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- EMDB-2418: Structure and activity of an RNA-targeting Type III-B CRISPER-Cas... -

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Entry
Database: EMDB / ID: 2418
TitleStructure and activity of an RNA-targeting Type III-B CRISPER-Cas complex
Map dataReconstruction of Type III-B CRISPR-Cas complex from Thermus thermophilus
SampleType III-B CRISPR-Cas complex of Thermus thermophilus:
(CRISPR-associated protein ...) x 4 / nucleic-acidNucleic acid / CRISPR system Cmr subunit Cmr5 / CRISPR-associated RAMP Cmr4
KeywordsCRISPR-Cas / RNA-targeting / Cmr complex
Function / homologyCRISPR-associated protein, Cmr3 / CRISPR-associated protein, TM1791 / CRISPR-associated protein (Cas_Cmr5) / CRISPR-associated protein (Cas_Cmr3) / RAMP superfamily / Cmr2, N-terminal domain superfamily / CRISPR-associated protein Cmr2, N-terminal / AF1862-like domain superfamily / CRISPR-associated RAMP Cmr4 / CRISPR-associated protein Cmr2 ...CRISPR-associated protein, Cmr3 / CRISPR-associated protein, TM1791 / CRISPR-associated protein (Cas_Cmr5) / CRISPR-associated protein (Cas_Cmr3) / RAMP superfamily / Cmr2, N-terminal domain superfamily / CRISPR-associated protein Cmr2, N-terminal / AF1862-like domain superfamily / CRISPR-associated RAMP Cmr4 / CRISPR-associated protein Cmr2 / CRISPR-associated protein, Cmr5 / GGDEF domain profile. / CRISPR-associated protein TM1795 / CRISPR type III-associated protein / GGDEF domain / CRISPR-associated protein / CRISPR-associated protein, TM1791 / CRISPR-associated protein TM1795 / CRISPR-associated protein, Cmr3 / CRISPR-associated RAMP Cmr4 / CRISPR-associated protein, Cmr5 / CRISPR-associated protein Cmr2 / regulation of defense response to virus / cytoplasm / Uncharacterized protein / CRISPR system Cmr subunit Cmr5 / Uncharacterized protein / Uncharacterized protein / Uncharacterized protein / Uncharacterized protein
Function and homology information
SourceThermus thermophilus (bacteria)
Methodsingle particle reconstruction / negative staining / 22 Å resolution
AuthorsStaals RHJ / Agari Y / Maki-Yonekura S / Zhu Y / Taylor DW / van Duijn E / Barendregt A / Vlot M / Koehorst JJ / Sakamoto K / Masuda A / Dohmae N / Schaap PJ / Doudna JA / Heck AJ / Yonekura K / van der Oost J / Shinkai A
CitationJournal: Mol. Cell / Year: 2013
Title: Structure and activity of the RNA-targeting Type III-B CRISPR-Cas complex of Thermus thermophilus.
Authors: Raymond H J Staals / Yoshihiro Agari / Saori Maki-Yonekura / Yifan Zhu / David W Taylor / Esther van Duijn / Arjan Barendregt / Marnix Vlot / Jasper J Koehorst / Keiko Sakamoto / Akiko Masuda / Naoshi Dohmae / Peter J Schaap / Jennifer A Doudna / Albert J R Heck / Koji Yonekura / John van der Oost / Akeo Shinkai
Abstract: The CRISPR-Cas system is a prokaryotic host defense system against genetic elements. The Type III-B CRISPR-Cas system of the bacterium Thermus thermophilus, the TtCmr complex, is composed of six ...The CRISPR-Cas system is a prokaryotic host defense system against genetic elements. The Type III-B CRISPR-Cas system of the bacterium Thermus thermophilus, the TtCmr complex, is composed of six different protein subunits (Cmr1-6) and one crRNA with a stoichiometry of Cmr112131445361:crRNA1. The TtCmr complex copurifies with crRNA species of 40 and 46 nt, originating from a distinct subset of CRISPR loci and spacers. The TtCmr complex cleaves the target RNA at multiple sites with 6 nt intervals via a 5' ruler mechanism. Electron microscopy revealed that the structure of TtCmr resembles a "sea worm" and is composed of a Cmr2-3 heterodimer "tail," a helical backbone of Cmr4 subunits capped by Cmr5 subunits, and a curled "head" containing Cmr1 and Cmr6. Despite having a backbone of only four Cmr4 subunits and being both longer and narrower, the overall architecture of TtCmr resembles that of Type I Cascade complexes.
DateDeposition: Jul 18, 2013 / Header (metadata) release: Aug 14, 2013 / Map release: Oct 16, 2013 / Last update: Nov 6, 2013

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 3.22
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 3.22
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

Fileemd_2418.map.gz (map file in CCP4 format, 11665 KB)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
144 pix
2.8 Å/pix.
= 403.2 Å
144 pix
2.8 Å/pix.
= 403.2 Å
144 pix
2.8 Å/pix.
= 403.2 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 2.8 Å
Density
Contour Level:3.22 (by author), 3.22 (movie #1):
Minimum - Maximum-9.27186775 - 17.75046158
Average (Standard dev.)0E-8 (1.00000000)
Details

EMDB XML:

Space Group Number1
Map Geometry
Axis orderXYZ
Dimensions144144144
Origin-72-72-72
Limit717171
Spacing144144144
CellA=B=C: 403.19998 Å
α=β=γ: 90.0 deg.

