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- EMDB-9066: CryoEM structure of AcrIIA2 in complex with CRISPR-Cas9 -

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Basic information

Entry
Database: EMDB / ID: 9066
TitleCryoEM structure of AcrIIA2 in complex with CRISPR-Cas9
Map dataSpyCas9-A2a complex
SampleAcrIIA2 interacting with sgRNA-bound Streptococcus pyogenes Cas9:
sgRNA-bound Streptococcus pyogenes Cas9 / AcrIIA2 / nucleic-acidNucleic acid / CRISPR-associated endonuclease Cas9 / Anti-CRISPR protein AcrIIA2
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 / defense response to virus / endonuclease activity / Hydrolases, Acting on ester bonds / RNA binding / DNA binding / metal ion binding / CRISPR-associated endonuclease Cas9
Function and homology information
SourceStreptococcus pyogenes M1 GAS (bacteria) / Listeria phage LP-101 (bacteriophage)
Methodsingle particle reconstruction / cryo EM / 3.4 Å resolution
AuthorsJiang F / Liu JJ / Doudna JA
CitationJournal: Mol. Cell / Year: 2019
Title: Temperature-Responsive Competitive Inhibition of CRISPR-Cas9.
Authors: Fuguo Jiang / Jun-Jie Liu / Beatriz A Osuna / Michael Xu / Joel D Berry / Benjamin J Rauch / Eva Nogales / Joseph Bondy-Denomy / Jennifer A Doudna
Abstract: CRISPR-Cas immune systems utilize RNA-guided nucleases to protect bacteria from bacteriophage infection. Bacteriophages have in turn evolved inhibitory "anti-CRISPR" (Acr) proteins, including six ...CRISPR-Cas immune systems utilize RNA-guided nucleases to protect bacteria from bacteriophage infection. Bacteriophages have in turn evolved inhibitory "anti-CRISPR" (Acr) proteins, including six inhibitors (AcrIIA1-AcrIIA6) that can block DNA cutting and genome editing by type II-A CRISPR-Cas9 enzymes. We show here that AcrIIA2 and its more potent homolog, AcrIIA2b, prevent Cas9 binding to DNA by occluding protein residues required for DNA binding. Cryo-EM-determined structures of AcrIIA2 or AcrIIA2b bound to S. pyogenes Cas9 reveal a mode of competitive inhibition of DNA binding that is distinct from other known Acrs. Differences in the temperature dependence of Cas9 inhibition by AcrIIA2 and AcrIIA2b arise from differences in both inhibitor structure and the local inhibitor-binding environment on Cas9. These findings expand the natural toolbox for regulating CRISPR-Cas9 genome editing temporally, spatially, and conditionally.
Validation ReportPDB-ID: 6mcb

SummaryFull reportAbout validation report
DateDeposition: Aug 31, 2018 / Header (metadata) release: Sep 26, 2018 / Map release: Jan 16, 2019 / Last update: Jan 16, 2019

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.28
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.28
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: : PDB-6mcb
  • Surface level: 0.28
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

Fileemd_9066.map.gz (map file in CCP4 format, 131073 KB)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
320 pix
0.83 Å/pix.
= 265.6 Å
320 pix
0.83 Å/pix.
= 265.6 Å
320 pix
0.83 Å/pix.
= 265.6 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.83 Å
Density
Contour Level:0.28 (by author), 0.28 (movie #1):
Minimum - Maximum-1.2549226 - 1.886544
Average (Standard dev.)0.00041811747 (0.049322553)
Details

EMDB XML:

Space Group Number1
Map Geometry
Axis orderXYZ
Dimensions320320320
Origin0.00.00.0
Limit319.0319.0319.0
Spacing320320320
CellA=B=C: 265.6 Å
α=β=γ: 90.0 deg.

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z0.830.830.83
M x/y/z320320320
origin x/y/z0.0000.0000.000
length x/y/z265.600265.600265.600
α/β/γ90.00090.00090.000
start NX/NY/NZ
NX/NY/NZ
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS320320320
D min/max/mean-1.2551.8870.000

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Supplemental data

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Sample components

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Entire AcrIIA2 interacting with sgRNA-bound Streptococcus pyogenes Cas9

EntireName: AcrIIA2 interacting with sgRNA-bound Streptococcus pyogenes Cas9
Number of components: 6

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Component #1: protein, AcrIIA2 interacting with sgRNA-bound Streptococcus pyoge...

ProteinName: AcrIIA2 interacting with sgRNA-bound Streptococcus pyogenes Cas9
Recombinant expression: No

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Component #2: protein, sgRNA-bound Streptococcus pyogenes Cas9

ProteinName: sgRNA-bound Streptococcus pyogenes Cas9 / Recombinant expression: No
SourceSpecies: Streptococcus pyogenes M1 GAS (bacteria)
Source (engineered)Expression System: Escherichia coli (E. coli)

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Component #3: protein, AcrIIA2

ProteinName: AcrIIA2 / Recombinant expression: No
SourceSpecies: Listeria phage LP-101 (bacteriophage)
Source (engineered)Expression System: Escherichia coli (E. coli)

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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 GUGCUU
MassTheoretical: 37.642258 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: 158.685828 kDa
SourceSpecies: Streptococcus pyogenes M1 GAS (bacteria)
Source (engineered)Expression System: Escherichia coli (E. coli)

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Component #6: protein, Anti-CRISPR protein AcrIIA2

ProteinName: Anti-CRISPR protein AcrIIA2 / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 14.167594 kDa
SourceSpecies: Listeria phage LP-101 (bacteriophage)
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 solutionBuffer solution: 30 mM Tris-HCl, pH 8.0, 150 mM sodium chloride, 5 mM DTT, 0.1% glycerol
pH: 8
VitrificationInstrument: FEI VITROBOT MARK II / Cryogen name: ETHANE / Temperature: 277 K / Humidity: 100 %

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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: 43.2 e/Å2 / Illumination mode: FLOOD BEAM
LensImaging mode: BRIGHT FIELD
Specimen HolderModel: OTHER
CameraDetector: GATAN K2 SUMMIT (4k x 4k)

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Image processing

ProcessingMethod: single particle reconstruction / Number of projections: 263270
3D reconstructionResolution: 3.4 Å / Resolution method: FSC 0.143 CUT-OFF

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Atomic model buiding

Modeling #1Refinement space: REAL
Input PDB model: 4ZT0
Output model

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