CryoEM-enabled visual proteomics reveals de novo structures of oligomeric protein complexes from Azotobacter vinelandii

bioRxiv (Cold Spring Harbor Laboratory), Journal Year: 2025, Volume and Issue: unknown

Published: Feb. 4, 2025

Abstract Single particle cryoelectron microscopy (cryoEM) and tomography (cryoET) are powerful methods for unveiling unique functionally relevant structural states. Aided by mass spectrometry machine learning, they promise to facilitate the visual exploration of proteomes. Leveraging proteomics, we interrogate structures isolated from a complex cellular milieu cryoEM identify classify molecular complexes de novo . That approach determines identity six distinct oligomeric protein partially purified extracts Azotobacter vinelandii using both anaerobic aerobic cryoEM. Identification first unknown species, phosphoglucoisomerase (Pgi1), is achieved comparing three automated model building programs: CryoID, DeepTracer, ModelAngelo with or without priori proteomics data. All programs Pgi1 protein, revealed be in new decameric state, as well additional globular identified glutamine synthetase (GlnA) bacterioferritin (Bfr). Large filamentous assemblies observed tomograms reconstructed cryoFIB milled lamellae nitrogen-fixing A. Enrichment these species cells centrifugation allows structure determination filament types helical reconstruction methods: Type 6 Secretion System non-contractile sheath tube (TssC), novel form soluble pyridine transhydrogenase (SthA), flagellar (FliC). The multimeric states SthA stand out contrast known crystallographic offer framework which evaluate their activities. Overall, allowing study near-native states, cryoEM-enabled reveals that correspond situ Figure

Language: Английский

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Artificial intelligence and deep learning in electron microscopy

Advances in imaging and electron physics, Journal Year: 2025, Volume and Issue: unknown

Published: Jan. 1, 2025

Language: Английский

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Simultaneous polyclonal antibody sequencing and epitope mapping by cryo electron microscopy and mass spectrometry – a perspective

Published: March 18, 2025

Antibodies are a major component of adaptive immunity against invading pathogens. Here we explore possibilities for an analytical approach to characterize the antigen-specific antibody repertoire directly from secreted proteins in convalescent serum. This aims perform simultaneous sequencing and epitope mapping using combination single particle cryo-electron microscopy (cryoEM) bottom-up proteomics techniques based on mass spectrometry (LC-MS/MS). We evaluate performance deep-learning tool ModelAngelo determining de novo sequences reconstructed 3D volumes antibody-antigen complexes. demonstrate that while map quality is critical bottleneck, it possible sequence variable domains cryoEM reconstructions with accuracies up 80-90%. While rate errors exceeds typical levels somatic hypermutation, show ModelAngelo-derived can be used assign V-genes. provides functional guide assemble peptides LC-MS/MS data more accurately improves tolerance background polyclonal sequences. Following this proof-of-principle, discuss feasibility future directions repertoires.

Language: Английский

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Simultaneous polyclonal antibody sequencing and epitope mapping by cryo electron microscopy and mass spectrometry

eLife, Journal Year: 2025, Volume and Issue: 14

Published: April 23, 2025

Antibodies are a major component of adaptive immunity against invading pathogens. Here, we explore possibilities for an analytical approach to characterize the antigen-specific antibody repertoire directly from secreted proteins in convalescent serum. This aims perform simultaneous sequencing and epitope mapping using combination single particle cryo-electron microscopy (cryoEM) bottom-up proteomics techniques based on mass spectrometry (LC-MS/MS). We evaluate performance deep-learning tool ModelAngelo determining de novo sequences reconstructed 3D volumes antibody-antigen complexes. demonstrate that while map quality is critical bottleneck, it possible sequence variable domains cryoEM reconstructions with accuracies up 80–90%. While rate errors exceeds typical levels somatic hypermutation, show ModelAngelo-derived can be used assign V-genes. provides functional guide assemble peptides LC-MS/MS data more accurately improves tolerance background polyclonal sequences. Following this proof-of-principle, discuss feasibility future directions repertoires.

Language: Английский

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Ring-stacked capsids of white spot syndrome virus and structural transitions with genome ejection

Science Advances, Journal Year: 2023, Volume and Issue: 9(8)

Published: Feb. 22, 2023

White spot syndrome virus (WSSV) is one of the largest DNA viruses and major pathogen responsible for white in crustaceans. The WSSV capsid critical genome encapsulation ejection exhibits rod-shaped oval-shaped structures during viral life cycle. However, detailed architecture structural transition mechanism remain unclear. Here, using cryo-electron microscopy (cryo-EM), we obtained a cryo-EM model were able to characterize its ring-stacked assembly mechanism. Furthermore, identified an from intact virions analyzed capsids induced by high salinity. These transitions, which decrease internal pressure, always accompany release mostly eliminate infection host cells. Our results demonstrate unusual offer insights into pressure-driven release.

Language: Английский

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