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Journal Article

A simple vapor-diffusion method enables protein crystallization inside the HARE serial crystallography chip

MPS-Authors
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Norton-Baker,  B.
Miller Group, Atomically Resolved Dynamics Department, Max Planck Institute for the Structure and Dynamics of Matter, Max Planck Society;
Department of Chemistry, University of California;

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Mehrabi,  P.
Miller Group, Atomically Resolved Dynamics Department, Max Planck Institute for the Structure and Dynamics of Matter, Max Planck Society;
Hamburg Centre for Ultrafast Imaging, Universität Hamburg;

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Schikora,  H.
Machine Physics, Scientific Service Units, Max Planck Institute for the Structure and Dynamics of Matter, Max Planck Society;

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Schulz,  E.-C.
Miller Group, Atomically Resolved Dynamics Department, Max Planck Institute for the Structure and Dynamics of Matter, Max Planck Society;
Hamburg Centre for Ultrafast Imaging, Universität Hamburg;

Fulltext (public)

nj5304.pdf
(Publisher version), 3MB

Supplementary Material (public)

suppl.zip
(Supplementary material), 8MB

Citation

Norton-Baker, B., Mehrabi, P., Boger, J., Schönherr, R., von Stetten, D., Schikora, H., et al. (2021). A simple vapor-diffusion method enables protein crystallization inside the HARE serial crystallography chip. Acta Crystallographica Section D: Structural Biology, 77(6): D77. doi:10.1107/S2059798321003855.


Cite as: http://hdl.handle.net/21.11116/0000-0008-9597-5
Abstract
Fixed-target serial crystallography has become an important method for the study of protein structure and dynamics at synchrotrons and X-ray free-electron lasers. However, sample homogeneity, consumption and the physical stress on samples remain major challenges for these high-throughput experiments, which depend on high-quality protein microcrystals. The batch crystallization procedures that are typically applied require time- and sample-intensive screening and optimization. Here, a simple protein crystallization method inside the features of the HARE serial crystallography chips is reported that circumvents batch crystallization and allows the direct transfer of canonical vapor-diffusion conditions to in-chip crystallization. Based on conventional hanging-drop vapor-diffusion experiments, the crystallization solution is distributed into the wells of the HARE chip and equilibrated against a reservoir with mother liquor. Using this simple method, high-quality microcrystals were generated with sufficient density for the structure determination of four different proteins. A new protein variant was crystallized using the protein concentrations encountered during canonical crystallization experiments, enabling structure determination from ∼55 µg of protein. Additionally, structure determination from intracellular crystals grown in insect cells cultured directly in the features of the HARE chips is demonstrated. In cellulo crystallization represents a comparatively un­explored space in crystallization, especially for proteins that are resistant to crystallization using conventional techniques, and eliminates any need for laborious protein purification. This in-chip technique avoids harvesting the sensitive crystals or any further physical handling of the crystal-containing cells. These proof-of-principle experiments indicate the potential of this method to become a simple alternative to batch crystallization approaches and also as a convenient extension to canonical crystallization screens.