Open AccessCloning, purification and crystallization of a Walker‐type Pyrococcus abyssi ATPase family member
Author(s) -
Uhring Muriel,
Bey Gilbert,
Lecompte Odile,
Cavarelli Jean,
Moras Dino,
Poch Olivier
Publication year - 2005
Publication title -
acta crystallographica section f
Language(s) - English
Resource type - Journals
ISSN - 1744-3091
DOI - 10.1107/s174430910502868x
Subject(s) - structural genomics , escherichia coli , cloning (programming) , atpase , orthorhombic crystal system , biology , gene , archaea , genome , chemistry , biochemistry , crystallography , genetics , protein structure , enzyme , crystal structure , computer science , programming language
Several ATPase proteins play essential roles in the initiation of chromosomal DNA replication in archaea. Walker‐type ATPases are defined by their conserved Walker A and B motifs, which are associated with nucleotide binding and ATP hydrolysis. A family of 28 ATPase proteins with non‐canonical Walker A sequences has been identified by a bioinformatics study of comparative genomics in Pyrococcus genomes. A high‐throughput structural study on P. abyssi has been started in order to establish the structure of these proteins. 16 genes have been cloned and characterized. Six out of the seven soluble constructs were purified in Escherichia coli and one of them, PABY2304, has been crystallized. X‐ray diffraction data were collected from selenomethionine‐derivative crystals using synchrotron radiation. The crystals belong to the orthorhombic space group C 2, with unit‐cell parameters a = 79.41, b = 48.63, c = 108.77 Å, and diffract to beyond 2.6 Å resolution.