Journal Article

FZJ-2024-06070

http://join2-wiki.gsi.de/foswiki/pub/Main/Artwork/join2_logo100x88.png Ice thickness control and measurement in the VitroJet for time-efficient single particle structure determination

Henderikx, R. J. M. (Corresponding author) ; Schotman, M. J. G. ; Shahzad, S.FZJ* ; Fromm, S. A. ; Mann, D.FZJ* ; Hennies, J. ; Heidler, T. V.FZJ* ; Ashtiani, D. ; Hagen, W. J. H. ; Jeurissen, R. J. M. ; Mattei, S. ; Peters, P. J. ; Sachse, C.FZJ* ; Beulen, B. W. A. M. M. (Corresponding author)

2024
Elsevier San Diego, Calif.

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Please use a persistent id in citations: doi:10.1016/j.jsb.2024.108139  doi:10.34734/FZJ-2024-06070

Abstract: Embedding biomolecules in vitreous ice of optimal thickness is critical for structure determination by cryoelectronmicroscopy. Ice thickness assessment and selection of suitable holes for data collection are currentlypart of time-consuming preparatory routines performed on expensive electron microscopes. To address thischallenge, a routine has been developed to measure ice thickness during sample preparation using an opticalcamera integrated in the VitroJet. This method allows to estimate the ice thickness with an error below ±20 nmfor ice layers in the range of 0–70 nm. Additionally, we characterized the influence of pin printing parametersand found that the median ice thickness can be reproduced with a standard deviation below ±11 nm forthicknesses up to 75 nm. Therefore, the ice thickness of buffer-suspended holes on an EM grid can be tuned andmeasured within the working range relevant for single particle cryo-EM. Single particle structures of apoferritinwere determined at two distinct thicknesses of 30 nm and 70 nm. These reconstructions demonstrate theimportance of ice thickness for time-efficient cryo-EM structure determination.

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Contributing Institute(s):

1. Strukturbiologie (ER-C-3)

Research Program(s):

1. 5352 - Understanding the Functionality of Soft Matter and Biomolecular Systems (POF4-535) (POF4-535)
2. 5241 - Molecular Information Processing in Cellular Systems (POF4-524) (POF4-524)

Appears in the scientific report 2024

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Database coverage:
; ; ; BIOSIS Previews ; Biological Abstracts ; Clarivate Analytics Master Journal List ; Current Contents - Life Sciences ; Ebsco Academic Search ; Essential Science Indicators ; IF < 5 ; JCR ; Nationallizenz ; SCOPUS ; Science Citation Index Expanded ; Web of Science Core Collection ; Zoological Record

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