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- PDB-5vzl: cryo-EM structure of the Cas9-sgRNA-AcrIIA4 anti-CRISPR complex -

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Basic information

Entry
Database: PDB / ID: 5vzl
Titlecryo-EM structure of the Cas9-sgRNA-AcrIIA4 anti-CRISPR complex
Components
  • CRISPR-associated endonuclease Cas9
  • phage anti-CRISPR AcrIIA4
  • single guide RNA (116-MER)
KeywordsImmune System/RNA / anti-CRISPR / Cas9 / CRISPR / gene editing / on-target / off-target / cryo-EM / Immune System-RNA complex
Function / homology
Function and homology information


symbiont-mediated suppression of host CRISPR-cas system / maintenance of CRISPR repeat elements / 3'-5' exonuclease activity / DNA endonuclease activity / defense response to virus / Hydrolases; Acting on ester bonds / DNA binding / RNA binding / metal ion binding
Similarity search - Function
CRISPR-associated endonuclease Cas9, PAM-interacting domain / CRISPR-associated endonuclease Cas9, bridge helix / CRISPR-associated endonuclease Cas9, REC lobe / REC lobe of CRISPR-associated endonuclease Cas9 / Bridge helix of CRISPR-associated endonuclease Cas9 / PAM-interacting domain of CRISPR-associated endonuclease Cas9 / CRISPR-associated endonuclease Cas9 / HNH endonuclease / Cas9-type HNH domain / Cas9-type HNH domain profile. ...CRISPR-associated endonuclease Cas9, PAM-interacting domain / CRISPR-associated endonuclease Cas9, bridge helix / CRISPR-associated endonuclease Cas9, REC lobe / REC lobe of CRISPR-associated endonuclease Cas9 / Bridge helix of CRISPR-associated endonuclease Cas9 / PAM-interacting domain of CRISPR-associated endonuclease Cas9 / CRISPR-associated endonuclease Cas9 / HNH endonuclease / Cas9-type HNH domain / Cas9-type HNH domain profile. / HNH nuclease / Ribonuclease H superfamily
Similarity search - Domain/homology
RNA / RNA (> 10) / RNA (> 100) / : / phage anti-CRISPR AcrIIA4 / CRISPR-associated endonuclease Cas9/Csn1
Similarity search - Component
Biological speciesStreptococcus pyogenes M1 GAS (bacteria)
unidentified phage (virus)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.9 Å
AuthorsJiang, F. / Liu, J.J. / Nogales, E. / Doudna, J.A.
Funding support United States, 1items
OrganizationGrant numberCountry
Howard Hughes Medical Institute (HHMI) United States
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 /
Abstract: CRISPR (clustered regularly interspaced short palindromic repeats)-Cas9 gene editing technology is derived from a microbial adaptive immune system, where bacteriophages are often the intended target. ...CRISPR (clustered regularly interspaced short palindromic repeats)-Cas9 gene editing technology is derived from a microbial adaptive immune system, where bacteriophages are often the intended target. Natural inhibitors of CRISPR-Cas9 enable phages to evade immunity and show promise in controlling Cas9-mediated gene editing in human cells. However, the mechanism of CRISPR-Cas9 inhibition is not known, and the potential applications for Cas9 inhibitor proteins in mammalian cells have not been fully established. We show that the anti-CRISPR protein AcrIIA4 binds only to assembled Cas9-single-guide RNA (sgRNA) complexes and not to Cas9 protein alone. A 3.9 Å resolution cryo-electron microscopy structure of the Cas9-sgRNA-AcrIIA4 complex revealed that the surface of AcrIIA4 is highly acidic and binds with a 1:1 stoichiometry to a region of Cas9 that normally engages the DNA protospacer adjacent motif. Consistent with this binding mode, order-of-addition experiments showed that AcrIIA4 interferes with DNA recognition but has no effect on preformed Cas9-sgRNA-DNA complexes. Timed delivery of AcrIIA4 into human cells as either protein or expression plasmid allows on-target Cas9-mediated gene editing while reducing off-target edits. These results provide a mechanistic understanding of AcrIIA4 function and demonstrate that inhibitors can modulate the extent and outcomes of Cas9-mediated gene editing.
History
DepositionMay 29, 2017Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jul 26, 2017Provider: repository / Type: Initial release
Revision 1.1Jan 31, 2018Group: Author supporting evidence / Data processing ...Author supporting evidence / Data processing / Database references / Experimental preparation
Category: em_sample_support / em_software ...em_sample_support / em_software / pdbx_audit_support / pdbx_database_related
Item: _em_sample_support.grid_type / _em_software.name / _pdbx_audit_support.funding_organization
Revision 1.2Mar 20, 2019Group: Data collection / Other / Structure summary / Category: cell / struct_keywords
Item: _cell.length_a / _cell.length_b ..._cell.length_a / _cell.length_b / _cell.length_c / _struct_keywords.pdbx_keywords / _struct_keywords.text
Revision 1.3Nov 20, 2019Group: Author supporting evidence / Category: pdbx_audit_support / Item: _pdbx_audit_support.funding_organization
Revision 1.4Dec 18, 2019Group: Other / Category: atom_sites
Item: _atom_sites.fract_transf_matrix[1][1] / _atom_sites.fract_transf_matrix[2][2] / _atom_sites.fract_transf_matrix[3][3]
Revision 1.5Mar 13, 2024Group: Data collection / Database references / Category: chem_comp_atom / chem_comp_bond / database_2
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession

