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- EMDB-6165: Essential Structural and Functional Roles of the Cmr4 Subunit in ... -

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Basic information

Entry
Database: EMDB / ID: 6165
TitleEssential Structural and Functional Roles of the Cmr4 Subunit in RNA Cleavage by the Cmr CRISPR-Cas Complex
Map dataReconstruction of Cmr1-6 with crRNA and target RNA
SampleCmr complex with crRNA and target RNA:
Cmr1 / Cmr2Mary River Aerodrome / Cmr3 / Cmr4 / Cmr5 / Cmr6 / (nucleic-acidNucleic acid) x 2
KeywordsCRISPR / Cmr complex / RNA interference / Cmr proteins
SourcePyrococcus furiosus (archaea) / unidentified (others)
Methodsingle particle reconstruction / negative staining / 13 Å resolution
AuthorsRamia NF / Spilman M / Tang L / Shao Y / Elmore J / Hale C / Cocozaki A / Bhattacharya N / Terns RM / Terns MP / Li H / Stagg SM
CitationJournal: Cell Rep / Year: 2014
Title: Essential structural and functional roles of the Cmr4 subunit in RNA cleavage by the Cmr CRISPR-Cas complex.
Authors: Nancy F Ramia / Michael Spilman / Li Tang / Yaming Shao / Joshua Elmore / Caryn Hale / Alexis Cocozaki / Nilakshee Bhattacharya / Rebecca M Terns / Michael P Terns / Hong Li / Scott M Stagg
Abstract: The Cmr complex is the multisubunit effector complex of the type III-B clustered regularly interspaced short palindromic repeats (CRISPR)-Cas immune system. The Cmr complex recognizes a target RNA ...The Cmr complex is the multisubunit effector complex of the type III-B clustered regularly interspaced short palindromic repeats (CRISPR)-Cas immune system. The Cmr complex recognizes a target RNA through base pairing with the integral CRISPR RNA (crRNA) and cleaves the target at multiple regularly spaced locations within the complementary region. To understand the molecular basis of the function of this complex, we have assembled information from electron microscopic and X-ray crystallographic structural studies and mutagenesis of a complete Pyrococcus furiosus Cmr complex. Our findings reveal that four helically packed Cmr4 subunits, which make up the backbone of the Cmr complex, act as a platform to support crRNA binding and target RNA cleavage. Interestingly, we found a hook-like structural feature associated with Cmr4 that is likely the site of target RNA binding and cleavage. Our results also elucidate analogies in the mechanisms of crRNA and target molecule binding by the distinct Cmr type III-A and Cascade type I-E complexes.
DateDeposition: Oct 27, 2014 / Header (metadata) release: Nov 26, 2014 / Map release: Dec 17, 2014 / Last update: Dec 24, 2014

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 5
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 5
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

Fileemd_6165.map.gz (map file in CCP4 format, 16001 KB)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
160 pix
2.78 Å/pix.
= 444.8 Å
160 pix
2.78 Å/pix.
= 444.8 Å
160 pix
2.78 Å/pix.
= 444.8 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
Contour Level:5 (by author), 5 (movie #1):
Minimum - Maximum-7.56332922 - 20.09210968
Average (Standard dev.)0E-8 (1)
Details

EMDB XML:

Space Group Number1
Map Geometry
Axis orderXYZ
Dimensions160160160
Origin-80-80-80
Limit797979
Spacing160160160
CellA=B=C: 444.8 Å
α=β=γ: 90 deg.

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z2.782.782.78
M x/y/z160160160
origin x/y/z0.0000.0000.000
length x/y/z444.800444.800444.800
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ969680
MAP C/R/S123
start NC/NR/NS-80-80-80
NC/NR/NS160160160
D min/max/mean-7.56320.0920.000

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Supplemental data

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Sample components

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Entire Cmr complex with crRNA and target RNA

EntireName: Cmr complex with crRNA and target RNA / Number of components: 8
Oligomeric State: 1 Cmr1 : 1 Cmr2 : 1 Cmr3 : 4 Cmr4 : 3 Cmr5 : 1 Cmr6 : 1 crRNA : 1 target RNA

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Component #1: protein, Cmr1

ProteinName: Cmr1 / Number of Copies: 1 / Recombinant expression: Yes
SourceSpecies: Pyrococcus furiosus (archaea)
Source (engineered)Expression System: Escherichia coli (E. coli)

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Component #2: protein, Cmr2

ProteinName: Cmr2Mary River Aerodrome / Number of Copies: 1 / Recombinant expression: Yes
SourceSpecies: Pyrococcus furiosus (archaea)
Source (engineered)Expression System: Escherichia coli (E. coli)

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Component #3: protein, Cmr3

ProteinName: Cmr3 / Recombinant expression: Yes / Number of Copies: 1
SourceSpecies: Pyrococcus furiosus (archaea)
Source (engineered)Expression System: Escherichia coli (E. coli)

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Component #4: protein, Cmr4

ProteinName: Cmr4 / Number of Copies: 4 / Recombinant expression: Yes
SourceSpecies: Pyrococcus furiosus (archaea)
Source (engineered)Expression System: Escherichia coli (E. coli)

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Component #5: protein, Cmr5

ProteinName: Cmr5 / Number of Copies: 3 / Recombinant expression: Yes
SourceSpecies: Pyrococcus furiosus (archaea)
Source (engineered)Expression System: Escherichia coli (E. coli)

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Component #6: protein, Cmr6

ProteinName: Cmr6 / Recombinant expression: Yes / Number of Copies: 1
SourceSpecies: Pyrococcus furiosus (archaea)
Source (engineered)Expression System: Escherichia coli (E. coli)

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Component #7: nucleic-acid, crRNA

Nucleic-acidName: crRNA / Class: RNA / Structure: SINGLE STRANDED / Synthetic: No
SourceSpecies: Pyrococcus furiosus (archaea)

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Component #8: nucleic-acid, target RNA

Nucleic-acidName: target RNA / Class: RNA / Structure: SINGLE STRANDED / Synthetic: No
SourceSpecies: unidentified (others)

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Experimental details

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Sample preparation

SpecimenSpecimen state: particle / Method: negative staining
Sample solutionBuffer solution: 20 mM Tris-HCl, pH 7.4, 500 mM NaCl, 5% v/v glycerol, 5 mM beta-mercaptoethanol
pH: 7.4
Support film400 mesh copper grid with thin carbon support
Staining2% w/v uranyl formate
VitrificationInstrument: NONE / Cryogen name: NONE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
ImagingMicroscope: FEI TITAN KRIOS / Date: Nov 13, 2013
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 120 kV / Electron dose: 15 e/Å2 / Electron beam tilt params: 0 / Illumination mode: FLOOD BEAM
LensMagnification: 59000 X (nominal)
Astigmatism: Objective lens astigmatism was corrected at 150,000 times magnification.
Cs: 2.7 mm / Imaging mode: BRIGHT FIELD / Defocus: 1000 - 2500 nm
Specimen HolderModel: FEI TITAN KRIOS AUTOGRID HOLDER
CameraDetector: GATAN ULTRASCAN 4000 (4k x 4k)

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Image acquisition

Image acquisitionNumber of digital images: 4478

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Image processing

ProcessingMethod: single particle reconstruction / Number of class averages: 345 / Applied symmetry: C1 (asymmetric) / Number of projections: 60293
3D reconstructionAlgorithm: iterative cross correlation against projections of a model
Software: EMAN1, Spider / CTF correction: phase flip of each particle / Resolution: 13 Å / Resolution method: FSC 0.143, semi-independent
FSC plot
(resolution estimation)

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