UCSF ChimeraX: Tools for structure building and analysis

Protein Science, Год журнала: 2023, Номер 32(11)

Опубликована: Сен. 29, 2023

Advances in computational tools for atomic model building are leading to accurate models of large molecular assemblies seen electron microscopy, often at challenging resolutions 3-4 Å. We describe new methods the UCSF ChimeraX modeling package that take advantage machine-learning structure predictions, provide likelihood-based fitting maps, and compute per-residue scores identify errors. Additional model-building assist analysis mutations, post-translational modifications, interactions with ligands. present latest capabilities, including several community-developed extensions. is available free charge noncommercial use https://www.rbvi.ucsf.edu/chimerax.

Язык: Английский

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Structure of the native myosin filament in the relaxed cardiac sarcomere

Nature, Год журнала: 2023, Номер 623(7988), С. 863 - 871

Опубликована: Ноя. 1, 2023

Abstract The thick filament is a key component of sarcomeres, the basic units striated muscle 1 . Alterations in proteins are associated with familial hypertrophic cardiomyopathy and other heart diseases 2 Despite central importance filament, its molecular organization remains unclear. Here we present architecture native cardiac sarcomeres relaxed state, determined by cryo-electron tomography. Our reconstruction reveals three-dimensional myosin, titin myosin-binding protein C (MyBP-C). arrangement myosin molecules dependent on their position along suggesting specialized capacities terms strain susceptibility force generation. Three pairs titin-α titin-β chains run axially intertwining tails probably orchestrating length-dependent activation sarcomere. Notably, whereas three entire length do not. structure also demonstrates that MyBP-C bridges thin filaments, carboxy-terminal region binding to directly stabilizing OFF state heads an unforeseen manner. These results provide foundation for future research investigating disorders involving sarcomeric components.

Язык: Английский

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ArtiaX: An electron tomography toolbox for the interactive handling of sub‐tomograms in UCSF ChimeraX

Protein Science, Год журнала: 2022, Номер 31(12)

Опубликована: Окт. 17, 2022

Abstract Cryo‐electron tomography analysis involves the selection of macromolecular complexes to be used for subsequent sub‐tomogram averaging and structure determination. Here, we describe a plugin developed UCSF ChimeraX that allows display, selection, editing particles within tomograms. Positions orientations selected can manually set, modified inspected in real time, both on screen virtual reality, exported various file formats. The parallel visualization stored several meta data lists, context any three‐dimensional image opened with ChimeraX. are rendered user‐defined colors or using colormaps, such individual classes groups particles, cross‐correlation coefficients, other types information highlighted user. implemented functions fast, reliable, intuitive, exploring broad range features They allow fluent human–machine interaction, which enables an effective understanding processing pipeline, even non‐specialist users.

Язык: Английский

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Translation dynamics in human cells visualized at high resolution reveal cancer drug action

Science, Год журнала: 2023, Номер 381(6653), С. 70 - 75

Опубликована: Июль 6, 2023

Ribosomes catalyze protein synthesis by cycling through various functional states. These states have been extensively characterized in vitro, but their distribution actively translating human cells remains elusive. We used a cryo-electron tomography-based approach and resolved ribosome structures inside with high resolution. revealed the of elongation cycle, Z transfer RNA binding site, dynamics expansion segments. Ribosome from treated Homoharringtonine, drug against chronic myeloid leukemia, how translation were altered situ resolve small molecules within active site ribosome. Thus, structural effects can be assessed at resolution cells.

Язык: Английский

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SARS-CoV-2 nsp3 and nsp4 are minimal constituents of a pore spanning replication organelle

Nature Communications, Год журнала: 2023, Номер 14(1)

Опубликована: Ноя. 30, 2023

Coronavirus replication is associated with the remodeling of cellular membranes, resulting in formation double-membrane vesicles (DMVs). A DMV-spanning pore was identified as a putative portal for viral RNA. However, exact components and structure SARS-CoV-2 DMV remain to be determined. Here, we investigate by situ cryo-electron tomography combined subtomogram averaging. We identify non-structural protein (nsp) 3 4 minimal required pore, which dependent on nsp3-4 proteolytic cleavage. In addition, show that Mac2-Mac3-DPUP-Ubl2 domains are critical nsp3 oligomerization crown integrity influences membrane curvature biogenesis DMVs. Altogether, have dual role driving organelles assembly pores propose here term replicopores.

