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- PDB-8ye6: Cryo-EM structure of Cas9-sgRNA-A32 complex -

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Basic information

Entry
Database: PDB / ID: 8ye6
TitleCryo-EM structure of Cas9-sgRNA-A32 complex
Components
  • A32
  • CRISPR-associated endonuclease Cas9/Csn1
  • RNA (92-MER)
KeywordsIMMUNE SYSTEM/RNA / CRISPR / Complex / IMMUNE SYSTEM-RNA complex
Function / homology
Function and homology information


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, bridge helix / Bridge helix of CRISPR-associated endonuclease Cas9 / CRISPR-associated endonuclease Cas9, PAM-interacting domain / CRISPR-associated endonuclease Cas9, REC lobe / REC lobe of CRISPR-associated endonuclease Cas9 / PAM-interacting domain of CRISPR-associated endonuclease Cas9 / : / Cas9 RuvC domain / HNH endonuclease / CRISPR-associated endonuclease Cas9 ...CRISPR-associated endonuclease Cas9, bridge helix / Bridge helix of CRISPR-associated endonuclease Cas9 / CRISPR-associated endonuclease Cas9, PAM-interacting domain / CRISPR-associated endonuclease Cas9, REC lobe / REC lobe of CRISPR-associated endonuclease Cas9 / PAM-interacting domain of CRISPR-associated endonuclease Cas9 / : / Cas9 RuvC domain / 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) / CRISPR-associated endonuclease Cas9/Csn1
Similarity search - Component
Biological speciesStreptococcus (bacteria)
Streptococcus pyogenes serotype M1 (bacteria)
Streptococcus pyogenes (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.95 Å
AuthorsZheng, J. / Zhu, Y. / Huang, Z.
Funding support China, 1items
OrganizationGrant numberCountry
National Natural Science Foundation of China (NSFC)31422014, 31450001, and 31300605 China
CitationJournal: Sci China Life Sci / Year: 2024
Title: Inhibition mechanisms of CRISPR-Cas9 by AcrIIA25.1 and AcrIIA32.
Authors: Jianlin Zheng / Yuwei Zhu / Tengjin Huang / Wenbo Gao / Jiale He / Zhiwei Huang /
Abstract: In the ongoing arms race between bacteria and bacteriophages, bacteriophages have evolved anti-CRISPR proteins to counteract bacterial CRISPR-Cas systems. Recently, AcrIIA25.1 and AcrIIA32 have been ...In the ongoing arms race between bacteria and bacteriophages, bacteriophages have evolved anti-CRISPR proteins to counteract bacterial CRISPR-Cas systems. Recently, AcrIIA25.1 and AcrIIA32 have been found to effectively inhibit the activity of SpyCas9 both in bacterial and human cells. However, their molecular mechanisms remain elusive. Here, we report the cryo-electron microscopy structures of ternary complexes formed by AcrIIA25.1 and AcrIIA32 bound to SpyCas9-sgRNA. Using structural analysis and biochemical experiments, we revealed that AcrIIA25.1 and AcrIIA32 recognize a novel, previously-unidentified anti-CRISPR binding site on SpyCas9. We found that both AcrIIA25.1 and AcrIIA32 directly interact with the WED domain, where they spatially obstruct conformational changes of the WED and PI domains, thereby inhibiting SpyCas9 from recognizing protospacer adjacent motif (PAM) and unwinding double-stranded DNA. In addition, they may inhibit nuclease activity by blocking the dynamic conformational changes of the SpyCas9 surveillance complex. In summary, our data elucidate the inhibition mechanisms of two new anti-CRISPR proteins, provide new strategies for the modulation of SpyCas9 activity, and expand our understanding of the diversity of anti-CRISPR protein inhibition mechanisms.
History
DepositionFeb 22, 2024Deposition site: PDBJ / Processing site: PDBC
Revision 1.0Sep 11, 2024Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
B: A32
A: CRISPR-associated endonuclease Cas9/Csn1
C: RNA (92-MER)


Theoretical massNumber of molelcules
Total (without water)205,2283
Polymers205,2283
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein A32


Mass: 16783.949 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Streptococcus (bacteria) / Production host: Escherichia coli (E. coli)
#2: Protein CRISPR-associated endonuclease Cas9/Csn1 / SpCas9 / SpyCas9


Mass: 158699.844 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Streptococcus pyogenes serotype M1 (bacteria)
Gene: cas9, csn1, SPy_1046 / Production host: Escherichia coli (E. coli)
References: UniProt: Q99ZW2, Hydrolases; Acting on ester bonds
#3: RNA chain RNA (92-MER)


Mass: 29743.719 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Streptococcus pyogenes (bacteria) / 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

ComponentName: cas9-sgRNA-A32 complex / Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
21Streptococcus pyogenes (bacteria)1314
31Streptococcus (bacteria)1301
41Streptococcus pyogenes serotype M1 (bacteria)301447
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 8
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: NITROGEN

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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: 2000 nm / Nominal defocus min: 1400 nm
Image recordingElectron dose: 50 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

EM softwareName: PHENIX / Version: 1.11.1_2575: / Category: model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 2.95 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 513280 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0114043
ELECTRON MICROSCOPYf_angle_d1.0619383
ELECTRON MICROSCOPYf_dihedral_angle_d11.3238341
ELECTRON MICROSCOPYf_chiral_restr0.062238
ELECTRON MICROSCOPYf_plane_restr0.0062145

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