Application of super-resolution and correlative double sampling in cryo-electron microscopy

DOI: 10.1039/D2FD00049K

Authors: Yuewen Sheng (Diamond Light Source) , Peter J. Harrison (Diamond Light Source) , Vinod Vogirala (Diamond Light Source) , Zhengyi Yang (Diamond Light Source) , Claire Strain-Damerell (Diamond Light Source; RCaH) , Thomas Frosio (Diamond Light Source) , Benjamin A. Himes (Wellcome Trust Centre for Human Genetics, University of Oxford) , C. Alistair Siebert (Diamond Light Source) , Peijun Zhang (Diamond Light Source; RCaH; Wellcome Trust Centre for Human Genetics, University of Oxford) , Daniel Clare (Diamond Light Source)
Co-authored by industrial partner: No

Type: Journal Paper
Journal: Faraday Discussions

State: Published (Approved)
Published: April 2022
Diamond Proposal Number(s): 26464 , 28151

Abstract: Developments in cryo-EM have allowed atomic or near-atomic resolution structure determination to become routine in single particle analysis (SPA). However, near-atomic resolution structures determined using cryo-electron tomography and sub-tomogram averaging (cryo-ET STA) are much less routine. In this paper, we show that by collecting cryo-ET STA data using the same conditions as SPA, with both Correlated Double Sampling (CDS) and super-resolution mode, allowed apoferritin to be reconstructed out to the physical Nyquist frequency of the images. Even with just two tilt series, STA yields an apoferritin map at 2.9 Å resolution. These results highlight the exciting potential of cryo-ET STA in the future of protein structure determination. While processing SPA data recorded in super-resolution mode may yield structures surpassing the physical Nyquist limit, processing cryo-ET STA data in super-resolution mode gave no additional resolution benefit. We further show that collecting SPA data in super-resolution mode, with CDS activated, reduces the estimated B-factor, leading to a reduction in the number of particles required to reach a target resolution without compromising data size on disk and area imaged in SerialEM. However, collecting SPA data in CDS does reduce throughput, given that a similar resolution structure, with a slightly larger B-factor, is achievable with optimised parameters for speed in EPU (without CDS).

Subject Areas: Technique Development, Biology and Bio-materials

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

Added On: 12/05/2022 09:50

Documents:
d2fd00049k.pdf

Discipline Tags:

Technique Development - Life Sciences & Biotech Structural biology Life Sciences & Biotech

Technical Tags:

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