Язык: Английский

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Extensive Angular Sampling Enables the Sensitive Localization of Macromolecules in Electron Tomograms

International Journal of Molecular Sciences, Год журнала: 2023, Номер 24(17), С. 13375 - 13375

Опубликована: Авг. 29, 2023

Cryo-electron tomography provides 3D images of macromolecules in their cellular context. To detect tomograms, template matching (TM) is often used, which uses models that are reliable for substantial parts the macromolecules. However, extent rotational searches particle detection has not been investigated due to computational limitations. Here, we provide a GPU implementation TM as part PyTOM software package, drastically speeds up orientational search and allows sampling beyond Crowther criterion within feasible timeframe. We quantify improvements sensitivity false-discovery rate examples ribosome identification detection. Sampling at criterion, was effectively impossible with CPU implementations extensive computation times, automated extraction high sensitivity. Consequently, also show an angular sample renders sensitive local alignment tilt series damage induced by focused ion beam milling. With this new release PyTOM, on integration other packages support more refined subtomogram-averaging workflows. The classification ribosomes appropriate locally corrected tomograms sufficiently low rate, allowing it be directly used high-resolution averaging adequate reveal polysome organization.

Язык: Английский

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High-confidence 3D template matching for cryo-electron tomography

Nature Communications, Год журнала: 2024, Номер 15(1)

Опубликована: Май 11, 2024

Abstract Visual proteomics attempts to build atlases of the molecular content cells but automated annotation cryo electron tomograms remains challenging. Template matching (TM) and methods based on machine learning detect structural signatures macromolecules. However, their applicability limited in terms both abundance size targets. Here we show that performance TM is greatly improved by using template-specific search parameter optimization including higher-resolution information. We establish a pipeline with systematically tuned parameters for automated, objective comprehensive identification structures confidence 10 100-fold above noise level. demonstrate high-fidelity high-confidence localizations nuclear pore complexes, vaults, ribosomes, proteasomes, fatty acid synthases, lipid membranes microtubules, individual subunits inside crowded eukaryotic cells. provide software tools generic implementation our method broadly applicable towards realizing visual proteomics.

Язык: Английский

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CryoET of β-amyloid and tau within postmortem Alzheimer’s disease brain

Nature, Год журнала: 2024, Номер 631(8022), С. 913 - 919

Опубликована: Июль 10, 2024

Abstract A defining pathological feature of most neurodegenerative diseases is the assembly proteins into amyloid that form disease-specific structures 1 . In Alzheimer’s disease, this characterized by deposition β-amyloid and tau with conformations. The in situ structure human brain unknown. Here, using cryo-fluorescence microscopy-targeted cryo-sectioning, cryo-focused ion beam-scanning electron microscopy lift-out cryo-electron tomography, we determined in-tissue architectures pathology a postmortem disease donor brain. plaques contained mixture fibrils, some which were branched, protofilaments, arranged parallel arrays lattice-like structures. Extracellular vesicles cuboidal particles defined non-amyloid constituents plaques. By contrast, inclusions formed clusters unbranched filaments. Subtomogram averaging cluster 136 filaments single tomogram revealed polypeptide backbone conformation filament polarity orientation paired helical within tissue. Filaments similar to each other, but different between clusters, showing heterogeneity spatially organized subcellular location. structural approaches outlined here for tissues have applications broad range diseases.

Язык: Английский

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Structure of the native γ-tubulin ring complex capping spindle microtubules

Nature Structural & Molecular Biology, Год журнала: 2024, Номер 31(7), С. 1134 - 1144

Опубликована: Апрель 12, 2024

Microtubule (MT) filaments, composed of α/β-tubulin dimers, are fundamental to cellular architecture, function and organismal development. They nucleated from MT organizing centers by the evolutionarily conserved γ-tubulin ring complex (γTuRC). However, molecular mechanism nucleation remains elusive. Here we used cryo-electron tomography determine structure native γTuRC capping minus end a in context enriched budding yeast spindles. In our structure, presents subunits seed exclusively 13-protofilament MTs, adopting an active closed conformation as perfect geometric template for nucleation. Our reconstruction revealed that coiled-coil protein staples first row alternating positions along γTuRC. This positioning onto suggests role augmenting γTuRC-mediated Based on results, describe model activation

Язык: Английский

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Passage of the HIV capsid cracks the nuclear pore

Cell, Год журнала: 2025, Номер unknown

Опубликована: Янв. 1, 2025

Язык: Английский

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