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Crystallization and preliminary X-ray diffraction analysis of the Cmr2–Cmr3 subcomplex in the CRISPR–Cas RNA-silencing effector complex

Abstract

The Cmr2–Cmr3 subcomplex from P. furiosus was co-crystallized with 3′-AMP. X-ray diffraction data for the crystals were collected to 2.6 Å resolution using a synchrotron-radiation source. Clustered, regularly interspaced, short palindromic repeat (CRISPR) loci, found in prokaryotes, are transcribed to produce CRISPR RNAs (crRNAs). The Cmr proteins (Cmr1–6) and crRNA form a ribonucleoprotein complex that degrades target RNAs derived from invading genetic elements. Cmr2dHD, a Cmr2 variant lacking the N-terminal putative HD nuclease domain, and Cmr3 were co-expressed in Escherichia coli cells and co-purified as a complex. The Cmr2dHD–Cmr3 complex was co-crystallized with 3′-AMP by the vapour-diffusion method. The crystals diffracted to 2.6 Å resolution using synchrotron radiation at the Photon Factory. The crystals belonged to the orthorhombic space group I222, with unit-cell parameters a = 103.9, b = 136.7, c = 192.0 Å. The asymmetric unit of the crystals is expected to contain one Cmr2dHD–Cmr3 complex with a Matthews coefficient of 3.0 Å{sup 3} Da{sup −1} and a solvent content of 59%.
Authors:
Osawa, Takuo; Inanaga, Hideko; Numata, Tomoyuki [1] 
  1. National Institute of Advanced Industrial Science and Technology (AIST), 1-1-1 Higashi, Tsukuba-shi, Ibaraki 305-8566 (Japan)
Publication Date:
Apr 30, 2013
Product Type:
Journal Article
Resource Relation:
Journal Name: Acta Crystallographica. Section F; Journal Volume: 69; Journal Issue: Pt 5; Other Information: PMCID: PMC3660908; PMID: 23695584; PUBLISHER-ID: pg5014; OAI: oai:pubmedcentral.nih.gov:3660908; Copyright (c) International Union of Crystallography 2013; Country of input: International Atomic Energy Agency (IAEA)
Subject:
75 CONDENSED MATTER PHYSICS, SUPERCONDUCTIVITY AND SUPERFLUIDITY; CRYSTALLIZATION; CRYSTALS; DIFFUSION; ESCHERICHIA COLI; PHOTONS; PROTEINS; RESOLUTION; SOLVENTS; SPACE GROUPS; SYNCHROTRON RADIATION; X-RAY DIFFRACTION
OSTI ID:
22376624
Country of Origin:
United Kingdom
Language:
English
Other Identifying Numbers:
Journal ID: ISSN 1744-3091; CODEN: ACSFCL; Other: PII: S1744309113011202; TRN: GB15$1807082086
Availability:
Available from http://dx.doi.org/10.1107/S1744309113011202; Available from http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3660908
Submitting Site:
INIS
Size:
page(s) 585-587
Announcement Date:
Aug 13, 2015

Citation Formats

Osawa, Takuo, Inanaga, Hideko, and Numata, Tomoyuki. Crystallization and preliminary X-ray diffraction analysis of the Cmr2–Cmr3 subcomplex in the CRISPR–Cas RNA-silencing effector complex. United Kingdom: N. p., 2013. Web. doi:10.1107/S1744309113011202.
Osawa, Takuo, Inanaga, Hideko, & Numata, Tomoyuki. Crystallization and preliminary X-ray diffraction analysis of the Cmr2–Cmr3 subcomplex in the CRISPR–Cas RNA-silencing effector complex. United Kingdom. doi:10.1107/S1744309113011202.
Osawa, Takuo, Inanaga, Hideko, and Numata, Tomoyuki. 2013. "Crystallization and preliminary X-ray diffraction analysis of the Cmr2–Cmr3 subcomplex in the CRISPR–Cas RNA-silencing effector complex." United Kingdom. doi:10.1107/S1744309113011202. https://www.osti.gov/servlets/purl/10.1107/S1744309113011202.
@misc{etde_22376624,
title = {Crystallization and preliminary X-ray diffraction analysis of the Cmr2–Cmr3 subcomplex in the CRISPR–Cas RNA-silencing effector complex}
author = {Osawa, Takuo, Inanaga, Hideko, and Numata, Tomoyuki}
abstractNote = {The Cmr2–Cmr3 subcomplex from P. furiosus was co-crystallized with 3′-AMP. X-ray diffraction data for the crystals were collected to 2.6 Å resolution using a synchrotron-radiation source. Clustered, regularly interspaced, short palindromic repeat (CRISPR) loci, found in prokaryotes, are transcribed to produce CRISPR RNAs (crRNAs). The Cmr proteins (Cmr1–6) and crRNA form a ribonucleoprotein complex that degrades target RNAs derived from invading genetic elements. Cmr2dHD, a Cmr2 variant lacking the N-terminal putative HD nuclease domain, and Cmr3 were co-expressed in Escherichia coli cells and co-purified as a complex. The Cmr2dHD–Cmr3 complex was co-crystallized with 3′-AMP by the vapour-diffusion method. The crystals diffracted to 2.6 Å resolution using synchrotron radiation at the Photon Factory. The crystals belonged to the orthorhombic space group I222, with unit-cell parameters a = 103.9, b = 136.7, c = 192.0 Å. The asymmetric unit of the crystals is expected to contain one Cmr2dHD–Cmr3 complex with a Matthews coefficient of 3.0 Å{sup 3} Da{sup −1} and a solvent content of 59%.}
doi = {10.1107/S1744309113011202}
journal = {Acta Crystallographica. Section F}
issue = {Pt 5}
volume = {69}
journal type = {AC}
place = {United Kingdom}
year = {2013}
month = {Apr}
}