Ice thickness control and measurement in the VitroJet for time-efficient single particle structure determination
Embedding biomolecules in vitreous ice of optimal thickness is critical for structure determination by cryo-electron microscopy. Ice thickness assessment and selection of suitable holes for data collection are currently part of time-consuming preparatory routines performed on expensive electron microscopes. To address this challenge, a routine has been developed to measure ice thickness during sample preparation using an optical camera integrated in the VitroJet. This method allows to estimate the ice thickness with an error below ±20 nm for ice layers in the range of 0-70 nm. Additionally, we characterized the influence of pin printing parameters and found that the median ice thickness can be reproduced with a standard deviation below ±11 nm for thicknesses up to 75 nm. Therefore, the ice thickness of buffer-suspended holes on an EM grid can be tuned and measured within the working range relevant for single particle cryo-EM. Single particle structures of apoferritin were determined at two distinct thicknesses of 30 nm and 70 nm. These reconstructions demonstrate the importance of ice thickness for time-efficient cryo-EM structure determination.
Authors
- Simone Matteï (ORCID: https://orcid.org/0000-0002-2005-8977)
- Roger Jeurissen
- Carsten Sachse (ORCID: https://orcid.org/0000-0002-1168-5143)
- Dariush Ashtiani
- Bart Beulen (ORCID: https://orcid.org/0000-0001-7673-2572)
- Peter J. Peters (ORCID: https://orcid.org/0000-0002-2964-5684)
- Wim J. H. Hagen (ORCID: https://orcid.org/0000-0001-6229-2692)
- Simon A. Fromm (ORCID: https://orcid.org/0000-0002-2094-1911)
- René Henderikx
- Maaike J. G. Schotman (ORCID: https://orcid.org/0000-0003-4826-3646)
- Daniel Mann (ORCID: https://orcid.org/0000-0002-9106-9358)
- Julian Hennies (ORCID: https://orcid.org/0000-0002-0555-151X)
- Thomas Heidler (ORCID: https://orcid.org/0000-0002-5997-3945)
- Saba Shahzad
Institutions
- Forschungszentrum Jülich (DE)
- Ernst Ruska Centre (DE)
- Maastricht University (NL)
- St.Jans Gasthuis Weert (NL)
- European Molecular Biology Laboratory (DE)
- European Molecular Biology Laboratory (DE)
- Heinrich Heine University Düsseldorf (DE)
- CryoLife (United States) (US)
- University of Twente (NL)
Publication Details
- Journal
- Journal of Structural Biology
- Published
- 2024-10-19
- DOI
- https://doi.org/10.1016/j.jsb.2024.108139
- Citations
- 3
- Primary Topic
- Icing and De-icing Technologies
- Type
- article
- Field-Weighted Citation Impact
- 3.90
Funders
- European Molecular Biology Laboratory
- Forschungszentrum Jülich
- Nederlandse Organisatie voor Wetenschappelijk Onderzoek
- Boehringer Ingelheim Stiftung