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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: phage anti-CRISPR AcrIIA4


Theoretical massNumber of molelcules
Total (without water)207,2483
Polymers207,2483
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area17050 Å2
ΔGint-115 kcal/mol
Surface area84820 Å2

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Components

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


Mass: 38292.645 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Streptococcus pyogenes M1 GAS (bacteria)
Gene: csn1, cas9, ERS445054_00848 / Production host: Escherichia coli (E. coli)
#2: Protein CRISPR-associated endonuclease Cas9


Mass: 158772.891 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Streptococcus pyogenes M1 GAS (bacteria)
Gene: csn1, cas9, ERS445054_00848 / Production host: Escherichia coli (E. coli)
References: UniProt: A0A0C6FZC2, UniProt: Q99ZW2*PLUS, Hydrolases; Acting on ester bonds
#3: Protein phage anti-CRISPR AcrIIA4


Mass: 10182.073 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) unidentified phage (virus) / Production host: Escherichia coli (E. coli) / References: UniProt: A0A2D0TCG7*PLUS

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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
IDNameTypeEntity IDParent-IDSource
1the complex of Streptococcus progenies Cas9 with sgRNA and AcrIIA4COMPLEXall0RECOMBINANT
2Streptococcus pyogenes M1 GASCOMPLEX#1-#21RECOMBINANT
3unidentified phageCOMPLEX#31RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
21Streptococcus pyogenes M1 GAS (bacteria)160490
32unidentified phage (virus)38018
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 8
SpecimenConc.: 1.25 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: spyCas9, sgRNA and AcrIIA4 complex
Specimen supportGrid material: COPPER / Grid mesh size: 400 divisions/in. / Grid type: C-flat-2/2
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 281 K
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
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 3000 nm / Nominal defocus min: 1000 nm / Calibrated defocus min: 900 nm / Calibrated defocus max: 4000 nm / Cs: 0.01 mm / C2 aperture diameter: 100 µm
Image recordingElectron dose: 46 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K2 SUMMIT (4k x 4k)

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Processing

SoftwareName: PHENIX / Version: 1.11.1_2575: / Classification: refinement
EM software
IDNameVersionCategoryDetails
4RELION2CTF correctionCTFFIND4
11RELION2final Euler assignment
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.9 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 185000 / Symmetry type: POINT
Atomic model building
IDProtocolSpace
1AB INITIO MODELREAL
2RIGID BODY FITREAL
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00614414
ELECTRON MICROSCOPYf_angle_d0.92720059
ELECTRON MICROSCOPYf_dihedral_angle_d12.1258452
ELECTRON MICROSCOPYf_chiral_restr0.052356
ELECTRON MICROSCOPYf_plane_restr0.0062153

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