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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
KeywordsCRISPR / Cmr complex / RNA interference / Cmr proteins
SampleCmr complex with crRNA and target RNA
SourcePyrococcus furiosus / archaea / thermophilic / ピロコッカス・フリオサス
Unidentified
Map dataReconstruction of Cmr1-6 with crRNA and target RNA
Methodsingle particle reconstruction, at 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
CitationCell Rep, 2014, 9, 1610-1617

Cell Rep, 2014, 9, 1610-1617 Yorodumi Papers
Essential structural and functional roles of the Cmr4 subunit in RNA cleavage by the Cmr CRISPR-Cas complex.
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

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
3D viewer


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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 package.

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 / thermophilic / ピロコッカス・フリオサス
Source (engineered)Expression System: Escherichia coli / bacteria / エシェリキア・コリ, 大腸菌 /

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Component #2: protein, Cmr2

ProteinName: Cmr2 / Number of Copies: 1 / Recombinant expression: Yes
SourceSpecies: Pyrococcus furiosus / archaea / thermophilic / ピロコッカス・フリオサス
Source (engineered)Expression System: Escherichia coli / bacteria / エシェリキア・コリ, 大腸菌 /

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Component #3: protein, Cmr3

ProteinName: Cmr3 / Recombinant expression: Yes / Number of Copies: 1
SourceSpecies: Pyrococcus furiosus / archaea / thermophilic / ピロコッカス・フリオサス
Source (engineered)Expression System: Escherichia coli / bacteria / エシェリキア・コリ, 大腸菌 /

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Component #4: protein, Cmr4

ProteinName: Cmr4 / Number of Copies: 4 / Recombinant expression: Yes
SourceSpecies: Pyrococcus furiosus / archaea / thermophilic / ピロコッカス・フリオサス
Source (engineered)Expression System: Escherichia coli / bacteria / エシェリキア・コリ, 大腸菌 /

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Component #5: protein, Cmr5

ProteinName: Cmr5 / Number of Copies: 3 / Recombinant expression: Yes
SourceSpecies: Pyrococcus furiosus / archaea / thermophilic / ピロコッカス・フリオサス
Source (engineered)Expression System: Escherichia coli / bacteria / エシェリキア・コリ, 大腸菌 /

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Component #6: protein, Cmr6

ProteinName: Cmr6 / Recombinant expression: Yes / Number of Copies: 1
SourceSpecies: Pyrococcus furiosus / archaea / thermophilic / ピロコッカス・フリオサス
Source (engineered)Expression System: Escherichia coli / bacteria / エシェリキア・コリ, 大腸菌 /

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Component #7: nucleic-acid, crRNA

Nucleic-acidName: crRNA / Class: RNA / Structure: SINGLE STRANDED / Synthetic: No
SourceSpecies: Pyrococcus furiosus / archaea / thermophilic / ピロコッカス・フリオサス

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Component #8: nucleic-acid, target RNA

Nucleic-acidName: target RNA / Class: RNA / Structure: SINGLE STRANDED / Synthetic: No
SourceSpecies: Unidentified

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Experimental details

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Sample preparation

Specimen stateparticle
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 assessment)

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