Beam image-shift accelerated data acquisition for near-atomic resolution single-particle cryo-electron tomography

Jonathan Bouvette,Hsuan-Fu Liu,Rick Huang,Alberto Bartesaghi,Mario J Borgnia,Ye Zhou,Roel M Schaaper,Xiaochen Du,Bradley P Klemm,Andrew P Sikkema,Juliana Da Fonseca Rezende E Mello

doi:10.1038/s41467-021-22251-8

Abstract

Tomographic reconstruction of cryopreserved specimens imaged in an electron microscope followed by extraction and averaging of sub-volumes has been successfully used to derive atomic models of macromolecules in their biological environment. Eliminating biochemical isolation steps required by other techniques, this method opens up the cell to in-situ structural studies. However, the need to compensate for errors in targeting introduced during mechanical navigation of the specimen significantly slows down tomographic data collection thus limiting its practical value. Here, we introduce protocols for tilt-series acquisition and processing that accelerate data collection speed by up to an order of magnitude and improve map resolution compared to existing approaches. We achieve this by using beam-image shift to multiply the number of areas imaged at each stage position, by integrating geometrical constraints during imaging to achieve high precision targeting, and by performing per-tilt astigmatic CTF estimation and data-driven exposure weighting to improve final map resolution. We validated our beam image-shift electron cryo-tomography (BISECT) approach by determining the structure of a low molecular weight target (~300 kDa) at 3.6 Å resolution where density for individual side chains is clearly resolved.

Highlights

Tomographic reconstruction of cryopreserved specimens imaged in an electron microscope followed by extraction and averaging of sub-volumes has been successfully used to derive atomic models of macromolecules in their biological environment
The first sub-nanometer resolution structures determined by cryo-electron tomography (CET) were obtained using the Constrained Single Particle Tomography (CSPT) approach[6], where the idea of working directly with the raw 2D projections extracted from the tilt-series and performing an Single-particle analysis (SPA)-like reconstruction was initially introduced
Achieving sub-nanometer resolution in CET requires the combination of multiple tightly overlapping projections of each regions of interest (ROI) recorded from different angles and with a well-defined defocus

Summary

Introduction

Tomographic reconstruction of cryopreserved specimens imaged in an electron microscope followed by extraction and averaging of sub-volumes has been successfully used to derive atomic models of macromolecules in their biological environment. While the raw projections still carry information from the surrounding environment, image alignment based on the features of the repetitive structure will cause the variable signal from the background to be averaged out during 3D reconstruction This hybrid strategy provides a convenient framework to analyze low contrast tomographic projections and produce high-resolution 3D maps by combining established principles for SPA image alignment and reconstruction, with enforcement of the tilt-geometry constraints. Despite these and other advances, fundamental technical barriers still exist that have limited the application of near-atomic resolution CET/SVA to either large molecular weight targets like ribosomes[7], or samples that display favorable spatial arrangements such as those present in quasi-crystalline formations of HIV-1 Gag polyprotein[8,9]. This method can be implemented on instruments without specialized stages, and by virtue of limiting the number of mechanical movements required for navigation, it reduces the wear on the stage

Methods

Results

Conclusion