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z2.82.82.8
M x/y/z144144144
origin x/y/z0.0000.0000.000
length x/y/z403.200403.200403.200
α/β/γ90.00090.00090.000
start NX/NY/NZ00-40
NX/NY/NZ555581
MAP C/R/S123
start NC/NR/NS-72-72-72
NC/NR/NS144144144
D min/max/mean-9.27217.750-0.000

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Supplemental data

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Sample components

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Entire Type III-B CRISPR-Cas complex of Thermus thermophilus

EntireName: Type III-B CRISPR-Cas complex of Thermus thermophilus / Number of components: 12
MassExperimental: 365 kDa / Measured by: MS/MS

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Component #1: protein, CRISPR-associated protein TM1791

ProteinName: CRISPR-associated protein TM1791 / a.k.a: Cmr6, TTHB165 / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 38 kDa / Experimental: 38 kDa
SourceSpecies: Thermus thermophilus (bacteria)
External referencesInterPro: CRISPR-associated protein, TM1791 / UniProt: Uncharacterized protein

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Component #2: nucleic-acid, CRISPR RNA

Nucleic-acidName: CRISPR RNA / a.k.a: crRNA / Class: RNA / Details: 40-46 nucleotide endogenous crRNA bound to complex / Structure: SINGLE STRANDED / Synthetic: No
SourceSpecies: Thermus thermophilus (bacteria)

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Component #3: protein, CRISPR system Cmr subunit Cmr5

ProteinName: CRISPR system Cmr subunit Cmr5
a.k.a: Cmr5, CRISPR type III-B/RAMP module-associated protein Cmr5
Recombinant expression: No / Number of Copies: 3
MassTheoretical: 12 kDa / Experimental: 12 kDa
SourceSpecies: Thermus thermophilus (bacteria)
External referencesUniProt: CRISPR system Cmr subunit Cmr5 / InterPro: CRISPR-associated protein, Cmr5 / Gene Ontology: regulation of defense response to virus

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Component #4: protein, CRISPR-associated protein Cmr3

ProteinName: CRISPR-associated protein Cmr3 / a.k.a: Cmr3, TTHB160 / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 39 kDa / Experimental: 39 kDa
SourceSpecies: Thermus thermophilus (bacteria)
External referencesUniProt: Uncharacterized protein / InterPro: CRISPR-associated protein, Cmr3

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Component #5: protein, CRISPR-associated protein Crm2

ProteinName: CRISPR-associated protein Crm2 / a.k.a: Cmr2, TTHB160Mary River Aerodrome / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 65 kDa / Experimental: 65 kDa
SourceSpecies: Thermus thermophilus (bacteria)
External referencesUniProt: Uncharacterized protein / InterPro: CRISPR-associated protein Cmr2

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Component #6: protein, CRISPR-associated RAMP Cmr4

ProteinName: CRISPR-associated RAMP Cmr4 / a.k.a: Cmr4, TTHB163 / Recombinant expression: No / Number of Copies: 4
MassTheoretical: 37 kDa / Experimental: 37 kDa
SourceSpecies: Thermus thermophilus (bacteria)
External referencesUniProt: Uncharacterized protein / InterPro: CRISPR-associated RAMP Cmr4

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Component #7: protein, CRISPR-associated protein TM1795

ProteinName: CRISPR-associated protein TM1795 / a.k.a: Cmr1, TTHB162 / Recombinant expression: No / Number of Copies: 1
MassExperimental: 44 kDa
SourceSpecies: Thermus thermophilus (bacteria)
External referencesInterPro: CRISPR-associated protein TM1795 / UniProt: Uncharacterized protein

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Experimental details

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Sample preparation

SpecimenSpecimen state: particle / Method: negative staining
Sample solutionSpecimen conc.: 0.03 mg/ml
Buffer solution: 25 mM Hepes, pH 7.5, 100 mM KCl, 1 mM TCEP, 5% glycerol
pH: 7.4
Support film200 mesh Cu grid
StainingSample applied to glow-discharged continuous carbon grids. After 1 min, grids were stained consecutively in 6 droplets of 2% (w/v) uranyl acetate solution and excess stain removed by gentle blotting with filter paper.
VitrificationInstrument: NONE / Cryogen name: NONE

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Electron microscopy imaging

ImagingMicroscope: FEI TECNAI 20 / Date: Jan 29, 2013 / Details: Data acquired using Leginon
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 120 kV / Electron dose: 20 e/Å2 / Illumination mode: FLOOD BEAM
LensMagnification: 80000 X (nominal)
Astigmatism: Objective astigmatism was corrected at 210,000 times magnification
Cs: 2.2 mm / Imaging mode: BRIGHT FIELD / Defocus: -500 - -1400 nm
Specimen HolderModel: OTHER
CameraDetector: GENERIC GATAN (4k x 4k)

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Image acquisition

Image acquisitionNumber of digital images: 300

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Image processing

ProcessingMethod: single particle reconstruction / Applied symmetry: C1 (asymmetric) / Number of projections: 45000
3D reconstructionAlgorithm: projection matching refinement / Software: EMAN2, SPARX / CTF correction: ACE2 micrograph / Resolution: 22 Å / Resolution method: FSC 0.5

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