Mass-selective and ice-free electron cryomicroscopy protein sample preparation via native electrospray ion-beam deposition.

Autor:	Esser TK; Department of Chemistry, University of Oxford, Mansfield Road, Oxford OX1 3TA, UK., Böhning J; Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford OX1 3RE, UK., Fremdling P; Department of Chemistry, University of Oxford, Mansfield Road, Oxford OX1 3TA, UK., Agasid MT; Department of Chemistry, University of Oxford, Mansfield Road, Oxford OX1 3TA, UK., Costin A; Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford OX1 3RE, UK., Fort K; Thermo Fisher Scientific, Hanna-Kunath-Straße 11, 28199 Bremen, Germany., Konijnenberg A; Thermo Fisher Scientific, Zwaanstraat 31G/H, 5651 CA Eindhoven, The Netherlands., Gilbert JD; Thermo Fisher Scientific, 5350 NE Dawson Creek Drive, Hillsboro, OR 97124, USA., Bahm A; Thermo Fisher Scientific, 5350 NE Dawson Creek Drive, Hillsboro, OR 97124, USA., Makarov A; Thermo Fisher Scientific, Hanna-Kunath-Straße 11, 28199 Bremen, Germany.; Biomolecular Mass Spectrometry and Proteomics, Bijvoet Center for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, University of Utrecht, Padualaan 8, 3584 CH Utrecht, The Netherlands., Robinson CV; Department of Chemistry, University of Oxford, Mansfield Road, Oxford OX1 3TA, UK., Benesch JLP; Department of Chemistry, University of Oxford, Mansfield Road, Oxford OX1 3TA, UK., Baker L; Division of Structural Biology, University of Oxford, Roosevelt Drive, Oxford OX3 7BN, UK., Bharat TAM; Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford OX1 3RE, UK.; Structural Studies Division, MRC Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge CB2 0QH, UK., Gault J; Department of Chemistry, University of Oxford, Mansfield Road, Oxford OX1 3TA, UK., Rauschenbach S; Department of Chemistry, University of Oxford, Mansfield Road, Oxford OX1 3TA, UK.; Max Planck Institute for Solid State Research, Heisenbergstrasse 1, DE-70569 Stuttgart, Germany.
Jazyk:	angličtina
Zdroj:	PNAS nexus [PNAS Nexus] 2022 Aug 06; Vol. 1 (4), pp. pgac153. Date of Electronic Publication: 2022 Aug 06 (Print Publication: 2022).
DOI:	10.1093/pnasnexus/pgac153
Abstrakt:	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. (© The Author(s) 2022. Published by Oxford University Press on behalf of National Academy of Sciences.)
Databáze:	MEDLINE
Externí odkaz:	Zobrazit plný text záznamu