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- EMDB-5740: Structure of an RNA silencing complex of the CRISPR-Cas immune system -

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Basic information

Entry
Database: EMDB / ID: EMD-5740
TitleStructure of an RNA silencing complex of the CRISPR-Cas immune system
Map dataSingle particle reconstruction of Cmr effector complex
Sample
  • Sample: Cmr1-6 effector complex
  • Protein or peptide: Cmr1
  • Protein or peptide: Cmr2
  • Protein or peptide: Cmr3
  • Protein or peptide: Cmr4
  • Protein or peptide: Cmr5
  • Protein or peptide: Cmr6
KeywordsEffector complex / RNA cleavage / bacterial immunity
Function / homology
Function and homology information


endonuclease activity / defense response to virus / Hydrolases; Acting on ester bonds / nucleotide binding / RNA binding / metal ion binding / cytoplasm
Similarity search - Function
: / Cmr1-like, C-terminal / : / : / CRISPR RNA silencing complex Cmr2 subunit, first helical domain / CRISPR RNA silencing complex Cmr2 subunit, Zn-binding domain / CRISPR-associated protein (Cas_Cmr5) / CRISPR-associated protein, TM1793 / CRISPR-associated protein TM1795 / CRISPR-associated protein, TM1791 ...: / Cmr1-like, C-terminal / : / : / CRISPR RNA silencing complex Cmr2 subunit, first helical domain / CRISPR RNA silencing complex Cmr2 subunit, Zn-binding domain / CRISPR-associated protein (Cas_Cmr5) / CRISPR-associated protein, TM1793 / CRISPR-associated protein TM1795 / CRISPR-associated protein, TM1791 / CRISPR-associated protein, Cmr3 / CRISPR-associated protein (Cas_Cmr3) / CRISPR-associated protein, Cmr5 / CRISPR-associated RAMP Cmr4 / AF1862-like domain superfamily / CRISPR-associated protein Cmr2 / CRISPR-associated protein Cmr2, N-terminal / CRISPR-Cas system, Cmr2 subunit, D1 domain, cysteine cluster / CRISPR-associated protein Cmr2, N-terminal / : / CRISPR RNA silencing complex Cmr2 subunit, second helical domain / : / : / Cas10/Cmr2, second palm domain / CRISPR type III-associated protein / RAMP superfamily / GGDEF domain profile. / GGDEF domain / Reverse transcriptase/Diguanylate cyclase domain
Similarity search - Domain/homology
CRISPR system Cmr subunit Cmr1-1 / CRISPR system Cmr subunit Cmr2 / CRISPR system Cmr subunit Cmr3 / CRISPR system Cmr endoribonuclease Cmr4 / CRISPR system Cmr subunit Cmr5 / CRISPR system Cmr subunit Cmr6
Similarity search - Component
Biological speciesPyrococcus furiosus (archaea)
Methodsingle particle reconstruction / cryo EM / Resolution: 12.0 Å
AuthorsSpilman MS / Cocozaki AI / Hale C / Shao Y / Ramia NF / Terns R / Terns M / Li H / Stagg SM
CitationJournal: Mol Cell / Year: 2013
Title: Structure of an RNA silencing complex of the CRISPR-Cas immune system.
Authors: Michael Spilman / Alexis Cocozaki / Caryn Hale / Yaming Shao / Nancy Ramia / Rebeca Terns / Michael Terns / Hong Li / Scott Stagg /
Abstract: Bacterial and archaeal clustered regularly interspaced short palindromic repeat (CRISPR) loci capture virus and plasmid sequences and use them to recognize and eliminate these invaders. CRISPR RNAs ...Bacterial and archaeal clustered regularly interspaced short palindromic repeat (CRISPR) loci capture virus and plasmid sequences and use them to recognize and eliminate these invaders. CRISPR RNAs (crRNAs) containing the acquired sequences are incorporated into effector complexes that destroy matching invader nucleic acids. The multicomponent Cmr effector complex cleaves RNA targets complementary to the crRNAs. Here, we report cryoelectron microscopy reconstruction of a functional Cmr complex bound with a target RNA at ~12 Å. Pairs of the Cmr4 and Cmr5 proteins form a helical core that is asymmetrically capped on each end by distinct pairs of the four remaining subunits: Cmr2 and Cmr3 at the conserved 5' crRNA tag sequence and Cmr1 and Cmr6 near the 3' end of the crRNA. The shape and organization of the RNA-targeting Cmr complex is strikingly similar to the DNA-targeting Cascade complex. Our results reveal a remarkably conserved architecture among very distantly related CRISPR-Cas complexes.
History
DepositionAug 12, 2013-
Header (metadata) releaseOct 23, 2013-
Map releaseOct 23, 2013-
UpdateNov 6, 2013-
Current statusNov 6, 2013Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 3.97
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 3.97
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_5740_1.jpg

