Sample-efficient time-resolved X-ray crystallography with serial small wedges

Bukhdruker, S.; Astashkin, R.; Bratanov, D.; Caramello, N.; Engilberge, S.; Baeken, C.; Balandin, T.; Popov, A.; Royant, A.; Mueller-Dieckmann, C.; Soler-Lopez, M.; Bamberg, E.; Gordeliy, V.; Melnikov, I.

doi:10.64898/2026.10.04.756486

bioRxiv · 10.64898/2026.10.04.756486

Sample-efficient time-resolved X-ray crystallography with serial small wedges

Bukhdruker, S.·Astashkin, R.·Bratanov, D.·Caramello, N.·Engilberge, S.·Baeken, C.·Balandin, T.·Popov, A.·Royant, A.·Mueller-Dieckmann, C.·Soler-Lopez, M.·Bamberg, E.·Gordeliy, V.·Melnikov, I.

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Abstract

Time-resolved crystallography can be inaccessible for membrane proteins and other difficult-to-crystallize targets for which suitable crystals are scarce. Here we present an acquisition strategy that records consecutive diffraction images during a small crystal rotation synchronized to a single activation pulse. Matching image segments are merged across crystals to reconstruct successive time windows, enabling a single crystal ensemble to capture an extended structural trajectory. Using the Mesh&Collect workflow at a synchrotron, we obtained ten consecutive 7.5-ms time windows at 2 [A] resolution from 1,255 microcrystals of a bacteriorhodopsin triple mutant at room temperature. An additional 790 crystals extended the observation window to 500 ms. Rotation-based processing, retrospective time binning and dose control maximize information extracted from each crystal, while compatible measurements can be pooled across acquisitions. This approach makes millisecond time-resolved crystallography practical for crystal-limited targets at synchrotrons, with further development potentially extending the temporal resolution down to 1 ms.

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Bukhdruker, S., Astashkin, R., Bratanov, D., Caramello, N., Engilberge, S., Baeken, C., Balandin, T., Popov, A., Royant, A., Mueller-Dieckmann, C., Soler-Lopez, M., Bamberg, E., Gordeliy, V., Melnikov, I.. 2026-10-09. Sample-efficient time-resolved X-ray crystallography with serial small wedges. https://doi.org/10.64898/2026.10.04.756486

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