Making the most of two crystals: structural analysis of a conserved hypothetical protein using native gel screening and SAD phasing.
The protein PAE2307 is a member of a protein family of unknown function which is conserved among a number of bacterial and archaeal species. The protein was overexpressed in Escherichia coli, purified and crystallized in two crystal forms. The prevalent form was twinned, but the other diffracted to 1.45 A resolution. The non-twinned crystals proved difficult to reproduce, so screening of potential heavy-atom derivatives by native polyacrylamide gel electrophoresis was used to establish suitable derivatization conditions. This process enabled the production of a K(2)Pt(NO2)4 derivative that was used to collect a single-wavelength anomalous diffraction (SAD) data set from the only available crystal. Phase information of high quality was obtained, enabling the calculation of an interpretable electron-density map.
Lott
J Shaun
JS
Laboratory of Structural Biology, School of Biological Sciences, University of Auckland, Private Bag 92-019, Auckland, New Zealand. s.lott@auckland.ac.nz
Banfield
Mark J
MJ
Sigrell
Jill
J
Baker
Edward N
EN
D000595:Amino Acid Sequence; D019843:Archaeal Proteins / Q000737:chemistry* / Q000235:genetics; D003460:Crystallization / Q000379:methods*; D018360:Crystallography, X-Ray / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel; D008958:Models, Molecular; D008969:Molecular Sequence Data; D041543:Pyrobaculum / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D055672:Static Electricity
D019843:Archaeal Proteins; D011994:Recombinant Proteins