synthetic cases to relate the resolution of a reconstruction to the alignment error as a function of the signal-to-noise ratio. I also administered the test to real cases of publicly available data. Adopting such a test can aid the microscopist in assessing the usefulness of the micrographs taken before committing to lengthy processing with questionable outcomes

The Ewald sphere/focus gradient does not limit the resolution of cryoEM reconstructions

In our quest to solve biomolecular structures to higher resolutions in cryoEM, care must be taken to deal with all aspects of image formation in the electron microscope. One of these is the Ewald sphere/focus gradient that derives from the scattering geometry in the microscope and its implications for recovering high resolution and handedness information. While several methods to deal with it has been proposed and implemented, there are still questions as to the correct approach. At the high acceleration voltages used for cryoEM, the traditional projection approximation that ignores the Ewald sphere breaks down around 2–3 Å and with large particles. This is likely not crucial for most biologically interesting molecules, but is required to understand detail about catalytic events, molecular orbitals, orientation of bound water molecules, etc. Through simulation I show that integration along the Ewald spheres in frequency space during reconstruction, the “simple insertion method” is adequate to reach resolutions to the Nyquist frequency. Both theory and simulations indicate that the handedness information encoded in such phases is irretrievably lost in the formation of real space images. The conclusion is that correct reconstruction along the Ewald spheres avoids the limitations of the projection approximation