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Challenges in solving structures from radiation-damaged tomograms of protein nanocrystals assessed by simulation

Peck, Ariana and Yao, Qing and Brewster, Aaron S. and Zwart, Petrus H. and Heumann, John M. and Sauter, Nicholas K. and Jensen, Grant J. (2021) Challenges in solving structures from radiation-damaged tomograms of protein nanocrystals assessed by simulation. Acta Crystallographica Section D: Biological Crystallography, D77 (5). pp. 572-586. ISSN 0907-4449. PMCID PMC8098477. doi:10.1107/S2059798321002369.

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Structure-determination methods are needed to resolve the atomic details that underlie protein function. X-ray crystallography has provided most of our knowledge of protein structure, but is constrained by the need for large, well ordered crystals and the loss of phase information. The rapidly developing methods of serial femtosecond crystallography, micro-electron diffraction and single-particle reconstruction circumvent the first of these limitations by enabling data collection from nanocrystals or purified proteins. However, the first two methods also suffer from the phase problem, while many proteins fall below the molecular-weight threshold required for single-particle reconstruction. Cryo-electron tomography of protein nanocrystals has the potential to overcome these obstacles of mainstream structure-determination methods. Here, a data-processing scheme is presented that combines routines from X-ray crystallography and new algorithms that have been developed to solve structures from tomograms of nanocrystals. This pipeline handles image-processing challenges specific to tomographic sampling of periodic specimens and is validated using simulated crystals. The tolerance of this workflow to the effects of radiation damage is also assessed. The simulations indicate a trade-off between a wider tilt range to facilitate merging data from multiple tomograms and a smaller tilt increment to improve phase accuracy. Since phase errors, but not merging errors, can be overcome with additional data sets, these results recommend distributing the dose over a wide angular range rather than using a finer sampling interval to solve the protein structure.

Item Type:Article
Related URLs:
URLURL TypeDescription CentralArticle Paper ItemCode
Peck, Ariana0000-0002-5940-3897
Yao, Qing0000-0003-3575-9909
Brewster, Aaron S.0000-0002-0908-7822
Zwart, Petrus H.0000-0003-3315-4092
Heumann, John M.0000-0001-6751-3028
Sauter, Nicholas K.0000-0003-2786-6552
Jensen, Grant J.0000-0003-1556-4864
Additional Information:© 2021 International Union of Crystallography. This is an open-access article distributed under the terms of the Creative Commons Attribution (CC-BY) Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original authors and source are cited. Received 19 September 2020; Accepted 2 March 2021. We thank Lauren Ann Metskas and Florian Schur for valuable discussions, in addition to David Stokes and Steven Ludtke for advice on the phase-splitting phenomenon. AP is The Mark Foundation for Cancer Research Fellow of the Damon Runyon Cancer Research Foundation (DRG 2361-19). This work was supported by NIH grants R35 GM122588 (to GJJ), AI150464 (to GJJ) and GM117126 (to NKS). Code availability: The code developed to process tomograms of nanocrystals is available at
Funding AgencyGrant Number
Damon Runyon Cancer Research FoundationDRG 2361-19
NIHR35 GM122588
Subject Keywords:cryo-electron tomography; diffraction methods; nanocrystals; radiation damage
Issue or Number:5
PubMed Central ID:PMC8098477
Record Number:CaltechAUTHORS:20200922-102800972
Persistent URL:
Official Citation:Challenges in solving structures from radiation-damaged tomograms of protein nanocrystals assessed by simulation. Peck, A., Yao, Q., Brewster, A. S., Zwart, P. H., Heumann, J. M., Sauter, N. K. & Jensen, G. J. (2021). Acta Cryst. D77, 572-586; DOI: 10.1107/S2059798321002369
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:105468
Deposited By: Tony Diaz
Deposited On:22 Sep 2020 17:43
Last Modified:26 May 2021 16:35

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