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- EMDB-8749: cryo-EM structure of the Cas9-sgRNA-AcrIIA4 anti-CRISPR complex -

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Basic information

Entry
Database: EMDB / ID: 8749
Titlecryo-EM structure of the Cas9-sgRNA-AcrIIA4 anti-CRISPR complex
Map dataCas9- sgRNA-AcrIIA4 anti-CRISPR complex
Samplethe complex of Streptococcus progenies Cas9 with sgRNA and AcrIIA4:
Streptococcus pyogenes M1 GAS / unidentified phage / nucleic-acidNucleic acid / CRISPR-associated endonuclease Cas9 / phage anti-CRISPR AcrIIA4
Function / homologyCRISPR-associated endonuclease Cas9, PAM-interacting domain / REC lobe of CRISPR-associated endonuclease Cas9 / Cas9-type HNH domain profile. / PAM-interacting domain of CRISPR-associated endonuclease Cas9 / HNH nuclease / CRISPR-associated endonuclease Cas9 / Bridge helix of CRISPR-associated endonuclease Cas9 / CRISPR-associated endonuclease Cas9, bridge helix / CRISPR-associated endonuclease Cas9, REC lobe / Cas9-type HNH domain ...CRISPR-associated endonuclease Cas9, PAM-interacting domain / REC lobe of CRISPR-associated endonuclease Cas9 / Cas9-type HNH domain profile. / PAM-interacting domain of CRISPR-associated endonuclease Cas9 / HNH nuclease / CRISPR-associated endonuclease Cas9 / Bridge helix of CRISPR-associated endonuclease Cas9 / CRISPR-associated endonuclease Cas9, bridge helix / CRISPR-associated endonuclease Cas9, REC lobe / Cas9-type HNH domain / HNH endonuclease / maintenance of CRISPR repeat elements / endodeoxyribonuclease activity / 3'-5' exonuclease activity / defense response to virus / Hydrolases, Acting on ester bonds / RNA binding / DNA binding / metal ion binding / un:a0a0c6fzc2: / CRISPR-associated endonuclease Cas9/Csn1
Function and homology information
SourceStreptococcus pyogenes M1 GAS (bacteria) / unidentified phage (virus)
Methodsingle particle reconstruction / cryo EM / 3.9 Å resolution
AuthorsJiang F / Liu JJ / Nogales E / Doudna JA
CitationJournal: Sci Adv / Year: 2017
Title: Disabling Cas9 by an anti-CRISPR DNA mimic.
Authors: Jiyung Shin / Fuguo Jiang / Jun-Jie Liu / Nicolas L Bray / Benjamin J Rauch / Seung Hyun Baik / Eva Nogales / Joseph Bondy-Denomy / Jacob E Corn / Jennifer A Doudna
Validation ReportPDB-ID: 5vzl

SummaryFull reportAbout validation report
DateDeposition: May 29, 2017 / Header (metadata) release: Jul 26, 2017 / Map release: Jul 26, 2017 / Last update: Mar 20, 2019

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.05
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.05
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: : PDB-5vzl
  • Surface level: 0.05
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

Fileemd_8749.map.gz (map file in CCP4 format, 67109 KB)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
256 pix
1.07 Å/pix.
= 273.92 Å
256 pix
1.07 Å/pix.
= 273.92 Å
256 pix
1.07 Å/pix.
= 273.92 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.07 Å
Density
Contour Level:0.05 (by author), 0.05 (movie #1):
Minimum - Maximum-0.1356536 - 0.21605033
Average (Standard dev.)0.00034119125 (0.004950442)
Details

EMDB XML:

Space Group Number1
Map Geometry
Axis orderXYZ
Dimensions256256256
Origin0.00.00.0
Limit255.0255.0255.0
Spacing256256256
CellA=B=C: 1.0 Å
α=β=γ: 90.0 deg.

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.071.071.07
M x/y/z256256256
origin x/y/z0.0000.0000.000
length x/y/z273.920273.920273.920
α/β/γ90.00090.00090.000
start NX/NY/NZ
NX/NY/NZ
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS256256256
D min/max/mean-0.1360.2160.000

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Supplemental data

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Sample components

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Entire the complex of Streptococcus progenies Cas9 with sgRNA and AcrIIA4

EntireName: the complex of Streptococcus progenies Cas9 with sgRNA and AcrIIA4
Number of components: 6

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Component #1: protein, the complex of Streptococcus progenies Cas9 with sgRNA a...

ProteinName: the complex of Streptococcus progenies Cas9 with sgRNA and AcrIIA4
Recombinant expression: No
SourceSpecies: Streptococcus pyogenes M1 GAS (bacteria)
Source (engineered)Expression System: Escherichia coli (E. coli)

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Component #2: protein, Streptococcus pyogenes M1 GAS

ProteinName: Streptococcus pyogenes M1 GAS / Recombinant expression: No
SourceSpecies: unidentified phage (virus)

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Component #3: protein, unidentified phage

ProteinName: unidentified phage / Recombinant expression: No

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Component #4: nucleic-acid, single guide RNA (116-MER)

Nucleic-acidName: single guide RNA (116-MER) / Class: RNA / Structure: OTHER / Synthetic: No
Sequence:
(GTP)GCGCAUAAA GAUGAGACGC GUUUUAGAGC UAUGCUGUUU UGAAAAAAAC AGCAUAGCAA GUUAAAAUAA GGCUAGUCCG UUAUCAACUU GAAAAAGUGG CACCGAGUCG GUGCUUCG
MassTheoretical: 38.292645 kDa
SourceSpecies: Streptococcus pyogenes M1 GAS (bacteria)

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Component #5: protein, CRISPR-associated endonuclease Cas9

ProteinName: CRISPR-associated endonuclease Cas9 / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 158.772891 kDa
SourceSpecies: Streptococcus pyogenes M1 GAS (bacteria)
Source (engineered)Expression System: Escherichia coli (E. coli)

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Component #6: protein, phage anti-CRISPR AcrIIA4

ProteinName: phage anti-CRISPR AcrIIA4 / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 10.182073 kDa
SourceSpecies: unidentified phage (virus)
Source (engineered)Expression System: Escherichia coli (E. coli)

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Experimental details

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Sample preparation

SpecimenSpecimen state: particle / Method: cryo EM
Sample solutionSpecimen conc.: 1.25 mg/ml / pH: 8
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Temperature: 281 K / Humidity: 100 %
Details: blot for 4.5 seconds with force of 12 before plunging.

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
ImagingMicroscope: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Electron dose: 46 e/Å2 / Illumination mode: FLOOD BEAM
LensCs: 0.01 mm / Imaging mode: BRIGHT FIELD / Defocus: 1000.0 - 3000.0 nm
Specimen HolderModel: OTHER
CameraDetector: GATAN K2 SUMMIT (4k x 4k)

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Image processing

ProcessingMethod: single particle reconstruction / Number of projections: 185000
3D reconstructionResolution: 3.9 Å / Resolution method: FSC 0.143 CUT-OFF

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Atomic model buiding

Modeling #1Refinement space: REAL
Modeling #2Refinement protocol: rigid body / Refinement space: REAL
Output model

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