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Cryo-Electron Tomography, Faster: Development of a Fast-Incremental Tilting Scheme for Rapid Tomogram Acquisition

Chreifi, Georges and Chen, Songye and Metskas, Lauren Ann and Mastronarde, David and Jensen, Grant J. (2019) Cryo-Electron Tomography, Faster: Development of a Fast-Incremental Tilting Scheme for Rapid Tomogram Acquisition. Biophysical Journal, 116 (3). 575a. ISSN 0006-3495. https://resolver.caltech.edu/CaltechAUTHORS:20190219-101505920

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Abstract

Cryo-electron microscopy enables high-resolution imaging for both protein structure determination and cellular imaging studies. When a sample's ultrastructure is unique, cryo-electron tomography can generate a volume image from a single sample, allowing the study of cellular architecture or polymorphic viruses. This technique requires tilting the microscope stage between each exposure, such that a collection of exposures is built into a stack that can be reconstructed into a three-dimensional volume. Proteins within these volumes can be averaged, with sub-tomogram averaged structures capable of reaching high resolution. However, each tomogram can require between 20-60 minutes of data collection time, restricting the number of tomograms that a user can collect in time-limited microscopy sessions. As the final resolution of sub-tomogram averages is dependent upon the number of particles in the dataset, the number of tomograms collected is severely limiting in cases where protein copy number is low. The Jensen lab has developed a “fast-incremental” tilt scheme, whereby a single tomogram can be collected in 3-10 minutes. An FEI Titan Krios cryo-electron microscope and Gatan K2 camera are operated in continuous acquisition mode, using SerialEM software to blank the electron beam during stage movement; the resulting acquisition is similar in geometry and exposure to a conventional tomogram. We find that the precision of a single-tilt stage and a camera with fast frame capture rates and high sensitivity compensate for the lack of time-consuming stage tracking and adjustments during data collection, to a resolution of roughly 2.5 nm. Further hardware and software development will improve the resolution and utility of this method.


Item Type:Article
Related URLs:
URLURL TypeDescription
https://doi.org/10.1016/j.bpj.2018.11.3091DOIArticle
ORCID:
AuthorORCID
Chen, Songye0000-0001-5407-5049
Metskas, Lauren Ann0000-0002-8073-6960
Jensen, Grant J.0000-0003-1556-4864
Additional Information:© 2019 Biophysical Society. Available online 15 February 2019.
Issue or Number:3
Record Number:CaltechAUTHORS:20190219-101505920
Persistent URL:https://resolver.caltech.edu/CaltechAUTHORS:20190219-101505920
Official Citation:Georges Chreifi, Songye Chen, Lauren Ann Metskas, David Mastronarde, Grant J. Jensen, Cryo-Electron Tomography, Faster: Development of a Fast-Incremental Tilting Scheme for Rapid Tomogram Acquisition, Biophysical Journal, Volume 116, Issue 3, Supplement 1, 2019, Page 575a, ISSN 0006-3495, https://doi.org/10.1016/j.bpj.2018.11.3091. (http://www.sciencedirect.com/science/article/pii/S000634951834356X)
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:92969
Collection:CaltechAUTHORS
Deposited By: Tony Diaz
Deposited On:19 Feb 2019 18:26
Last Modified:03 Oct 2019 20:50

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