7NKF

Hen egg white lysozyme (HEWL) Grown inside (Not centrifuged) HARE serial crystallography chip.

7NKF の概要

• エントリーDOI: 10.2210/pdb7nkf/pdb
• 分子名称: Lysozyme, SODIUM ION (3 entities in total)
• 機能のキーワード: serial crystallography, lysozyme, vapour diffusion, silicon chip, hydrolase
• 由来する生物種: Gallus gallus
• タンパク質・核酸の鎖数: 1
• 化学式量合計: 14354.15

構造登録者

Norton-Baker, B.,Mehrabi, P.,Boger, J.,Schonherr, R.,von Stetten, D.,Schikora, H.,Martin, R.W.,Miller, R.J.D.,Redecke, L.,Schulz, E.C. (登録日: 2021-02-17, 公開日: 2021-06-16, 最終更新日: 2024-11-06)

主引用文献

Norton-Baker, B.,Mehrabi, P.,Boger, J.,Schonherr, R.,von Stetten, D.,Schikora, H.,Kwok, A.O.,Martin, R.W.,Miller, R.J.D.,Redecke, L.,Schulz, E.C.
A simple vapor-diffusion method enables protein crystallization inside the HARE serial crystallography chip.
Acta Crystallogr D Struct Biol, 77:820-834, 2021

PubMed 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.

PubMed: 34076595
DOI: 10.1107/S2059798321003855

実験手法

X-RAY DIFFRACTION (1.7 Å)