Heavy-atom derivatization.
Most of the standard methods of solving macromolecular structures involve producing a protein crystal that is derivatized by an anomalous scatterer or heavy atom (MIR, SIRAS, MAD, SAD etc.). The theoretical methodology which underpins the extraction of phase information from such derivatives is widely available in the literature. In addition, there are comprehensive sources of information on the chemistry of heavy-atom compounds and the ligands with which they are known to interact, as well as the Heavy Atom Databank accessible on the World Wide Web. This contribution therefore aims to provide some information on the less well documented practical problems of firstly deciding on an overall strategy for derivatization and secondly performing the physical manipulations involved in producing heavy-atom derivatives from native protein crystals and then cryocooling them. Ways to optimize the chances of isomorphous unit cells are suggested. Methods of determining whether or not the heavy atom is bound are outlined, including the powerful technique of PIXE (particle-induced X-ray emission).
Department of Biochemistry, Oxford University, Rex Richards Building, South Parks Road, Oxford OX1 3QU, England. elspeth@biop.ox.ac.uk
Elspeth
E
Garman
James W
JW
Murray
D003460:Crystallization; D018360:Crystallography, X-Ray / Q000379:methods*; D016208:Databases, Factual; D006801:Humans; D013058:Mass Spectrometry; D011506:Proteins / Q000737:chemistry*; D012643:Selenium / Q000737:chemistry; D012996:Solutions / Q000737:chemistry
D011506:Proteins; D012996:Solutions; D012643:Selenium