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- PDB-8ja0: Cryo-EM structure of the NmeCas9-sgRNA-AcrIIC4 ternary complex -

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Basic information

Entry
Database: PDB / ID: 8ja0
TitleCryo-EM structure of the NmeCas9-sgRNA-AcrIIC4 ternary complex
Components
  • CRISPR-associated endonuclease Cas9
  • RNA (117-MER)
  • Uncharacterized protein
KeywordsRNA BINDING PROTEIN/RNA / a protein complex / VIRAL PROTEIN / RNA BINDING PROTEIN-RNA complex
Function / homology
Function and homology information


maintenance of CRISPR repeat elements / endonuclease activity / defense response to virus / Hydrolases; Acting on ester bonds / DNA binding / RNA binding / metal ion binding
Similarity search - Function
RuvC endonuclease subdomain 3 / RuvC endonuclease subdomain 3 / HNH endonuclease / CRISPR-associated endonuclease Cas9 / Cas9-type HNH domain / Cas9-type HNH domain profile. / HNH nuclease / Ribonuclease H superfamily
Similarity search - Domain/homology
RNA / RNA (> 10) / RNA (> 100) / Uncharacterized protein / CRISPR-associated endonuclease Cas9
Similarity search - Component
Biological speciesNeisseria meningitidis (bacteria)
Haemophilus parainfluenzae (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.52 Å
AuthorsYin, H. / Li, Z. / Yu, G.M. / Li, X.Z.
CitationJournal: Nucleic Acids Res / Year: 2023
Title: Inhibitory mechanism of CRISPR-Cas9 by AcrIIC4.
Authors: Xuzichao Li / Fumeng Liao / Jiaqi Gao / Guangyong Song / Chendi Zhang / Nan Ji / Xiaoshen Wang / Jing Wen / Jia He / Yong Wei / Heng Zhang / Zhuang Li / Guimei Yu / Hang Yin /
Abstract: CRISPR-Cas systems act as the adaptive immune systems of bacteria and archaea, targeting and destroying invading foreign mobile genetic elements (MGEs) such as phages. MGEs have also evolved anti- ...CRISPR-Cas systems act as the adaptive immune systems of bacteria and archaea, targeting and destroying invading foreign mobile genetic elements (MGEs) such as phages. MGEs have also evolved anti-CRISPR (Acr) proteins to inactivate the CRISPR-Cas systems. Recently, AcrIIC4, identified from Haemophilus parainfluenzae phage, has been reported to inhibit the endonuclease activity of Cas9 from Neisseria meningitidis (NmeCas9), but the inhibition mechanism is not clear. Here, we biochemically and structurally investigated the anti-CRISPR activity of AcrIIC4. AcrIIC4 folds into a helix bundle composed of three helices, which associates with the REC lobe of NmeCas9 and sgRNA. The REC2 domain of NmeCas9 is locked by AcrIIC4, perturbing the conformational dynamics required for the target DNA binding and cleavage. Furthermore, mutation of the key residues in the AcrIIC4-NmeCas9 and AcrIIC4-sgRNA interfaces largely abolishes the inhibitory effects of AcrIIC4. Our study offers new insights into the mechanism of AcrIIC4-mediated suppression of NmeCas9 and provides guidelines for the design of regulatory tools for Cas9-based gene editing applications.
History
DepositionMay 5, 2023Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Aug 9, 2023Provider: repository / Type: Initial release
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Revision 1.1Jul 3, 2024Group: Data collection / Category: chem_comp_atom / chem_comp_bond / em_admin / Item: _em_admin.last_update
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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: CRISPR-associated endonuclease Cas9
D: Uncharacterized protein
B: RNA (117-MER)


Theoretical massNumber of molelcules
Total (without water)171,9663
Polymers171,9663
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein CRISPR-associated endonuclease Cas9


Mass: 124613.883 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Neisseria meningitidis (bacteria) / Gene: cas9, NMV_1993 / Production host: Escherichia coli (E. coli)
References: UniProt: C9X1G5, Hydrolases; Acting on ester bonds
#2: Protein Uncharacterized protein


Mass: 9985.316 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Haemophilus parainfluenzae (bacteria) / Gene: NCTC10672_00033, NCTC10672_02354 / Production host: Escherichia coli (E. coli) / References: UniProt: A0A377JKY9
#3: RNA chain RNA (117-MER)


Mass: 37367.059 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Neisseria meningitidis (bacteria) / Production host: Escherichia coli (E. coli)
Has protein modificationN

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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

ComponentName: a protein complex / Type: COMPLEX / Entity ID: all / Source: MULTIPLE SOURCES
Source (natural)Organism: Neisseria meningitidis (bacteria)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7.5
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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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 FIELD / Nominal defocus max: 2500 nm / Nominal defocus min: 500 nm
Image recordingElectron dose: 40 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

SoftwareName: PHENIX / Version: 1.20.1_4487: / Classification: refinement
CTF correctionType: PHASE FLIPPING ONLY
3D reconstructionResolution: 3.52 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 251996 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00211739
ELECTRON MICROSCOPYf_angle_d0.5616380
ELECTRON MICROSCOPYf_dihedral_angle_d12.9262600
ELECTRON MICROSCOPYf_chiral_restr0.0351901
ELECTRON MICROSCOPYf_plane_restr0.0041707

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