TY  - JOUR
AU  - Norton-Baker, Brenna
AU  - Mehrabi, Pedram
AU  - Boger, Juliane
AU  - Schoenherr, Robert Frank
AU  - von Stetten, David
AU  - Schikora, Hendrik
AU  - Kwok, Ashley O.
AU  - Martin, Rachel W.
AU  - Miller, R. J. Dwayne
AU  - Redecke, Lars
AU  - Schulz, Eike C.
TI  - A simple vapor-diffusion method enables protein crystallization inside the HARE serial crystallography chip
JO  - Acta crystallographica / Section D
VL  - 77
IS  - 6
SN  - 2059-7983
CY  - Bognor Regis
PB  - Wiley
M1  - PUBDB-2021-02689
SP  - 820 - 834
PY  - 2021
AB  - 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.
LB  - PUB:(DE-HGF)16
C6  - pmid:34076595
UR  - <Go to ISI:>//WOS:000659143800009
DO  - DOI:10.1107/S2059798321003855
UR  - https://bib-pubdb1.desy.de/record/459716
ER  -