Journal Article

PUBDB-2021-02689

http://join2-wiki.gsi.de/foswiki/pub/Main/Artwork/join2_logo100x88.png A simple vapor-diffusion method enables protein crystallization inside the HARE serial crystallography chip

Norton-Baker, B.CFEL*MPG*EMBL* ; Mehrabi, P.CFEL*MPG*EMBL*Extern* ; Boger, J. ; Schoenherr, R. F.Extern*DESY* ; von Stetten, D.EMBL* ; Schikora, H.CFEL*MPG* ; Kwok, A. O. ; Martin, R. W.Extern* ; Miller, R. J. D.CFEL*MPG* ; Redecke, L.Extern*DESY* ; Schulz, E. C. (Corresponding author)

2021
Wiley Bognor Regis

This record in other databases:        

Please use a persistent id in citations: doi:10.1107/S2059798321003855  doi:10.3204/PUBDB-2021-02689

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.

---

Contributing Institute(s):

1. FS-Photon Science (FS-PS)
2. EMBL-User (EMBL-User)
3. Universität zu Lübeck (U Lübeck)
4. DOOR-User (DOOR ; HAS-User)

Research Program(s):

1. 632 - Materials – Quantum, Complex and Functional Materials (POF4-632) (POF4-632)
2. 633 - Life Sciences – Building Blocks of Life: Structure and Function (POF4-633) (POF4-633)
3. 6G3 - PETRA III (DESY) (POF4-6G3) (POF4-6G3)
4. DFG project 194651731 - EXC 1074: Hamburger Zentrum für ultraschnelle Beobachtung (CUI): Struktur, Dynamik und Kontrolle von Materie auf atomarer Skala (194651731) (194651731)
5. DFG project 390715994 - EXC 2056: CUI: Advanced Imaging of Matter (390715994) (390715994)

Experiment(s):

1. PETRA Beamline P14 (PETRA III)

Appears in the scientific report 2021

---

Database coverage:
; ; ; BIOSIS Previews ; Biological Abstracts ; Clarivate Analytics Master Journal List ; Current Contents - Life Sciences ; DEAL Wiley ; Ebsco Academic Search ; Essential Science Indicators ; IF >= 5 ; JCR ; PubMed Central ; SCOPUS ; Science Citation Index Expanded ; Web of Science Core Collection

---