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Single particle cryo-electron microscopy: From sample to structure

DOI: 10.3791/62415 DOI Help

Authors: Joshua B. R. White (University of Leeds) , Daniel P. Maskell (University of Leeds) , Andrew Howe (Diamond Light Source) , Martin Harrow (Diamond Light Source) , Daniel K. Clare (Diamond Light Source) , C. Alistair Siebert (Diamond Light Source) , Emma L. Hesketh (University of Leeds) , Rebecca F. Thompson (University of Leeds)
Co-authored by industrial partner: No

Type: Journal Paper
Journal: Journal Of Visualized Experiments

State: Published (Approved)
Published: May 2021
Diamond Proposal Number(s): 23047

Open Access Open Access

Abstract: Cryo-electron microscopy (cryoEM) is a powerful technique for structure determination of macromolecular complexes, via single particle analysis (SPA). The overall process involves i) vitrifying the specimen in a thin film supported on a cryoEM grid; ii) screening the specimen to assess particle distribution and ice quality; iii) if the grid is suitable, collecting a single particle dataset for analysis; and iv) image processing to yield an EM density map. In this protocol, an overview for each of these steps is provided, with a focus on the variables which a user can modify during the workflow and the troubleshooting of common issues. With remote microscope operation becoming standard in many facilities, variations on imaging protocols to assist users in efficient operation and imaging when physical access to the microscope is limited will be described.

Subject Areas: Biology and Bio-materials

Diamond Offline Facilities: Electron Bio-Imaging Centre (eBIC)
Instruments: Krios I-Titan Krios I at Diamond , Krios II-Titan Krios II at Diamond , Krios III-Titan Krios III at Diamond , Krios IV-Titan Krios IV at Diamond , Talos-Talos Arctica at Diamond

Documents:
jove-protocol-62415-single-particle-cryo-electron-microscopy-from-sample-to-structure.pdf

Discipline Tags:

Life Sciences & Biotech Structural biology

Technical Tags:

Microscopy Electron Microscopy (EM) Cryo Electron Microscopy (Cryo EM)