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Qualitative analyses of polishing and pre-coating FIB milled crystals for MicroED

Martynowycz, Michael W. and Zhao, Wei and Hattne, Johan and Jensen, Grant J. and Gonen, Tamir (2019) Qualitative analyses of polishing and pre-coating FIB milled crystals for MicroED. . (Submitted) http://resolver.caltech.edu/CaltechAUTHORS:20190418-123951811

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Abstract

Microcrystal electron diffraction (MicroED) leverages the strong interaction between matter and electrons to determine protein structures from vanishingly small crystals. This strong interaction limits the thickness of crystals that can be investigated by MicroED, mainly due to absorption. Recent studies have demonstrated that focused ion beam (FIB) can thin even very large crystals into ideal sized lamellae however it is not clear how to best apply FIB-milling for MicroED. Here, The effects of polishing the lamellae, whereby the last few nanometers are milled away using a low-current gallium beam, are explored in both platinum pre-coated and uncoated samples. Our results suggest that pre-coating samples with a thin layer of platinum followed by polishing the crystal surfaces prior to data collection consistently led to superior results as indicated by higher signal/noise ratio, higher resolution and better refinement statistics. This study lays the foundation for routine and reproducible methodology for sample preparation in MicroED.


Item Type:Report or Paper (Discussion Paper)
Related URLs:
URLURL TypeDescription
https://doi.org/10.1101/613042DOIDiscussion Paper
https://www.biorxiv.org/content/10.1101/613042v1OrganizationDiscussion Paper
ORCID:
AuthorORCID
Jensen, Grant J.0000-0003-1556-4864
Additional Information:The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. All rights reserved. No reuse allowed without permission. bioRxiv preprint first posted online Apr. 18, 2019. The Gonen lab and the Jensen lab are supported by funds from the Howard Hughes Medical Institute. Part of the work was done in the Beckman Institute Resource Center for Transmission Electron Microscopy at Caltech. The structures presented in this investigation were deposited in the protein data bank (PDB) under the accession codes: XXXX, and in the electron microscopy data bank (EMDB) under accession codes EMDXXXX.
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Funding AgencyGrant Number
Howard Hughes Medical Institute (HHMI)UNSPECIFIED
Record Number:CaltechAUTHORS:20190418-123951811
Persistent URL:http://resolver.caltech.edu/CaltechAUTHORS:20190418-123951811
Official Citation:Qualitative analyses of polishing and pre-coating FIB milled crystals for MicroED. Michael Martynowycz, Wei Zhao, Johan Hattne, Grant Jensen, Tamir Gonen. bioRxiv 613042; doi: https://doi.org/10.1101/613042
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:94790
Collection:CaltechAUTHORS
Deposited By: Tony Diaz
Deposited On:18 Apr 2019 20:15
Last Modified:18 Apr 2019 20:15

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