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Benchmarking ideal sample thickness in cryo-EM using MicroED

Michael W. Martynowycz, Max T.B. Clabbers, Johan Unge, Johan Hattne, Tamir Gonen
doi: https://doi.org/10.1101/2021.07.02.450941
Michael W. Martynowycz
1Howard Hughes Medical Institute, University of California Los Angeles, Los Angeles CA 90095
2Department of Biological Chemistry, University of California Los Angeles, Los Angeles CA 90095
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Max T.B. Clabbers
2Department of Biological Chemistry, University of California Los Angeles, Los Angeles CA 90095
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Johan Unge
2Department of Biological Chemistry, University of California Los Angeles, Los Angeles CA 90095
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Johan Hattne
1Howard Hughes Medical Institute, University of California Los Angeles, Los Angeles CA 90095
2Department of Biological Chemistry, University of California Los Angeles, Los Angeles CA 90095
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Tamir Gonen
1Howard Hughes Medical Institute, University of California Los Angeles, Los Angeles CA 90095
2Department of Biological Chemistry, University of California Los Angeles, Los Angeles CA 90095
3Department of Physiology, University of California Los Angeles, Los Angeles CA 90095
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  • For correspondence: tgonen@g.ucla.edu
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Abstract

The relationship between sample thickness and quality of data obtained by microcrystal electron diffraction (MicroED) is investigated. Several EM grids containing proteinase K microcrystals of similar sizes from the same crystallization batch were prepared. Each grid was transferred into a focused ion-beam scanning electron microscope (FIB/SEM) where the crystals were then systematically thinned into lamellae between 95 nm and 1650 nm thick. MicroED data were collected at either 120, 200, or 300 kV accelerating voltages. Lamellae thicknesses were converted to multiples of the calculated inelastic mean free path (MFP) of electrons at each accelerating voltage to allow the results to be compared on a common scale. The quality of the data and subsequently determined structures were assessed using standard crystallographic measures. Structures were reliably determined from crystalline lamellae only up to twice the inelastic mean free path. Lower resolution diffraction was observed at three times the mean free path for all three accelerating voltages but the quality was insufficient to yield structures. No diffraction data were observed from lamellae thicker than four times the calculated inelastic mean free path. The quality of the determined structures and crystallographic statistics were similar for all lamellae up to 2x the inelastic mean free path in thickness, but quickly deteriorated at greater thicknesses. This study provides a benchmark with respect to the ideal limit for biological specimen thickness with implications for all cryo-EM methods.

Significance A systematic investigation of the effects of thickness on electron scattering from protein crystals was previously not feasible, because there was no accurate method to control sample thickness. Here, the recently developed methods for preparing protein crystals into lamellae of precise thickness by ion-beam milling are used to investigate the effects of increasing sample thickness on MicroED data quality. These experiments were conducted using the three most common accelerating voltages in cryo-EM. Data across these accelerating voltages and thicknesses were compared on a common scale using their calculated inelastic mean free path lengths. It is found that structures may accurately be determined from crystals up to twice the inelastic mean free path length in thickness, regardless of the acceleration voltage.

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 July 03, 2021.
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Benchmarking ideal sample thickness in cryo-EM using MicroED
Michael W. Martynowycz, Max T.B. Clabbers, Johan Unge, Johan Hattne, Tamir Gonen
bioRxiv 2021.07.02.450941; doi: https://doi.org/10.1101/2021.07.02.450941
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Benchmarking ideal sample thickness in cryo-EM using MicroED
Michael W. Martynowycz, Max T.B. Clabbers, Johan Unge, Johan Hattne, Tamir Gonen
bioRxiv 2021.07.02.450941; doi: https://doi.org/10.1101/2021.07.02.450941

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