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Title: Crystallization and preliminary X-ray analysis of the complex between a Bacillus subtilis α/β-type small acid-soluble spore protein and DNA

Abstract

An α/β-type small, acid-soluble spore protein (SASP) from Bacillus subtilis, a major source of DNA protection against damaging effects in spores, was crystallized in a functionally relevant complex with a double-stranded DNA. This report provides insights into initial characterization of the complex and its structure elucidation. An engineered variant of an α/β-type small acid-soluble spore protein (SASP) from Bacillus subtilis was crystallized in a complex with a ten-base-pair double-stranded DNA by the hanging-drop vapor-diffusion method using ammonium sulfate as a precipitating agent. Crystals grew at 281 K using sodium cacodylate buffer pH 5.5 and these crystals diffracted X-rays to beyond 2.4 Å resolution using synchrotron radiation. The crystallized complex contains two or three SASP molecules bound to one DNA molecule. The crystals belong to the hexagonal space group P6{sub 1}22 or P6{sub 5}22, with unit-cell parameters a = b = 87.0, c = 145.4 Å, α = β = 90.0, γ = 120.0°. Diffraction data were 96.6% complete to 2.4 Å resolution, with an R{sub sym} of 8.5%. Structure solution by the multiwavelength/single-wavelength anomalous dispersion method using isomorphous crystals of selenomethionine-labeled protein is in progress.

Authors:
 [1]; ;  [2];  [3];  [1]
  1. Children’s Hospital Oakland Research Institute, Oakland, CA 94609 (United States)
  2. Department of Molecular, Microbial and Structural Biology, University of Connecticut Health Center, Farmington, CT 06032 (United States)
  3. Berkeley Center for Structural Biology, Lawrence Berkeley National Laboratory, Berkeley, CA 94720 (United States)
Publication Date:
OSTI Identifier:
22360347
Resource Type:
Journal Article
Resource Relation:
Journal Name: Acta Crystallographica. Section F; Journal Volume: 63; Journal Issue: Pt 6; Other Information: PMCID: PMC2335083; PMID: 17554173; PUBLISHER-ID: ll5117; OAI: oai:pubmedcentral.nih.gov:2335083; Copyright (c) International Union of Crystallography 2007; Country of input: International Atomic Energy Agency (IAEA)
Country of Publication:
United Kingdom
Language:
English
Subject:
75 CONDENSED MATTER PHYSICS, SUPERCONDUCTIVITY AND SUPERFLUIDITY; AMMONIUM SULFATES; BUFFERS; CRYSTALLIZATION; CRYSTALS; DIFFRACTION; DIFFUSION; DNA; MOLECULES; RESOLUTION; SAFETY; SODIUM; SPACE GROUPS; SYNCHROTRON RADIATION; WAVELENGTHS

