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Title: Purification, crystallization and preliminary X-ray diffraction analysis of the histone chaperone cia1 from fission yeast

Abstract

The histone chaperone cia1 from fission yeast has been overexpressed in E. coli, purified and crystallized using the vapour-diffusion method. In fission yeast, cia1{sup +} is an essential gene that encodes a histone chaperone, a homologue of human CIA (CCG1-interacting factor A) and budding yeast Asf1p (anti-silencing function-1), which both facilitate nucleosome assembly by interacting with the core histones H3/H4. The conserved domain (residues 1–161) of the cia1{sup +}-encoded protein was expressed in Escherichia coli, purified to near-homogeneity and crystallized by the sitting-drop vapour-diffusion method. The protein was crystallized in the monoclinic space group C2, with unit-cell parameters a = 79.16, b = 40.53, c = 69.79 Å, β = 115.93° and one molecule per asymmetric unit. The crystal diffracted to beyond 2.10 Å resolution using synchrotron radiation.

Authors:
; ; ; ;  [1];  [2];  [3];  [1];  [1];  [4];  [4]
  1. RIKEN Genomic Sciences Center, 1-7-22 Suehiro, Tsurumi, Yokohama 230-0045 (Japan)
  2. Laboratory of Developmental Biology, Institute of Molecular and Cellular Biosciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 111-0032 (Japan)
  3. (ERATO), Japan Science and Technology Corporation (JST), 5-9-6 Tokodai, Tsukuba, Ibaraki 300-2635 (Japan)
  4. (Japan)
Publication Date:
OSTI Identifier:
22356171
Resource Type:
Journal Article
Resource Relation:
Journal Name: Acta Crystallographica. Section F; Journal Volume: 61; Journal Issue: Pt 11; Other Information: PMCID: PMC1978123; PMID: 16511210; PUBLISHER-ID: pu5100; OAI: oai:pubmedcentral.nih.gov:1978123; Copyright (c) International Union of Crystallography 2005; Country of input: International Atomic Energy Agency (IAEA)
Country of Publication:
United Kingdom
Language:
English
Subject:
75 CONDENSED MATTER PHYSICS, SUPERCONDUCTIVITY AND SUPERFLUIDITY; CRYSTALLIZATION; CRYSTALS; DIFFUSION; ESCHERICHIA COLI; MOLECULES; RESOLUTION; SPACE GROUPS; SYNCHROTRON RADIATION; X-RAY DIFFRACTION

