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

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.

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Supporting information: 3D-Files to re-generate the HARE-chip crystallization tray (txt), Crystal hit-maps (pdf)
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 Creators:
Norton-Baker, B.1, 2, Author              
Mehrabi, P.1, 3, Author              
Boger, J.4, Author
Schönherr, R.4, 5, Author
von Stetten, D.6, Author
Schikora, H.7, Author              
Kwok, A. O.2, Author
Martin, R. W.2, 8, Author
Miller, R. J. Dwayne9, 10, Author
Redecke, L.4, 5, Author
Schulz, E.-C.1, 3, Author              
Affiliations:
1Miller Group, Atomically Resolved Dynamics Department, Max Planck Institute for the Structure and Dynamics of Matter, Max Planck Society, ou_1938288              
2Department of Chemistry, University of California, ou_persistent22              
3Hamburg Centre for Ultrafast Imaging, Universität Hamburg, ou_persistent22              
4Institute of Biochemistry, Center for Structural and Cell Biology in Medicine, University of Lübeck, ou_persistent22              
5Photon Science, Deutsches Elektronen-Synchrotron (DESY), ou_persistent22              
6European Molecular Biology Laboratory, Hamburg Unit c/o Deutsches Elektronen-Synchrotron, ou_persistent22              
7Machine Physics, Scientific Service Units, Max Planck Institute for the Structure and Dynamics of Matter, Max Planck Society, ou_2074322              
8Department of Molecular Biology and Biochemistry, University of California, ou_persistent22              
9Department of Physics, Universität Hamburg, ou_persistent22              
10Departments of Chemistry and Physics, University of Toronto, ou_persistent22              

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Free keywords: fixed-target crystallography; serial crystallography; protein crystallization; in cellulo crystallization; in vivo crystals
 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.

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Language(s): eng - English
 Dates: 2021-02-182021-04-102021-05-19
 Publication Status: Published online
 Pages: -
 Publishing info: -
 Table of Contents: -
 Rev. Type: Peer
 Identifiers: DOI: 10.1107/S2059798321003855
 Degree: -

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Title: Acta Crystallographica Section D: Structural Biology
  Abbreviation : Acta Cryst. D
Source Genre: Journal
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Publ. Info: Chester, England : International Union of Crystallography
Pages: - Volume / Issue: 77 (6) Sequence Number: D77 Start / End Page: - Identifier: ISSN: 2059-7983
CoNE: https://pure.mpg.de/cone/journals/resource/2059-7983