Away from the edge II: in-house Se-SAS phasing with chromium radiation.
Recently, the demands of high-throughput macromolecular crystallography have driven continuous improvements in phasing methods, data-collection protocols and many other technologies. Single-wavelength anomalous scattering (SAS) phasing with chromium X-ray radiation opens a new possibility for phasing a protein with data collected in-house and has led to several successful examples of de novo structure solution using only weak anomalous scatterers such as sulfur. To further reduce data-collection time and make SAS phasing more robust, it is natural to combine selenomethionine-derivatized protein (SeMet protein) with Cr Kalpha radiation to take advantage of the larger anomalous scattering signal from selenium (f'' = 2.28 e(-)) compared with sulfur (f'' = 1.14 e(-)). As reported herein, the crystal structure of a putative chorismate mutase from Clostridium thermocellum was determined using Se-SAS with Cr Kalpha radiation. Each protein molecule contains eight selenomethionine residues in 148 amino-acid residues, providing a calculated Bijvoet ratio of about 3.5% at the Cr Kalpha wavelength. A single data set to 2.2 A resolution with approximately ninefold redundancy was collected using an imaging-plate detector coupled with a Cr source. Structure solution, refinement and deposition to the Protein Data Bank were performed within 9 h of the availability of the scaled diffraction data. The procedure used here is applicable to many other proteins and promises to become a routine pathway for in-house high-throughput crystallography.
Xu
Hao
H
Southeast Collaboratory for Structural Genomics, Department of Biochemistry and Molecular Biology, The University of Georgia, Athens, GA 30605, USA.
Yang
Cheng
C
Chen
Lirong
L
Kataeva
Irina A
IA
Tempel
Wolfram
W
Lee
Doowon
D
Habel
Jeff E
JE
Nguyen
Duong
D
Pflugrath
James W
JW
Ferrara
Joseph D
JD
Arendall
W Bryan
WB
Richardson
Jane S
JS
Richardson
David C
DC
Liu
Zhi Jie
ZJ
Newton
M Gary
MG
Rose
John P
JP
Wang
Bi Cheng
BC
D002826:Chorismate Mutase / Q000737:chemistry*; D002857:Chromium / Q000737:chemistry*; D048013:Acetivibrio thermocellus / Q000201:enzymology; D018360:Crystallography, X-Ray / Q000379:methods*; D017433:Protein Structure, Secondary; D011506:Proteins / Q000737:chemistry*; D012542:Scattering, Radiation; D051140:Selenoproteins; D014961:X-Ray Diffraction
D011506:Proteins; D051140:Selenoproteins; D002857:Chromium; D002826:Chorismate Mutase