Cryo-electron tomography remote data collection and subtomogram averaging

DOI: 10.3791/63923

Authors: Yuewen Sheng (Diamond Light Source) , Kyle Morris (Diamond Light Source) , Julika Radecke (Diamond Light Source) , Peijun Zhang (Diamond Light Source; Wellcome Trust Centre for Human Genetics, University of Oxford; Chinese Academy of Medical Sciences Oxford Institute, University of Oxford)
Co-authored by industrial partner: No

Type: Journal Paper
Journal: Journal Of Visualized Experiments

State: Published (Approved)
Published: July 2022

Abstract: Cryo-electron tomography (cryo-ET) has been gaining momentum in recent years, especially since the introduction of direct electron detectors, improved automated acquisition strategies, preparative techniques that expand the possibilities of what the electron microscope can image at high-resolution using cryo-ET and new subtomogram averaging software. Additionally, data acquisition has become increasingly streamlined, making it more accessible to many users. The SARS-CoV-2 pandemic has further accelerated remote cryo-electron microscopy (cryo-EM) data collection, especially for single-particle cryo-EM, in many facilities globally, providing uninterrupted user access to state-of-the-art instruments during the pandemic. With the recent advances in Tomo5 (software for 3D electron tomography), remote cryo-ET data collection has become robust and easy to handle from anywhere in the world. This article aims to provide a detailed walk-through, starting from the data collection setup in the tomography software for the process of a (remote) cryo-ET data collection session with detailed troubleshooting. The (remote) data collection protocol is further complemented with the workflow for structure determination at near-atomic resolution by subtomogram averaging with emClarity, using apoferritin as an example.

Subject Areas: Biology and Bio-materials

Diamond Offline Facilities: Electron Bio-Imaging Centre (eBIC)
Instruments: Krios I-Titan Krios I at Diamond

Added On: 27/07/2022 14:06

Documents:
jove-protocol-63923-cryo-electron-tomography-remote-data-collection-subtomogram.pdf

Discipline Tags:

Structural biology Life Sciences & Biotech

Technical Tags:

Imaging Tomography Cryo Electron Tomography (Cryo ET)