Mass-selective and ice-free electron cryomicroscopy protein sample preparation via native electrospray ion-beam deposition.
cryo-EM
ice-free sample preparation
ion-beam deposition
native mass spectrometry
structural biology
Journal
PNAS nexus
ISSN: 2752-6542
Titre abrégé: PNAS Nexus
Pays: England
ID NLM: 9918367777906676
Informations de publication
Date de publication:
Sep 2022
Sep 2022
Historique:
received:
19
05
2022
accepted:
03
08
2022
entrez:
30
1
2023
pubmed:
31
1
2023
medline:
31
1
2023
Statut:
epublish
Résumé
Despite tremendous advances in sample preparation and classification algorithms for electron cryomicroscopy (cryo-EM) and single-particle analysis (SPA), sample heterogeneity remains a major challenge and can prevent access to high-resolution structures. In addition, optimization of preparation conditions for a given sample can be time-consuming. In the current work, it is demonstrated that native electrospray ion-beam deposition (native ES-IBD) is an alternative, reliable approach for the preparation of extremely high-purity samples, based on mass selection in vacuum. Folded protein ions are generated by native electrospray ionization, separated from other proteins, contaminants, aggregates, and fragments, gently deposited on cryo-EM grids, frozen in liquid nitrogen, and subsequently imaged by cryo-EM. We demonstrate homogeneous coverage of ice-free cryo-EM grids with mass-selected protein complexes. SPA reveals that the complexes remain folded and assembled, but variations in secondary and tertiary structures are currently limiting information in 2D classes and 3D EM density maps. We identify and discuss challenges that need to be addressed to obtain a resolution comparable to that of the established cryo-EM workflow. Our results show the potential of native ES-IBD to increase the scope and throughput of cryo-EM for protein structure determination and provide an essential link between gas-phase and solution-phase protein structures.
Identifiants
pubmed: 36714824
doi: 10.1093/pnasnexus/pgac153
pii: pgac153
pmc: PMC9802471
doi:
Types de publication
Journal Article
Langues
eng
Pagination
pgac153Subventions
Organisme : Medical Research Council
ID : MC_UP_1201/31
Pays : United Kingdom
Informations de copyright
© The Author(s) 2022. Published by Oxford University Press on behalf of National Academy of Sciences.
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