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- EMDB-6167: Essential Structural and Functional Roles of the Cmr4 Subunit in ... -

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Basic information

Entry
Database: EMDB / ID: EMD-6167
TitleEssential Structural and Functional Roles of the Cmr4 Subunit in RNA Cleavage by the Cmr CRISPR-Cas Complex
Map dataHelical reconstruction of Cmr4-5 filament
Sample
  • Sample: Filament of Cmr4 and Cmr5
  • Protein or peptide: Cmr4
  • Protein or peptide: Cmr5
KeywordsCRISPR / Cmr complex / RNA interference / Cmr proteins
Biological speciesPyrococcus furiosus (archaea)
Methodhelical reconstruction / cryo EM / Resolution: 13.0 Å
AuthorsRamia NF / Spilman M / Tang L / Shao Y / Elmore J / Hale C / Cocozaki A / Bhattacharya N / Terns RM / Terns MP ...Ramia 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.
History
DepositionOct 27, 2014-
Header (metadata) releaseNov 26, 2014-
Map releaseDec 17, 2014-
UpdateDec 24, 2014-
Current statusDec 24, 2014Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 3
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 3
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

400_6167.gif

Downloads & links

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Map

FileDownload / File: emd_6167.map.gz / Format: CCP4 / Size: 3.3 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationHelical reconstruction of Cmr4-5 filament
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
2.78 Å/pix.
x 96 pix.
= 266.88 Å		2.78 Å/pix.
x 96 pix.
= 266.88 Å		2.78 Å/pix.
x 96 pix.
= 266.88 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 EMDB: 3.3 / Movie #1: 3
Minimum - Maximum-5.21389723 - 7.38952255
Average (Standard dev.)0.03401719 (±1.03925931)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-48-48-48
Dimensions969696
Spacing969696
CellA=B=C: 266.88 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z2.782.782.78
M x/y/z969696
origin x/y/z0.0000.0000.000
length x/y/z266.880266.880266.880
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ969680
MAP C/R/S123
start NC/NR/NS-48-48-48
NC/NR/NS969696
D min/max/mean-5.2147.3900.034

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Supplemental data

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Sample components

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Entire : Filament of Cmr4 and Cmr5

EntireName: Filament of Cmr4 and Cmr5
Components
  • Sample: Filament of Cmr4 and Cmr5
  • Protein or peptide: Cmr4
  • Protein or peptide: Cmr5

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Supramolecule #1000: Filament of Cmr4 and Cmr5

SupramoleculeName: Filament of Cmr4 and Cmr5 / type: sample / ID: 1000 / Number unique components: 2

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Macromolecule #1: Cmr4

MacromoleculeName: Cmr4 / type: protein_or_peptide / ID: 1 / Recombinant expression: Yes
Source (natural)Organism: Pyrococcus furiosus (archaea)
Recombinant expressionOrganism: Escherichia coli (E. coli)

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Macromolecule #2: Cmr5

MacromoleculeName: Cmr5 / type: protein_or_peptide / ID: 2 / Recombinant expression: Yes
Source (natural)Organism: Pyrococcus furiosus (archaea)
Recombinant expressionOrganism: Escherichia coli (E. coli)

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Experimental details

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Structure determination

Methodcryo EM
Processinghelical reconstruction
Aggregation statehelical array

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Sample preparation

BufferpH: 7.4
Details: 20 mM Tris-HCl, pH 7.4, 500 mM NaCl, 5% v/v glycerol, 5 mM beta-mercaptoethanol
GridDetails: 400 mesh Quantifoil grid with 2-micron holes
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Instrument: FEI VITROBOT MARK IV
DetailsFilaments were prepared for cryoEM by heating a sample of Cmr4-5 to 70 degrees C for 15 minutes and cooling to room temperature over 10 minutes.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Alignment procedureLegacy - Astigmatism: Objective lens astigmatism was corrected at 150,000 times magnification.
Legacy - Electron beam tilt params: 0
DateJun 19, 2013
Image recordingCategory: CCD / Film or detector model: GATAN ULTRASCAN 4000 (4k x 4k) / Number real images: 1866 / Average electron dose: 15 e/Å2
Electron beamAcceleration voltage: 120 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 3.3 µm / Nominal defocus min: 1.7 µm / Nominal magnification: 59000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

DetailsFeatureless cylinder was refined by projection and cross correlation with EMAN1, correspondence analysis with Spider, and helical symmetry refinement with IHRSR.
Final reconstructionApplied symmetry - Helical parameters - Δz: 24.7 Å
Applied symmetry - Helical parameters - Δ&Phi: 48.2 °
Applied symmetry - Helical parameters - Axial symmetry: C1 (asymmetric)
Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 13.0 Å / Resolution method: OTHER / Software - Name: EMAN1, Spider, IHRSR
CTF correctionDetails: phase flip of each particle
FSC plot (resolution estimation)

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