5WJK

2.0-Angstrom In situ Mylar structure of sperm whale myoglobin (SWMb) at 293 K

5WJK の概要

• エントリーDOI: 10.2210/pdb5wjk/pdb
• 分子名称: Myoglobin, PROTOPORPHYRIN IX CONTAINING FE, CHLORIDE ION, ... (5 entities in total)
• 機能のキーワード: heme-bound, soluble protein, oxygen transport
• 由来する生物種: Physeter catodon (Sperm whale)
• タンパク質・核酸の鎖数: 1
• 化学式量合計: 18447.10

構造登録者

Broecker, J.,Ou, W.-L.,Ernst, O.P. (登録日: 2017-07-23, 公開日: 2017-12-13, 最終更新日: 2023-10-04)

主引用文献

Broecker, J.,Morizumi, T.,Ou, W.L.,Klingel, V.,Kuo, A.,Kissick, D.J.,Ishchenko, A.,Lee, M.Y.,Xu, S.,Makarov, O.,Cherezov, V.,Ogata, C.M.,Ernst, O.P.
High-throughput in situ X-ray screening of and data collection from protein crystals at room temperature and under cryogenic conditions.
Nat Protoc, 13:260-292, 2018

PubMed Abstract: Protein crystallography has significantly advanced in recent years, with in situ data collection, in which crystals are placed in the X-ray beam within their growth medium, being a major point of focus. In situ methods eliminate the need to harvest crystals, a previously unavoidable drawback, particularly for often small membrane-protein crystals. Here, we present a protocol for the high-throughput in situ X-ray screening of and data collection from soluble and membrane-protein crystals at room temperature (20-25°C) and under cryogenic conditions. The Mylar in situ method uses Mylar-based film sandwich plates that are inexpensive, easy to make, and compatible with automated imaging, and that show very low background scattering. They support crystallization in microbatch and vapor-diffusion modes, as well as in lipidic cubic phases (LCPs). A set of 3D-printed holders for differently sized patches of Mylar sandwich films makes the method robust and versatile, allows for storage and shipping of crystals, and enables automated mounting at synchrotrons, as well as goniometer-based screening and data collection. The protocol covers preparation of in situ plates and setup of crystallization trials; 3D printing and assembly of holders; opening of plates, isolation of film patches containing crystals, and loading them onto holders; basic screening and data-collection guidelines; and unloading of holders, as well as reuse and recycling of them. In situ plates are prepared and assembled in 1 h; holders are 3D-printed and assembled in ≤90 min; and an in situ plate is opened, and a film patch containing crystals is isolated and loaded onto a holder in 5 min.

PubMed: 29300389
DOI: 10.1038/nprot.2017.135

実験手法

X-RAY DIFFRACTION (2 Å)