Shaikhqasem, A., Hamdi, F., Machner, L., Parthier, C., Breithaupt, C., Kyrilis, F. L., Feller, S. M., Kastritis, P. L., & Stubbs, M. T.
Strategies for mitigating radiation damage and improving data completeness in 3D electron diffraction of protein crystals. Acta Crystallographica. Section D, Structural Biology, 82(Pt 1), 11–22. https://doi.org/10.1107/S2059798325011258
Published: January 2026
Abstract: While 3D electron diffraction (3D-ED or microcrystal electron diffraction; MicroED) has emerged as a promising method for protein structure determination, its applicability is hindered by a high susceptibility to radiation damage, leading to a decreasing signal-to-noise ratio in consecutive diffraction patterns that limits the quality (resolution and redundancy) of the data. In addition, data completeness may be restricted due to the geometrical limitations of current sample holders and stages. Although specialized equipment can overcome these challenges, many laboratories do not have access to such instrumentation. In this work, we introduce an approach that addresses these issues using a commonly available 200 keV cryo-electron microscope. The multi-position acquisition technique that we present here combines (i) multiple data acquisitions from a single crystal over several tilt ranges and (ii) merging data from a small number of crystals each tilted about a different axis. The robustness of this approach is demonstrated by the de novo elucidation of a protein–peptide complex structure from only two orthorhombic microcrystals.
Keywords: electron diffraction; radiation damage; data completeness; 3D-ED; MicroED.
© 2026 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. (http://creativecommons.org/licenses/by/4.0/).