Downloads & links

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Map

FileDownload / File: emd_5740.map.gz / Format: CCP4 / Size: 6.4 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationSingle particle reconstruction of Cmr effector complex
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
2.78 Å/pix.
x 120 pix.
= 333.6 Å		2.78 Å/pix.
x 120 pix.
= 333.6 Å		2.78 Å/pix.
x 120 pix.
= 333.6 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 2.78 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 3.97 / Movie #1: 3.97
Minimum - Maximum-8.774785039999999 - 15.44263649
Average (Standard dev.)0.0 (±0.84591085)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-20-20-20
Dimensions120120120
Spacing120120120
CellA=B=C: 333.6 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z2.782.782.78
M x/y/z120120120
origin x/y/z0.0000.0000.000
length x/y/z333.600333.600333.600
α/β/γ90.00090.00090.000
start NX/NY/NZ-132-122-147
NX/NY/NZ250274261
MAP C/R/S123
start NC/NR/NS-20-20-20
NC/NR/NS120120120
D min/max/mean-8.77515.443-0.000

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Supplemental data

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Sample components

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Entire : Cmr1-6 effector complex

EntireName: Cmr1-6 effector complex
Components
  • Sample: Cmr1-6 effector complex
  • Protein or peptide: Cmr1
  • Protein or peptide: Cmr2
  • Protein or peptide: Cmr3
  • Protein or peptide: Cmr4
  • Protein or peptide: Cmr5
  • Protein or peptide: Cmr6

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Supramolecule #1000: Cmr1-6 effector complex

SupramoleculeName: Cmr1-6 effector complex / type: sample / ID: 1000 / Number unique components: 6
Molecular weightTheoretical: 350 KDa

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Macromolecule #1: Cmr1

MacromoleculeName: Cmr1 / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Recombinant expression: Yes
Source (natural)Organism: Pyrococcus furiosus (archaea)
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceUniProtKB: CRISPR system Cmr subunit Cmr1-1

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Macromolecule #2: Cmr2

MacromoleculeName: Cmr2 / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Recombinant expression: Yes
Source (natural)Organism: Pyrococcus furiosus (archaea)
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceUniProtKB: CRISPR system Cmr subunit Cmr2

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Macromolecule #3: Cmr3

MacromoleculeName: Cmr3 / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Recombinant expression: Yes
Source (natural)Organism: Pyrococcus furiosus (archaea)
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceUniProtKB: CRISPR system Cmr subunit Cmr3

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Macromolecule #4: Cmr4

MacromoleculeName: Cmr4 / type: protein_or_peptide / ID: 4 / Number of copies: 3 / Recombinant expression: Yes
Source (natural)Organism: Pyrococcus furiosus (archaea)
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceUniProtKB: CRISPR system Cmr endoribonuclease Cmr4

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Macromolecule #5: Cmr5

MacromoleculeName: Cmr5 / type: protein_or_peptide / ID: 5 / Number of copies: 3 / Recombinant expression: Yes
Source (natural)Organism: Pyrococcus furiosus (archaea)
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceUniProtKB: CRISPR system Cmr subunit Cmr5

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Macromolecule #6: Cmr6

MacromoleculeName: Cmr6 / type: protein_or_peptide / ID: 6 / Number of copies: 1 / Recombinant expression: Yes
Source (natural)Organism: Pyrococcus furiosus (archaea)
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceUniProtKB: CRISPR system Cmr subunit Cmr6

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration2 mg/mL
BufferpH: 7.5 / Details: 20 mM Tris-HCl, 100 mM NaCl
GridDetails: C-flat 2/2 400 mesh holey carbon grid
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 93 K / Instrument: FEI VITROBOT MARK IV
Method: Plasma clean grids for 5 seconds, apply 3 uL sample, and blot for 4 seconds before plunging.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
TemperatureAverage: 94 K
Alignment procedureLegacy - Astigmatism: Objective lens astigmatism was corrected at 120,000 times magnification
DateNov 2, 2012
Image recordingCategory: CCD / Film or detector model: GATAN ULTRASCAN 4000 (4k x 4k) / Number real images: 4182 / Average electron dose: 20 e/Å2
Electron beamAcceleration voltage: 120 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 107913 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 3.0 µm / Nominal defocus min: 1.5 µm / Nominal magnification: 59000
Sample stageSpecimen holder: Nitrogen cooled / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

DetailsImages were collected automatically using Leginon software. Particles were automatically picked using a template for single particle reconstruction.
CTF correctionDetails: Phase flip each particle
Final reconstructionAlgorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 12.0 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: EMAN, Spider / Number images used: 43749
Final two d classificationNumber classes: 388

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Atomic model buiding 1

Initial modelPDB ID:

4h4k


Chain - #0 - Chain ID: A / Chain - #1 - Chain ID: C
SoftwareName: Chimera
DetailsCmr2-Cmr3 crystal structure was fit into the EM density using the "fit in map" feature from Chimera. EM density corresponding to Cmr2-Cmr3 was segmented out. The Cmr2-Cmr3 structure was refined in the Cmr2-Cmr3 density map using MDFF with an EM scaling factor of 0.3 and implicit solvent.
RefinementSpace: REAL / Protocol: FLEXIBLE FIT

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