Citation Formats

Bumbaca, Daniela, Kosman, Jeffrey, Setlow, Peter, Henderson, R. Keith, and Jedrzejas, Mark J., E-mail: mjedrzejas@chori.org. Crystallization and preliminary X-ray analysis of the complex between a Bacillus subtilis α/β-type small acid-soluble spore protein and DNA. United Kingdom: N. p., 2007. Web. doi:10.1107/S1744309107022750.
Bumbaca, Daniela, Kosman, Jeffrey, Setlow, Peter, Henderson, R. Keith, & Jedrzejas, Mark J., E-mail: mjedrzejas@chori.org. Crystallization and preliminary X-ray analysis of the complex between a Bacillus subtilis α/β-type small acid-soluble spore protein and DNA. United Kingdom. doi:10.1107/S1744309107022750.
Bumbaca, Daniela, Kosman, Jeffrey, Setlow, Peter, Henderson, R. Keith, and Jedrzejas, Mark J., E-mail: mjedrzejas@chori.org. Fri . "Crystallization and preliminary X-ray analysis of the complex between a Bacillus subtilis α/β-type small acid-soluble spore protein and DNA". United Kingdom. doi:10.1107/S1744309107022750.
@article{osti_22360347,
title = {Crystallization and preliminary X-ray analysis of the complex between a Bacillus subtilis α/β-type small acid-soluble spore protein and DNA},
author = {Bumbaca, Daniela and Kosman, Jeffrey and Setlow, Peter and Henderson, R. Keith and Jedrzejas, Mark J., E-mail: mjedrzejas@chori.org},
abstractNote = {An α/β-type small, acid-soluble spore protein (SASP) from Bacillus subtilis, a major source of DNA protection against damaging effects in spores, was crystallized in a functionally relevant complex with a double-stranded DNA. This report provides insights into initial characterization of the complex and its structure elucidation. An engineered variant of an α/β-type small acid-soluble spore protein (SASP) from Bacillus subtilis was crystallized in a complex with a ten-base-pair double-stranded DNA by the hanging-drop vapor-diffusion method using ammonium sulfate as a precipitating agent. Crystals grew at 281 K using sodium cacodylate buffer pH 5.5 and these crystals diffracted X-rays to beyond 2.4 Å resolution using synchrotron radiation. The crystallized complex contains two or three SASP molecules bound to one DNA molecule. The crystals belong to the hexagonal space group P6{sub 1}22 or P6{sub 5}22, with unit-cell parameters a = b = 87.0, c = 145.4 Å, α = β = 90.0, γ = 120.0°. Diffraction data were 96.6% complete to 2.4 Å resolution, with an R{sub sym} of 8.5%. Structure solution by the multiwavelength/single-wavelength anomalous dispersion method using isomorphous crystals of selenomethionine-labeled protein is in progress.},
doi = {10.1107/S1744309107022750},
journal = {Acta Crystallographica. Section F},
number = Pt 6,
volume = 63,
place = {United Kingdom},
year = {Fri Jun 01 00:00:00 EDT 2007},
month = {Fri Jun 01 00:00:00 EDT 2007}
}
  • Small, acid-soluble proteins (SASP) of the alpha/beta-type are associated with DNA in spores of Bacillus subtilis. Induction of synthesis of alpha/beta-type SASP in Escherichia coli resulted in rapid cessation of DNA synthesis, followed by a halt in RNA and then protein accumulation, although significant mRNA and protein synthesis continued. There was a significant loss in viability associated with SASP synthesis in E. coli: recA+ cells became extremely long filaments, whereas recA mutant cells became less filamentous. The nucleoids of cells with alpha/beta-type SASP were extremely condensed, as viewed in both light and electron microscopes, and immunoelectron microscopy showed that themore » alpha/beta-type SASP were associated with the cell DNA. Induction of alpha/beta-type SASP synthesis in E. coli increased the negative superhelical density of plasmid DNA by approximately 20%; UV irradiation of E. coli with alpha/beta-type SASP gave reduced yields of thymine dimers but significant amounts of the spore photoproduct. These changes in E. coli DNA topology and photochemistry due to alpha/beta-type SASP are similar to the effects of alpha/beta-type SASP on the DNA in Bacillus spores, further suggesting that alpha/beta-type SASP are a major factor determining DNA properties in bacterial spores.« less
  • A preparation of replication terminator protein (RTP) of B. subtilis and a 37-base-pair TerI sequence (comprising two binding sites for RTP) has been purified and crystallized. The replication terminator protein (RTP) of Bacillus subtilis binds to specific DNA sequences that halt the progression of the replisome in a polar manner. These terminator complexes flank a defined region of the chromosome into which they allow replication forks to enter but not exit. Forcing the fusion of replication forks in a specific zone is thought to allow the coordination of post-replicative processes. The functional terminator complex comprises two homodimers each of 29more » kDa bound to overlapping binding sites. A preparation of RTP and a 37-base-pair TerI sequence (comprising two binding sites for RTP) has been purified and crystallized. A data set to 3.9 Å resolution with 97.0% completeness and an R{sub sym} of 12% was collected from a single flash-cooled crystal using synchrotron radiation. The diffraction data are consistent with space group P622, with unit-cell parameters a = b = 118.8, c = 142.6 Å.« less
  • Crystallization and preliminary X-ray diffraction analysis of the two domains of DnaD from B. subtilis is reported. The DnaD protein is an essential component of the chromosome-replication machinery of the Gram-positive bacterium Bacillus subtilis and is part of the primosomal cascade that ultimately loads the replicative ring helicase DnaC onto DNA. Moreover, DnaD is a global regulator of DNA architecture, as it forms higher order nucleoprotein structures in order to open supercoiled DNA. Here, the crystallization and preliminary X-ray diffraction analysis of the two domains of DnaD from B. subtilis are reported. Crystals of the N-terminal domain are trigonal, withmore » either P3{sub 1}21 or P3{sub 2}21 space-group symmetry, and diffracted X-rays to 2.0 Å resolution; crystals of the C-terminal domain are hexagonal, with space group P6{sub 1} or P6{sub 5}, and diffracted X-rays to 2.9 Å resolution in-house. Determination of the structure of the DnaD domains will provide insight into how remodelling of the nucleoid is associated with priming of replication in the model Gram-positive organism B. subtilis.« less
  • B. subtilis YjcG protein was expressed, purified and crystallized. A complete diffraction data set was collected at BSRF beamline 3W1A and processed to 2.3 Å resolution. Bacillus subtilis YjcG is a functionally uncharacterized protein with 171 residues that has no structural homologue in the Protein Data Bank. However, it shows sequence homology to bacterial and archaeal 2′–5′ RNA ligases. In order to identify its exact function via structural studies, the yjcG gene was amplified from B. subtilis genomic DNA and cloned into the expression vector pET21-DEST. The protein was expressed in a soluble form in Escherichia coli and was purifiedmore » to homogeneity. Crystals suitable for X-ray analysis were obtained that diffracted to 2.3 Å and belonged to space group C2, with unit-cell parameters a = 99.66, b = 73.93, c = 61.77 Å, β = 113.56°.« less
  • The crystallization and preliminary X-ray crystallographic analysis of protein YtlP from B. subtilis is reported. Bacillus subtilis YtlP is a protein that is predicted to belong to the bacterial and archael 2′-5′ RNA-ligase family. It contains 183 residues and two copies of the HXTX sequence motif conserved among proteins belonging to this family. In order to determine the structure of YtlP and to compare it with the paralogue YjcG and identified 2′-5′ RNA ligases, the gene ytlP was amplified from B. subtilis genomic DNA and cloned into expression vector pET-21a. The soluble protein was produced in Escherichia coli, purified tomore » homogeneity and crystals suitable for X-ray analysis were obtained. The crystal diffracted to 2.0 Å and belonged to space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 34.16, b = 48.54, c = 105.75 Å.« less