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:
- Children’s Hospital Oakland Research Institute, Oakland, CA 94609 (United States)
- Department of Molecular, Microbial and Structural Biology, University of Connecticut Health Center, Farmington, CT 06032 (United States)
- 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 »
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