Crystallization and crystallographic analysis of yeast Sec2p, a guanine nucleotide-exchange factor for the yeast Rab GTPase Sec4p
- Department of Biological Information, Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, 4259 Nagatsuta-cho, Midori-ku, Yokohama-shi, Kanagawa 226-8501 (Japan)
Crystal structure of a triple mutant (M115L, K121M, T142M) of SeMet-labelled Sec2p, a GEF for Rab GTPase, has been solved by SAD. Control of the selenium sites by mutagenesis much improved the resolution of the SeMet-labelled crystals. Sec2p is a guanine nucleotide-exchange factor (GEF) for the yeast Rab GTPase Sec4p. Sec2p accelerates GDP release from Sec4p and promotes GDP–GTP exchange for Sec4p activation. In order to elucidate this nucleotide-exchange mechanism using X-ray crystallography, three constructs of native Sec2p (Sec2{sub 1–160}p, Sec2{sub 18–160}p and Sec2{sub 31–160}p) and three constructs of selenomethionine-labelled Sec2p [Sec2{sub 31–160}p, Sec2{sub 31–160}p (M115L) and Sec2{sub 31–160}p (M115L, K121M, T142M)] were crystallized. These six crystals diffracted to 8.8, 4.8, 2.6, 4.0, 3.3 and 3.0 Å resolution, respectively. The data set from the SeMet-labelled Sec2{sub 31–160}p (M115L, K121M, T142M) crystal was processed for SAD phasing; the crystal belonged to space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 101.9, b = 176.6, c = 181.5 Å.
- OSTI ID:
- 22360373
- Journal Information:
- Acta Crystallographica. Section F, Vol. 63, Issue Pt 7; Other Information: PMCID: PMC2335144; PMID: 17620726; PUBLISHER-ID: ll5113; OAI: oai:pubmedcentral.nih.gov:2335144; Copyright (c) International Union of Crystallography 2007; Country of input: International Atomic Energy Agency (IAEA); ISSN 1744-3091
- Country of Publication:
- United Kingdom
- Language:
- English
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