| Autor: |
Kyle L. Fort, P. Fremdling, Adam Costin, Mark T. Agasid, Lindsay A Baker, Alexander Makarov, Justin L. P. Benesch, A. Bahm, Joseph Gault, Albert Konijnenberg, S. Rauschenbach, T. K. Esser, Tanmay A.M. Bharat, J. Boehning, Carol V. Robinson |
| Rok vydání: |
2021 |
| Předmět: |
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| DOI: |
10.1101/2021.10.18.464782 |
| Popis: |
Electron cryomicroscopy (cryo-EM) and single-particle analysis (SPA) have revolutionized structure determination of homogeneous proteins. However, obtaining high-resolution structures from heterogeneous samples remains a major challenge, as the various protein states embedded in thin films of vitreous ice may be classified incorrectly, resulting in detrimental averaging of features. Here we present native electrospray ion-beam deposition (native ES-IBD) for the preparation of extremely high-purity cryo-EM samples, based on mass selection in vacuum. Folded protein ions are generated by native electrospray ionization, mass-filtered, and gently deposited on cryo-EM grids, and subsequently frozen in liquid nitrogen. We demonstrate homogeneous coverage of ice-free cryo-EM grids with mass-selected proteins and protein assemblies. SPA reveals that they remain structurally intact, but variations in secondary and tertiary structure are currently limiting information in 2D classes and 3D EM density maps. Our results show the potential of native ES-IBD to increase the scope and throughput of cryo-EM structure determination. |
| Databáze: |
OpenAIRE |
| Externí odkaz: |
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