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- PDB-6mcc: CryoEM structure of AcrIIA2 homolog in complex with CRISPR-Cas9 -

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Basic information

Entry
Database: PDB / ID: 6mcc
TitleCryoEM structure of AcrIIA2 homolog in complex with CRISPR-Cas9
Components
  • Anti-CRISPR AcrIIA2 Homolog
  • CRISPR-associated endonuclease Cas9
  • Single guide RNA (116-MER)
KeywordsHYDROLASE/RNA/VIRAL PROTEIN / CRISPR-Cas / Cas9 / Cas9 inhibitors / anti-CRISPR / AcrIIA2 / bacteriophage / gene editing / HYDROLASE-RNA-VIRAL PROTEIN complex
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
Specimen sourceStreptococcus pyogenes M1 GAS (bacteria)
Listeria phage LP-101 (bacteriophage)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / 3.9 Å resolution
AuthorsJiang, F. / Liu, J.J. / Doudna, J.A.
Funding supportUnited States , 1 items
OrganizationGrant numberCountry
Howard Hughes Medical InstituteUnited States
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 Report
SummaryFull reportAbout validation report
DateDeposition: Aug 31, 2018 / Release: Jan 16, 2019
RevisionDateData content typeGroupCategoryItemProviderType
1.0Jan 16, 2019Structure modelrepositoryInitial release
1.1Feb 20, 2019Structure modelData collection / Database referencescitation_citation.journal_volume / _citation.page_first / _citation.year

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Assembly

Deposited unit
B: Single guide RNA (116-MER)
A: CRISPR-associated endonuclease Cas9
C: Anti-CRISPR AcrIIA2 Homolog


Theoretical massNumber of molelcules
Total (without water)211,6263
Polyers211,6263
Non-polymers00
Water0
1


TypeNameSymmetry operationNumber
identity operation1_5551
Buried area (Å2)17330
ΔGint (kcal/M)-137
Surface area (Å2)85660

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Components

#1: RNA chain Single guide RNA (116-MER)


Mass: 37642.258 Da / Num. of mol.: 1
Source: (gene. exp.) Streptococcus pyogenes M1 GAS (bacteria)
Production host: Escherichia coli (E. coli)
#2: Protein/peptide CRISPR-associated endonuclease Cas9


Mass: 158685.828 Da / Num. of mol.: 1
Source: (gene. exp.) Streptococcus pyogenes M1 GAS (bacteria)
Gene: cas9, M1GAS476_0830 / Production host: Escherichia coli (E. coli)
References: UniProt: J7M7J1, Hydrolases, Acting on ester bonds
#3: Protein/peptide Anti-CRISPR AcrIIA2 Homolog / AcrIIA2b.3


Mass: 15297.688 Da / Num. of mol.: 1 / Source: (gene. exp.) Listeria phage LP-101 (bacteriophage) / Production host: Escherichia coli (E. coli)

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / Reconstruction method: single particle reconstruction

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Sample preparation

Component
IDNameTypeEntity IDParent IDSource
1AcrIIA2 homolog (AcrIIA2b) bound to sgRNA-bound Streptococcus pyogenes Cas9COMPLEX1, 2, 30MULTIPLE SOURCES
2sgRNA-bound Streptococcus pyogenes Cas9COMPLEX1, 21RECOMBINANT
3AcrIIA2 homolog (AcrIIA2b)COMPLEX31RECOMBINANT
Source (natural)
IDEntity assembly IDNcbi tax IDOrganism
12160490Streptococcus pyogenes M1 GAS (bacteria)
231458856Listeria phage LP-101 (bacteriophage)
Source (recombinant)
IDEntity assembly IDNcbi tax IDOrganism
12562Escherichia coli (E. coli)
23562Escherichia coli (E. coli)
Buffer solutionDetails: 30 mM Tris-HCl, pH 8.0, 150 mM sodium chloride, 5 mM DTT, 0.1% glycerol
pH: 8
SpecimenDetails: SpyCas9-sgRNA-AcrIIA2b complex / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: unspecified
VitrificationInstrument: FEI VITROBOT MARK II / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 kelvins

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyMicroscope model: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy
Image recordingElectron dose: 46.2 e/Å2 / Film or detector model: GATAN K2 SUMMIT (4k x 4k)

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Processing

SoftwareName: PHENIX / Version: 1.12_2829: / Classification: refinement
EM software
IDNameCategory
7Cootmodel fitting
13PHENIXmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.9 Å / Resolution method: FSC 0.143 CUT-OFF / Number of particles: 167464 / Symmetry type: POINT
Atomic model buildingRef protocol: OTHER / Ref space: REAL
Atomic model buildingPDB-ID: 4ZT0
Refine LS restraints
Refine IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00614683
ELECTRON MICROSCOPYf_angle_d1.00220439
ELECTRON MICROSCOPYf_dihedral_angle_d11.3318607
ELECTRON MICROSCOPYf_chiral_restr0.0562411
ELECTRON MICROSCOPYf_plane_restr0.0062194

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