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

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Entry
Database: EMDB / ID: EMD-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
Sample
  • Sample: Cmr complex with crRNA and target RNA
  • Protein or peptide: Cmr1
  • Protein or peptide: Cmr2
  • Protein or peptide: Cmr3
  • Protein or peptide: Cmr4
  • Protein or peptide: Cmr5
  • Protein or peptide: Cmr6
  • RNA: crRNA
  • RNA: target RNA
KeywordsCRISPR / Cmr complex / RNA interference / Cmr proteins
Biological speciesPyrococcus furiosus (archaea) / unidentified (others)
Methodsingle particle reconstruction / negative staining / 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: 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

400_6165.gif

Downloads & links

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Map

FileDownload / File: emd_6165.map.gz / Format: CCP4 / Size: 15.3 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationReconstruction of Cmr1-6 with crRNA and target RNA
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
2.78 Å/pix.
x 160 pix.
= 444.8 Å		2.78 Å/pix.
x 160 pix.
= 444.8 Å		2.78 Å/pix.
x 160 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

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 5.0 / Movie #1: 5
Minimum - Maximum-7.56332922 - 20.09210968
Average (Standard dev.)0.0 (±1.0)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-80-80-80
Dimensions160160160
Spacing160160160
CellA=B=C: 444.8 Å
α=β=γ: 90.0 °

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
Components
  • Sample: Cmr complex with crRNA and target RNA
  • Protein or peptide: Cmr1
  • Protein or peptide: Cmr2
  • Protein or peptide: Cmr3
  • Protein or peptide: Cmr4
  • Protein or peptide: Cmr5
  • Protein or peptide: Cmr6
  • RNA: crRNA
  • RNA: target RNA

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Supramolecule #1000: Cmr complex with crRNA and target RNA

SupramoleculeName: Cmr complex with crRNA and target RNA / type: sample / ID: 1000
Oligomeric state: 1 Cmr1 : 1 Cmr2 : 1 Cmr3 : 4 Cmr4 : 3 Cmr5 : 1 Cmr6 : 1 crRNA : 1 target RNA
Number unique components: 8

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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)

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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)

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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)

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Macromolecule #4: Cmr4

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

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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)

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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)

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Macromolecule #7: crRNA

MacromoleculeName: crRNA / type: rna / ID: 7 / Classification: OTHER / Structure: SINGLE STRANDED / Synthetic?: No
Source (natural)Organism: Pyrococcus furiosus (archaea)

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Macromolecule #8: target RNA

MacromoleculeName: target RNA / type: rna / ID: 8 / Classification: OTHER / Structure: SINGLE STRANDED / Synthetic?: No
Source (natural)Organism: unidentified (others)

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Experimental details

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Structure determination

Methodnegative staining
Processingsingle particle reconstruction
Aggregation stateparticle

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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
StainingType: NEGATIVE / Details: 2% w/v uranyl formate
GridDetails: 400 mesh copper grid with thin carbon support
VitrificationCryogen name: NONE / Instrument: OTHER

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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
DateNov 13, 2013
Image recordingCategory: CCD / Film or detector model: GATAN ULTRASCAN 4000 (4k x 4k) / Number real images: 4478 / 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: 2.5 µm / Nominal defocus min: 1.0 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

CTF correctionDetails: phase flip of each particle
Final reconstructionAlgorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 13.0 Å / Resolution method: OTHER / Software - Name: EMAN1, Spider / Number images used: 60293
Final two d classificationNumber classes: 345
FSC plot (resolution estimation)

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