Automated cryo-lamella preparation for high-throughput in-situ structural biology

Genevieve Buckley; Gediminas Gervinskas; Cyntia Taveneau; Hariprasad Venugopal; James C Whisstock; Alex de Marco

doi:10.1016/j.jsb.2020.107488

Automated cryo-lamella preparation for high-throughput in-situ structural biology

J Struct Biol. 2020 May 1;210(2):107488. doi: 10.1016/j.jsb.2020.107488. Epub 2020 Feb 29.

Authors

Genevieve Buckley¹, Gediminas Gervinskas², Cyntia Taveneau¹, Hariprasad Venugopal², James C Whisstock³, Alex de Marco⁴

Affiliations

¹ ARC Centre of Excellence in Advanced Molecular Imaging, Monash University, Australia; Biomedicine Discovery Institute, Department of Biochemistry and Molecular Biology, Monash University, Clayton, Australia.
² Clive and Vera Ramaciotti Centre for Cryo-Electron Microscopy, Monash University, Clayton, Australia.
³ ARC Centre of Excellence in Advanced Molecular Imaging, Monash University, Australia; Biomedicine Discovery Institute, Department of Biochemistry and Molecular Biology, Monash University, Clayton, Australia; University of Warwick, Coventry CV4, 7AL, United Kingdom; EMBL Australia, Monash University, Clayton, Australia.
⁴ ARC Centre of Excellence in Advanced Molecular Imaging, Monash University, Australia; Biomedicine Discovery Institute, Department of Biochemistry and Molecular Biology, Monash University, Clayton, Australia; University of Warwick, Coventry CV4, 7AL, United Kingdom. Electronic address: alex.demarco@monash.edu.

PMID: 32126263
DOI: 10.1016/j.jsb.2020.107488

Abstract

Cryo-transmission electron tomography (cryo-ET) in association with cryo-focused ion beam (cryo-FIB) milling enables structural biology studies to be performed directly within the cellular environment. Cryo-preserved cells are milled and a lamella with a typical thickness of 200-300 nm provides an electron transparent window suitable for cryo-ET imaging. Cryo-FIB milling is an effective method, but it is a tedious and time-consuming process, which typically results in ~10 lamellae per day. Here, we introduce an automated method to reproducibly prepare cryo-lamellae on a grid and reduce the amount of human supervision. We tested the routine on cryo-preserved Saccharomyces cerevisiae, mammalian 293 T cells, and lysozyme protein crystals. Here we demonstrate that our method allows an increased throughput, achieving a rate of 5 lamellae/hour without the need to supervise the FIB milling. We demonstrate that the quality of the lamellae is consistent throughout the preparation and their compatibility with cryo-ET analyses.

Keywords: Automation; Cryo-FIB; Cryo-lamella; In situ structural biology; cryo-EM.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Animals
Cryoelectron Microscopy / methods*
Electron Microscope Tomography / methods
Humans
Microscopy, Electron, Transmission / methods*