Strategies for Mitigating Radiation Damage and Improving Data Completeness in 3D Electron Diffraction of Protein Crystals

Shaikhqasem, A.; Hamdi, F.; Machner, L.; Parthier, C.; Breithaupt, C.; Kyrilis, F. L.; Feller, S. M.; Kastritis, P. L.; Stubbs, M. T.

doi:10.1101/2025.02.06.636927

bioRxiv · 10.1101/2025.02.06.636927

Strategies for Mitigating Radiation Damage and Improving Data Completeness in 3D Electron Diffraction of Protein Crystals

Shaikhqasem, A.·Hamdi, F.·Machner, L.·Parthier, C.·Breithaupt, C.·Kyrilis, F. L.·Feller, S. M.·Kastritis, P. L.·Stubbs, M. T.

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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 high susceptibility to radiation damage, leading to 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. In this work, we introduce an approach that addresses these issues using a commonly available 200keV cryo-electron microscope. The multi-position acquisition technique we present here combines (a) multiple data acquisitions from a single crystal over several tilt ranges and (b) 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.

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Shaikhqasem, A., Hamdi, F., Machner, L., Parthier, C., Breithaupt, C., Kyrilis, F. L., Feller, S. M., Kastritis, P. L., Stubbs, M. T.. 2025-02-06. Strategies for Mitigating Radiation Damage and Improving Data Completeness in 3D Electron Diffraction of Protein Crystals. https://doi.org/10.1101/2025.02.06.636927

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