title: Practical experience with the use of halides for phasing macromolecular structures: a powerful tool for structural genomics.
abstract: The crystal structure of pepstatin-insensitive carboxyl proteinase (PCP) from Pseudomonas sp. 101, an enzyme with no overall sequence similarity to any other proteinases of known structure, was solved using crystals soaked in sodium bromide solution and then cryocooled. A data set collected at the bromine peak absorption wavelength was sufficient for calculation of an excellent map and the entire process of phasing and tracing the maps required almost no direct human intervention. The process of structure solution using single-wavelength data was compared with three-wavelength multiwavelength anomalous diffraction (MAD); although the latter resulted in slightly better maps, the use of this much more labor-intensive approach did not significantly improve the ability to solve the structure. The successful phasing approaches are compared with several less successful attempts utilizing other crystal forms of the enzyme and the practical aspects of the use of bromine as a heavy-atom derivative are discussed. In conclusion, the use of halides with single-wavelength diffraction data fulfills the requirements of being a first-choice method of high-throughput structure solution for the emerging field of structural genomics.
authors.affiliation: Synchrotron Radiation Research Section, Macromolecular Crystallography Laboratory, Program in Structural Biology, National Cancer Institute and NSLS, Brookhaven National Laboratory, Building 725A-X9, Upton, NY 11973, USA. dauter@bnl.gov
authors.forename: Z
authors.initials: Z
authors.lastname: Dauter
authors.forename: M
authors.initials: M
authors.lastname: Li
authors.forename: A
authors.initials: A
authors.lastname: Wlodawer
mesh_terms: D016282:Aspartic Acid Endopeptidases / Q000737:chemistry*; D001426:Bacterial Proteins; D001965:Bromides; D018360:Crystallography, X-Ray; D023281:Genomics / Q000379:methods*; D007202:Indicators and Reagents; D008958:Models, Molecular; D011487:Protein Conformation; D011549:Pseudomonas / Q000201:enzymology*; D012697:Serine Endopeptidases; D017670:Sodium Compounds
chemical_list: D001426:Bacterial Proteins; D001965:Bromides; D007202:Indicators and Reagents; D017670:Sodium Compounds; D012697:Serine Endopeptidases; C104058:xanthomonapepsin; D016282:Aspartic Acid Endopeptidases; C027938:sodium bromide