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- EMDB-2900: Structures of the CRISPR-Cmr complex reveal mode of RNA target po... -

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Basic information

Entry
Database: EMDB / ID: 2900
TitleStructures of the CRISPR-Cmr complex reveal mode of RNA target positioning
Map dataReconstruction of target-bound CRISPR-Cas complex
Sampletarget-bound CRISPR-Cmr complex:
(CRISPR-associated protein ...) x 4 / (nucleic-acidNucleic acid) x 2 / CRISPR system Cmr subunit Cmr5 / CRISPR-associated RAMP Cmr4
KeywordsCRISPR-Cas
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 RAMP 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 / cryo EM / 4.4 Å resolution
AuthorsTaylor DW / Zhu Y / Staals RHJ / Kornfeld JE / Shinkai A / van der Oost J / Nogales E / Doudna JA
CitationJournal: Science / Year: 2015
Title: Structural biology. Structures of the CRISPR-Cmr complex reveal mode of RNA target positioning.
Authors: David W Taylor / Yifan Zhu / Raymond H J Staals / Jack E Kornfeld / Akeo Shinkai / John van der Oost / Eva Nogales / Jennifer A Doudna
Abstract: Adaptive immunity in bacteria involves RNA-guided surveillance complexes that use CRISPR (clustered regularly interspaced short palindromic repeats)-associated (Cas) proteins together with CRISPR ...Adaptive immunity in bacteria involves RNA-guided surveillance complexes that use CRISPR (clustered regularly interspaced short palindromic repeats)-associated (Cas) proteins together with CRISPR RNAs (crRNAs) to target invasive nucleic acids for degradation. Whereas type I and type II CRISPR-Cas surveillance complexes target double-stranded DNA, type III complexes target single-stranded RNA. Near-atomic resolution cryo-electron microscopy reconstructions of native type III Cmr (CRISPR RAMP module) complexes in the absence and presence of target RNA reveal a helical protein arrangement that positions the crRNA for substrate binding. Thumblike β hairpins intercalate between segments of duplexed crRNA:target RNA to facilitate cleavage of the target at 6-nucleotide intervals. The Cmr complex is architecturally similar to the type I CRISPR-Cascade complex, suggesting divergent evolution of these immune systems from a common ancestor.
DateDeposition: Feb 10, 2015 / Header (metadata) release: Apr 1, 2015 / Map release: Apr 29, 2015 / Last update: May 13, 2015

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.013
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.013
  • 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_2900.map.gz (map file in CCP4 format, 128001 KB)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
320 pix
1 Å/pix.
= 320. Å
320 pix
1 Å/pix.
= 320. Å
320 pix
1 Å/pix.
= 320. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1 Å
Density
Contour Level:0.013 (by author), 0.013 (movie #1):
Minimum - Maximum-0.02361985 - 0.05433919
Average (Standard dev.)3.12E-6 (0.00238488)
Details

EMDB XML:

Space Group Number1
Map Geometry
Axis orderXYZ
Dimensions320320320
Origin000
Limit319319319
Spacing320320320
CellA=B=C: 320 Å
α=β=γ: 90 deg.

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z111
M x/y/z320320320
origin x/y/z0.0000.0000.000
length x/y/z320.000320.000320.000
α/β/γ90.00090.00090.000
start NX/NY/NZ
NX/NY/NZ
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS320320320
D min/max/mean-0.0240.0540.000

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Supplemental data

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Sample components

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Entire target-bound CRISPR-Cmr complex

EntireName: target-bound CRISPR-Cmr complex / Number of components: 13
MassExperimental: 381 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, target RNA

Nucleic-acidName: target RNA / Class: RNA / Details: 50 nucleotide target RNA / Structure: SINGLE STRANDED / Synthetic: Yes
MassTheoretical: 16.2 kDa
SourceSpecies: Thermus thermophilus (bacteria)

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Component #3: nucleic-acid, CRISPR RNA

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

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Component #4: 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
Number of Copies: 3 / Recombinant expression: No
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 #5: 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 #6: protein, CRISPR-associated protein Crm2

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

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Component #7: 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 referencesInterPro: CRISPR-associated RAMP Cmr4 / UniProt: Uncharacterized protein

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Component #8: 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: cryo EM
Sample solutionSpecimen conc.: 1 mg/ml / Buffer solution: 20 mM Tris-HCl, pH 8.0, 150 mM NaCl / pH: 8
Support film4/2 C-flat grids with a thin-layer of carbon over the holes
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Temperature: 100 K / Humidity: 100 %
Method: Grids were rapidly plunged into liquid ethane using an FEI Vitrobot MarkIV maintained at 4 degrees C after being blotted for 4-4.5 seconds with a blotting force of 15-20.

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
ImagingMicroscope: FEI TITAN KRIOS / Date: Jul 31, 2014
Details: Data acquired using Leginon. We collected a 6 s exposure fractionated into 20, 300 ms frames with a dose of 8 electrons per square Angstrom per second.
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Electron dose: 48 e/Å2 / Illumination mode: FLOOD BEAM
LensMagnification: 29000 X (nominal)
Astigmatism: Objective astigmatism was corrected at 210,000 times magnification
Cs: 2.7 mm / Imaging mode: BRIGHT FIELD / Defocus: -2000 - -4500 nm
Specimen HolderModel: FEI TITAN KRIOS AUTOGRID HOLDER
CameraDetector: GATAN K2 (4k x 4k)

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Image acquisition

Image acquisitionNumber of digital images: 4000

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Image processing

ProcessingMethod: single particle reconstruction / Applied symmetry: C1 (asymmetric) / Number of projections: 175000
3D reconstructionAlgorithm: 3D Autorefine / Software: Relion / CTF correction: CTFFind3 / Resolution: 4.4 Å / Resolution method: FSC 0.143, gold-standard

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