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

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Basic information

Entry
Database: PDB / ID: 6mcb
TitleCryoEM structure of AcrIIA2 in complex with CRISPR-Cas9
Components
  • Anti-CRISPR protein AcrIIA2
  • 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 / homology
Function and homology information


maintenance of CRISPR repeat elements / defense response to virus / endonuclease activity / Hydrolases, Acting on ester bonds / RNA binding / DNA binding / metal ion binding
PAM-interacting domain of CRISPR-associated endonuclease Cas9 / CRISPR-associated endonuclease Cas9, bridge helix / Bridge helix of CRISPR-associated endonuclease Cas9 / REC lobe of CRISPR-associated endonuclease Cas9 / HNH endonuclease / Cas9-type HNH domain / CRISPR-associated endonuclease Cas9, REC lobe / CRISPR-associated endonuclease Cas9, PAM-interacting domain / CRISPR-associated endonuclease Cas9 / HNH nuclease
CRISPR-associated endonuclease Cas9
Biological speciesStreptococcus pyogenes M1 GAS (bacteria)
Listeria phage LP-101 (bacteriophage)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.4 Å
AuthorsJiang, F. / Liu, J.J. / Doudna, J.A.
Funding support United States, 1items
OrganizationGrant numberCountry
Howard Hughes Medical Institute (HHMI) United 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
History
DepositionAug 31, 2018Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jan 16, 2019Provider: repository / Type: Initial release
Revision 1.1Feb 20, 2019Group: Data collection / Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first / _citation.year
Revision 1.2Nov 20, 2019Group: Author supporting evidence / Category: pdbx_audit_support / Item: _pdbx_audit_support.funding_organization

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Structure visualization

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Assembly

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


Theoretical massNumber of molelcules
Total (without water)210,4963
Polymers210,4963
Non-polymers00
Water0
1


TypeNameSymmetry operationNumber
identity operation1_5551
Buried area18560 Å2
ΔGint-134 kcal/mol
Surface area78800 Å2

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Components

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


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


Mass: 158685.828 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
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 Anti-CRISPR protein AcrIIA2 /


Mass: 14167.594 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
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 / 3D reconstruction method: single particle reconstruction

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Sample preparation

Component

Type: COMPLEX

IDNameEntity IDParent-IDSource
1AcrIIA2 interacting with sgRNA-bound Streptococcus pyogenes Cas91, 2, 30MULTIPLE SOURCES
2sgRNA-bound Streptococcus pyogenes Cas91, 21RECOMBINANT
3AcrIIA231RECOMBINANT
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
12Streptococcus pyogenes M1 GAS (bacteria)160490
23Listeria phage LP-101 (bacteriophage)1458856
Source (recombinant)

Ncbi tax-ID: 562 / Organism: Escherichia coli (E. coli)

IDEntity assembly-ID
12
23
Buffer solutionpH: 8
Details: 30 mM Tris-HCl, pH 8.0, 150 mM sodium chloride, 5 mM DTT, 0.1% glycerol
SpecimenDetails: SpyCas9-sgRNA-AcrIIA2 complex / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid type: C-flat-2/0.5 4C
VitrificationInstrument: FEI VITROBOT MARK II / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: 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: 43.2 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
IDNameVersionCategory
7Coot0.8.9model fitting
13PHENIX1.12-2829model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.4 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 263270 / Symmetry type: POINT
Atomic model buildingProtocol: OTHER / Space: REAL
Atomic model buildingPDB-ID: 4ZT0
Refine LS restraints

Refinement-ID: ELECTRON MICROSCOPY

TypeDev idealNumber
f_bond_d0.0114693
f_angle_d1.08820446
f_dihedral_angle_d11.9598623
f_chiral_restr0.0612407
f_plane_restr0.0072197

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