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- PDB-8hj4: CryoEM structure of an anti-CRISPR protein AcrIIC5 bound to Nme1C... -

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Basic information

Entry
Database: PDB / ID: 8hj4
TitleCryoEM structure of an anti-CRISPR protein AcrIIC5 bound to Nme1Cas9-sgRNA complex
Components
  • CRISPR-associated endonuclease Cas9
  • Phage protein
  • sgRNA
KeywordsHYDROLASE/RNA/ANTIMICROBIAL PROTEIN / Cas9 / anti-CRISPR proteins / cleavage inhibition / ANTIMICROBIAL PROTEIN / HYDROLASE-RNA-ANTIMICROBIAL PROTEIN 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) / CRISPR-associated endonuclease Cas9 / Phage protein
Similarity search - Component
Biological speciesNeisseria meningitidis serogroup C (bacteria)
Simonsiella muelleri ATCC 29453 (bacteria)
Neisseria meningitidis 8013 (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.1 Å
AuthorsWang, Y. / Sun, W.
Funding support China, 1items
OrganizationGrant numberCountry
National Natural Science Foundation of China (NSFC) China
CitationJournal: Nucleic Acids Res / Year: 2023
Title: Anti-CRISPR AcrIIC5 is a dsDNA mimic that inhibits type II-C Cas9 effectors by blocking PAM recognition.
Authors: Wei Sun / Xiaolong Zhao / Jinlong Wang / Xiaoqi Yang / Zhi Cheng / Shuo Liu / Jiuyu Wang / Gang Sheng / Yanli Wang /
Abstract: Anti-CRISPR proteins are encoded by phages to inhibit the CRISPR-Cas systems of the hosts. AcrIIC5 inhibits several naturally high-fidelity type II-C Cas9 enzymes, including orthologs from Neisseria ...Anti-CRISPR proteins are encoded by phages to inhibit the CRISPR-Cas systems of the hosts. AcrIIC5 inhibits several naturally high-fidelity type II-C Cas9 enzymes, including orthologs from Neisseria meningitidis (Nme1Cas9) and Simonsiella muelleri (SmuCas9). Here, we solve the structure of AcrIIC5 in complex with Nme1Cas9 and sgRNA. We show that AcrIIC5 adopts a novel fold to mimic the size and charge distribution of double-stranded DNA, and uses its negatively charged grooves to bind and occlude the protospacer adjacent motif (PAM) binding site in the target DNA cleft of Cas9. AcrIIC5 is positioned into the crevice between the WED and PI domains of Cas9, and one end of the anti-CRISPR interacts with the phosphate lock loop and a linker between the RuvC and BH domains. We employ biochemical and mutational analyses to build a model for AcrIIC5's mechanism of action, and identify residues on both the anti-CRISPR and Cas9 that are important for their interaction and inhibition. Together, the structure and mechanism of AcrIIC5 reveal convergent evolution among disparate anti-CRISPR proteins that use a DNA-mimic strategy to inhibit diverse CRISPR-Cas surveillance complexes, and provide new insights into a tool for potent inhibition of type II-C Cas9 orthologs.
History
DepositionNov 22, 2022Deposition site: PDBJ / Processing site: PDBJ
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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: CRISPR-associated endonuclease Cas9
B: sgRNA
C: Phage protein


Theoretical massNumber of molelcules
Total (without water)183,1603
Polymers183,1603
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein CRISPR-associated endonuclease Cas9


Mass: 124700.961 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Neisseria meningitidis serogroup C (strain 8013) (bacteria)
Gene: cas9, NMV_1993 / Production host: Escherichia coli BL21(DE3) (bacteria)
References: UniProt: C9X1G5, Hydrolases; Acting on ester bonds
#2: RNA chain sgRNA


Mass: 43059.352 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Neisseria meningitidis 8013 (bacteria)
#3: Protein Phage protein


Mass: 15399.833 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Simonsiella muelleri ATCC 29453 (bacteria)
Gene: HMPREF9021_01755 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: V9H5N5
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: Complex of AcrIIC5 with sgRNA-bound Nme1Cas9 / Type: COMPLEX / Entity ID: all / Source: MULTIPLE SOURCES
Molecular weightValue: 183.04 kDa/nm / Experimental value: NO
Source (natural)Organism: Neisseria meningitidis 8013 (bacteria)
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria)
Buffer solutionpH: 7.5
Buffer component
IDConc.NameFormulaBuffer-ID
1150 mMsodium chlorideNaCl1
220 mMtris(hydroxymethyl)aminomethaneTris1
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen 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 FIELD / Nominal defocus max: 1600 nm / Nominal defocus min: 1000 nm
Image recordingElectron dose: 60 e/Å2 / Film or detector model: GATAN K2 QUANTUM (4k x 4k)

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Processing

SoftwareName: PHENIX / Version: 1.19.2_4158: / Classification: refinement
EM softwareName: PHENIX / Category: model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.1 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 291265 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00312103
ELECTRON MICROSCOPYf_angle_d0.52516863
ELECTRON MICROSCOPYf_dihedral_angle_d12.2132626
ELECTRON MICROSCOPYf_chiral_restr0.0351944
ELECTRON MICROSCOPYf_plane_restr0.0041775

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