Use of complementary cation and anion heavy-atom salt derivatives to solve the structure of cytochrome P450 46A1.
Human cytochrome P450 46A1 (CYP46A1) is one of the key enzymes in cholesterol homeostasis in the brain. The crystallization and heavy-atom structure solution of an active truncated CYP46A1 in complex with the high-affinity substrate analogue cholesterol-3-sulfate (CH-3S) is reported. The 2.6 angstroms structure of CYP46A1-CH-3S was solved using both anion and cation heavy-atom salts. In addition to the native anomalous signal from the haem iron, an NaI anion halide salt derivative and a complementary CsCl alkali-metal cation salt derivative were used. The general implications of the use of halide and alkali-metal quick soaks are discussed. The importance of using isoionic strength buffers, the titration of heavy-atom salts into different ionic species and the role of concentration are considered. It was observed that cation/anion-binding sites will occasionally overlap, which could negatively impact upon mixed RbBr soaks used for multiple anomalous scatterer MAD (MMAD). The use of complementary cation and anion heavy-atom salt derivatives is a convenient and powerful tool for MIR(AS) structure solution.
White
Mark Andrew
MA
Sealy Center for Structural and Molecular Biophysics, UTMB Galveston, TX 77555, USA. white@xray.utmb.edu
Mast
Natalia
N
Bjorkhem
Ingemar
I
Johnson
Eric F
EF
Stout
C David
CD
Pikuleva
Irina A
IA
D000838:Anions / Q000737:chemistry*; D002412:Cations / Q000737:chemistry*; D018360:Crystallography, X-Ray / Q000379:methods; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry*; D008958:Models, Molecular
D000838:Anions; D002412:Cations; D003577:Cytochrome P-450 Enzyme System