Citation Formats

Umehara, Takashi, Otta, Yumi, Tsuganezawa, Keiko, Matsumoto, Takehisa, Tanaka, Akiko, Horikoshi, Masami, Horikoshi Gene Selector Project, Exploratory Research for Advanced Technology, Padmanabhan, Balasundaram, E-mail: paddy@gsc.riken.jp, Yokoyama, Shigeyuki, E-mail: paddy@gsc.riken.jp, RIKEN Harima Institute at SPring-8, 1-1-1 Kouto, Mikazuki-cho, Sayo, Hyogo 679-5148, and Department of Biophysics and Biochemistry, Graduate School of Science, The University of Tokyo, Bunkyo-ku, Tokyo 113-0033. Purification, crystallization and preliminary X-ray diffraction analysis of the histone chaperone cia1 from fission yeast. United Kingdom: N. p., 2005. Web. doi:10.1107/S1744309105030927.
Umehara, Takashi, Otta, Yumi, Tsuganezawa, Keiko, Matsumoto, Takehisa, Tanaka, Akiko, Horikoshi, Masami, Horikoshi Gene Selector Project, Exploratory Research for Advanced Technology, Padmanabhan, Balasundaram, E-mail: paddy@gsc.riken.jp, Yokoyama, Shigeyuki, E-mail: paddy@gsc.riken.jp, RIKEN Harima Institute at SPring-8, 1-1-1 Kouto, Mikazuki-cho, Sayo, Hyogo 679-5148, & Department of Biophysics and Biochemistry, Graduate School of Science, The University of Tokyo, Bunkyo-ku, Tokyo 113-0033. Purification, crystallization and preliminary X-ray diffraction analysis of the histone chaperone cia1 from fission yeast. United Kingdom. doi:10.1107/S1744309105030927.
Umehara, Takashi, Otta, Yumi, Tsuganezawa, Keiko, Matsumoto, Takehisa, Tanaka, Akiko, Horikoshi, Masami, Horikoshi Gene Selector Project, Exploratory Research for Advanced Technology, Padmanabhan, Balasundaram, E-mail: paddy@gsc.riken.jp, Yokoyama, Shigeyuki, E-mail: paddy@gsc.riken.jp, RIKEN Harima Institute at SPring-8, 1-1-1 Kouto, Mikazuki-cho, Sayo, Hyogo 679-5148, and Department of Biophysics and Biochemistry, Graduate School of Science, The University of Tokyo, Bunkyo-ku, Tokyo 113-0033. Tue . "Purification, crystallization and preliminary X-ray diffraction analysis of the histone chaperone cia1 from fission yeast". United Kingdom. doi:10.1107/S1744309105030927.
@article{osti_22356171,
title = {Purification, crystallization and preliminary X-ray diffraction analysis of the histone chaperone cia1 from fission yeast},
author = {Umehara, Takashi and Otta, Yumi and Tsuganezawa, Keiko and Matsumoto, Takehisa and Tanaka, Akiko and Horikoshi, Masami and Horikoshi Gene Selector Project, Exploratory Research for Advanced Technology and Padmanabhan, Balasundaram, E-mail: paddy@gsc.riken.jp and Yokoyama, Shigeyuki, E-mail: paddy@gsc.riken.jp and RIKEN Harima Institute at SPring-8, 1-1-1 Kouto, Mikazuki-cho, Sayo, Hyogo 679-5148 and Department of Biophysics and Biochemistry, Graduate School of Science, The University of Tokyo, Bunkyo-ku, Tokyo 113-0033},
abstractNote = {The histone chaperone cia1 from fission yeast has been overexpressed in E. coli, purified and crystallized using the vapour-diffusion method. In fission yeast, cia1{sup +} is an essential gene that encodes a histone chaperone, a homologue of human CIA (CCG1-interacting factor A) and budding yeast Asf1p (anti-silencing function-1), which both facilitate nucleosome assembly by interacting with the core histones H3/H4. The conserved domain (residues 1–161) of the cia1{sup +}-encoded protein was expressed in Escherichia coli, purified to near-homogeneity and crystallized by the sitting-drop vapour-diffusion method. The protein was crystallized in the monoclinic space group C2, with unit-cell parameters a = 79.16, b = 40.53, c = 69.79 Å, β = 115.93° and one molecule per asymmetric unit. The crystal diffracted to beyond 2.10 Å resolution using synchrotron radiation.},
doi = {10.1107/S1744309105030927},
journal = {Acta Crystallographica. Section F},
number = Pt 11,
volume = 61,
place = {United Kingdom},
year = {Tue Nov 01 00:00:00 EST 2005},
month = {Tue Nov 01 00:00:00 EST 2005}
}
  • A soluble variant of the monoglyceride lipase Yju3p was successfully expressed, purified and crystallized. Diffraction data were collected to 2.4 Å resolution. The protein Yju3p is the orthologue of monoglyceride lipases in the yeast Saccharomyces cerevisiae. A soluble variant of this lipase termed s-Yju3p (38.3 kDa) was generated and purified to homogeneity by affinity and size-exclusion chromatography. s-Yju3p was crystallized in a vapour-diffusion setup at 293 K and a complete data set was collected to 2.4 Å resolution. The crystal form was orthorhombic (space group P2{sub 1}2{sub 1}2{sub 1}), with unit-cell parameters a = 77.2, b = 108.6, c =more » 167.7 Å. The asymmetric unit contained four molecules with a solvent content of 46.4%.« less
  • In Escherichia coli, the common pilus (Ecp) belongs to an alternative chaperone–usher pathway that plays a major role in both early biofilm formation and host-cell adhesion. Initial attempts at crystallizing the chaperone EcpB using natively purified protein from the bacterial periplasm were not successful; however, after the isolation of EcpB under denaturing conditions and subsequent refolding, crystals were obtained at pH 8.0 using the sitting-drop method of vapour diffusion. This is the first time that this refolding strategy has been used to purify CU chaperones. Pili are key cell-surface components that allow the attachment of bacteria to both biological andmore » abiotic solid surfaces, whilst also mediating interactions between themselves. In Escherichia coli, the common pilus (Ecp) belongs to an alternative chaperone–usher (CU) pathway that plays a major role in both early biofilm formation and host-cell adhesion. The chaperone EcpB is involved in the biogenesis of the filament, which is composed of EcpA and EcpD. Initial attempts at crystallizing EcpB using natively purified protein from the bacterial periplasm were not successful; however, after the isolation of EcpB under denaturing conditions and subsequent refolding, crystals were obtained at pH 8.0 using the sitting-drop method of vapour diffusion. Diffraction data have been processed to 2.4 Å resolution. These crystals belonged to the trigonal space group P3{sub 1}21 or P3{sub 2}21, with unit-cell parameters a = b = 62.65, c = 121.14 Å and one monomer in the asymmetric unit. Molecular replacement was unsuccessful, but selenomethionine-substituted protein and heavy-atom derivatives are being prepared for phasing. The three-dimensional structure of EcpB will provide invaluable information on the subtle mechanistic differences in biogenesis between the alternative and classical CU pathways. Furthermore, this is the first time that this refolding strategy has been used to purify CU chaperones, and it could be implemented in similar systems where it has not been possible to obtain highly ordered crystals.« less
  • YidC, a membrane-protein chaperone/insertase from B. halodurans, was expressed, purified and crystallized in the lipidic cubic phase. An X-ray diffraction data set was collected to 2.4 Å resolution. YidC, a member of the YidC/Oxa1/Alb3 family, inserts proteins into the membrane and facilitates membrane-protein folding in bacteria. YidC plays key roles in both Sec-mediated integration and Sec-independent insertion of membrane proteins. Here, Bacillus halodurans YidC2, which has five transmembrane helices conserved among the other family members, was identified as a target protein for structure determination by a fluorescent size-exclusion chromatography analysis. The protein was overexpressed, purified and crystallized in the lipidicmore » cubic phase. The crystals diffracted X-rays to 2.4 Å resolution and belonged to space group P2{sub 1}, with unit-cell parameters a = 43.9, b = 60.6, c = 58.9 Å, β = 100.3°. The experimental phases were determined by the multiwavelength anomalous diffraction method using a mercury-derivatized crystal.« less
  • No abstract prepared.
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