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Qualitative Analyses of Polishing and Precoating 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 Precoating FIB Milled Crystals for MicroED. Structure, 27 (10). pp. 1594-1600. ISSN 0969-2126. https://resolver.caltech.edu/CaltechAUTHORS:20190815-100854997

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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) milling can thin 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 the platinum-precoated and uncoated samples. Our results suggest that precoating 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-to-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:Article
Related URLs:
URLURL TypeDescription
https://doi.org/10.1016/j.str.2019.07.004DOIArticle
ORCID:
AuthorORCID
Jensen, Grant J.0000-0003-1556-4864
Additional Information:© 2019 Elsevier Ltd. Received 17 April 2019, Revised 12 June 2019, Accepted 15 July 2019, Available online 15 August 2019. The Gonen lab and the Jensen lab are supported by funds from the Howard Hughes Medical Institute. This study was supported grants from the US National Institutes of Health nos. 2P50GM082545 and R35 GM122588 to G.J.J. Author Contributions: M.W.M. prepared the protein crystals. W.Z. and M.W.M. collected the FIB-SEM data and milled the crystals. M.W.M. collected the MicroED data. M.W.M. and J.H. processed the data. The manuscript was written by M.W.M. and T.G. with contributions from all authors. M.W.M., G.J.J., and T.G. designed the experiments. Figures were prepared by M.W.M. and T.G. The authors declare no competing interests.
Funders:
Funding AgencyGrant Number
Howard Hughes Medical Institute (HHMI)UNSPECIFIED
NIH2P50GM082545
NIHR35 GM122588
Subject Keywords:cryoelectron microscopy; cryo-EM; MicroED; FIB-SEM; electron diffraction; electron crystallography
Issue or Number:10
Record Number:CaltechAUTHORS:20190815-100854997
Persistent URL:https://resolver.caltech.edu/CaltechAUTHORS:20190815-100854997
Official Citation:Michael W. Martynowycz, Wei Zhao, Johan Hattne, Grant J. Jensen, Tamir Gonen, Qualitative Analyses of Polishing and Precoating FIB Milled Crystals for MicroED, Structure, Volume 27, Issue 10, 2019, Pages 1594-1600.e2, ISSN 0969-2126, https://doi.org/10.1016/j.str.2019.07.004. (http://www.sciencedirect.com/science/article/pii/S0969212619302369)
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:97917
Collection:CaltechAUTHORS
Deposited By: Tony Diaz
Deposited On:15 Aug 2019 17:14
Last Modified:03 Oct 2019 21:36

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