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Ab initio phasing macromolecular structures using electron-counted MicroED data

Michael W. Martynowycz, Max T.B. Clabbers, Johan Hattne, Tamir Gonen
doi: https://doi.org/10.1101/2021.10.16.464672
Michael W. Martynowycz
1Howard Hughes Medical Institute, University of California, Los Angeles CA 90095
2Department of Biological Chemistry, University of California, Los Angeles CA 90095
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Max T.B. Clabbers
2Department of Biological Chemistry, University of California, Los Angeles CA 90095
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Johan Hattne
1Howard Hughes Medical Institute, University of California, Los Angeles CA 90095
2Department of Biological Chemistry, University of California, Los Angeles CA 90095
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Tamir Gonen
1Howard Hughes Medical Institute, University of California, Los Angeles CA 90095
2Department of Biological Chemistry, University of California, Los Angeles CA 90095
3Department of Physiology, University of California, Los Angeles CA 90095
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  • For correspondence: tgonen@g.ucla.edu
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Abstract

Structures of two globular proteins were determined ab initio using microcrystal electron diffraction (MicroED) data that was collected on a direct electron detector in counting mode. Microcrystals were identified using a scanning electron microscope (SEM) and thinned with a focused ion-beam (FIB) to produce crystalline lamellae of ideal thickness. Continuous rotation data were collected using an ultra-low exposure rate on a Falcon 4 direct electron detector in electron-counting mode. For the first sample, triclinic lysozyme extending to 0.87 Å resolution, an ideal helical fragment of only three alanine residues provided initial phases. These phases were improved using density modification, allowing the entire atomic structure to be built automatically. A similar approach was successful on a second macromolecular sample, proteinase K, which is much larger and diffracted to a modest 1.5 Å resolution. These results demonstrate that macromolecules can be determined to sub-Ångström resolution by MicroED and that ab initio phasing can be successfully applied to counting data collected on a direct electron detector.

Competing Interest Statement

The authors have declared no competing interest.

Copyright 
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.
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Posted October 17, 2021.
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Ab initio phasing macromolecular structures using electron-counted MicroED data
Michael W. Martynowycz, Max T.B. Clabbers, Johan Hattne, Tamir Gonen
bioRxiv 2021.10.16.464672; doi: https://doi.org/10.1101/2021.10.16.464672
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Ab initio phasing macromolecular structures using electron-counted MicroED data
Michael W. Martynowycz, Max T.B. Clabbers, Johan Hattne, Tamir Gonen
bioRxiv 2021.10.16.464672; doi: https://doi.org/10.1101/2021.10.16.464672

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