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Optical monitoring of freeze‐trapped reaction intermediates in protein crystals: a microspectrophotometer for cryogenic protein crystallography
Author(s) -
Sakai Keisuke,
Matsui Yasuhiro,
Kouyama Tsutomu,
Shiro Yoshitsugu,
Adachi Shinichi
Publication year - 2002
Publication title -
journal of applied crystallography
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.429
H-Index - 162
ISSN - 1600-5767
DOI - 10.1107/s0021889801021331
Subject(s) - protein crystallization , bacteriorhodopsin , monochromator , crystal (programming language) , absorption spectroscopy , materials science , absorption (acoustics) , microscope , optical microscope , pinhole (optics) , optics , analytical chemistry (journal) , chemistry , crystallography , optoelectronics , crystallization , chromatography , membrane , wavelength , physics , scanning electron microscope , biochemistry , organic chemistry , computer science , composite material , programming language
A microspectrophotometer for cryogenic protein crystallography is described. It is capable of measuring visible absorption spectra (350–800 nm) of a single crystal during X‐ray data collection. The microspectrophotometer is designed to minimize the level of stray light by using a double monochromator and an optical microscope equipped with two field diaphragms and a pinhole. In this system, a thick crystal with an optical density of ∼5 is measurable. In order to demonstrate the performance of the system, the absorption spectra of the unphotolyzed state and the primary photoreaction intermediate of bacteriorhodopsin in the P622 crystal have been measured under a flow of cold nitrogen gas at 100 K.