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- PDB-6rj9: Cryo-EM structure of St1Cas9-sgRNA-tDNA20-AcrIIA6 monomeric assembly. -

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Basic information

Entry
Database: PDB / ID: 6rj9
TitleCryo-EM structure of St1Cas9-sgRNA-tDNA20-AcrIIA6 monomeric assembly.
Components
  • AcrIIA6
  • CRISPR-associated endonuclease Cas9 1
  • sgRNA
  • tDNA20
KeywordsHYDROLASE / CRISPR-Cas9 / anti-CRISPR protein / bacteriophages / Streptococcus thermophilus Cas9 / St1Cas9
Function / homology
Function and homology information


symbiont-mediated suppression of host CRISPR-cas system / maintenance of CRISPR repeat elements / defense response to virus / endonuclease activity / Hydrolases; Acting on ester bonds / DNA binding / RNA binding / metal ion binding
Similarity search - Function
: / Cas9, PI domain / Cas9, WED domain / CRISPR-Cas9 WED domain / CRISPR-Cas9 PI domain / Cas9, alpha-helical lobe domain / Cas9 alpha-helical lobe domain / RuvC endonuclease subdomain 3 / RuvC endonuclease subdomain 3 / CRISPR-associated endonuclease Cas9 ...: / Cas9, PI domain / Cas9, WED domain / CRISPR-Cas9 WED domain / CRISPR-Cas9 PI domain / Cas9, alpha-helical lobe domain / Cas9 alpha-helical lobe domain / RuvC endonuclease subdomain 3 / RuvC endonuclease subdomain 3 / CRISPR-associated endonuclease Cas9 / HNH endonuclease / Cas9-type HNH domain / Cas9-type HNH domain profile. / HNH nuclease / Ribonuclease H superfamily
Similarity search - Domain/homology
DNA / DNA (> 10) / RNA / RNA (> 10) / RNA (> 100) / AcrIIA6 / CRISPR-associated endonuclease Cas9 1
Similarity search - Component
Biological speciesStreptococcus phage D1811 (virus)
Streptococcus thermophilus (bacteria)
Streptococcus virus 2972
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.2 Å
AuthorsGoulet, A. / Chaves-Sanjuan, A. / Cambillau, C.
Funding support France, 1items
OrganizationGrant numberCountry
French National Research Agency18-CE11-0016-01 France
CitationJournal: Mol Cell / Year: 2019
Title: Cas9 Allosteric Inhibition by the Anti-CRISPR Protein AcrIIA6.
Authors: Olivier Fuchsbauer / Paolo Swuec / Claire Zimberger / Béatrice Amigues / Sébastien Levesque / Daniel Agudelo / Alexis Duringer / Antonio Chaves-Sanjuan / Silvia Spinelli / Geneviève M ...Authors: Olivier Fuchsbauer / Paolo Swuec / Claire Zimberger / Béatrice Amigues / Sébastien Levesque / Daniel Agudelo / Alexis Duringer / Antonio Chaves-Sanjuan / Silvia Spinelli / Geneviève M Rousseau / Minja Velimirovic / Martino Bolognesi / Alain Roussel / Christian Cambillau / Sylvain Moineau / Yannick Doyon / Adeline Goulet /
Abstract: In the arms race against bacteria, bacteriophages have evolved diverse anti-CRISPR proteins (Acrs) that block CRISPR-Cas immunity. Acrs play key roles in the molecular coevolution of bacteria with ...In the arms race against bacteria, bacteriophages have evolved diverse anti-CRISPR proteins (Acrs) that block CRISPR-Cas immunity. Acrs play key roles in the molecular coevolution of bacteria with their predators, use a variety of mechanisms of action, and provide tools to regulate Cas-based genome manipulation. Here, we present structural and functional analyses of AcrIIA6, an Acr from virulent phages, exploring its unique anti-CRISPR action. Our cryo-EM structures and functional data of AcrIIA6 binding to Streptococcus thermophilus Cas9 (St1Cas9) show that AcrIIA6 acts as an allosteric inhibitor and induces St1Cas9 dimerization. AcrIIA6 reduces St1Cas9 binding affinity for DNA and prevents DNA binding within cells. The PAM and AcrIIA6 recognition sites are structurally close and allosterically linked. Mechanistically, AcrIIA6 affects the St1Cas9 conformational dynamics associated with PAM binding. Finally, we identify a natural St1Cas9 variant resistant to AcrIIA6 illustrating Acr-driven mutational escape and molecular diversification of Cas9 proteins.
History
DepositionApr 26, 2019Deposition site: PDBE / Processing site: PDBE
Revision 1.0Oct 2, 2019Provider: repository / Type: Initial release
Revision 1.1Oct 23, 2019Group: Data collection / Database references / Category: citation / citation_author
Item: _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.name
Revision 1.2Nov 6, 2019Group: Data collection / Refinement description / Category: software / Item: _software.name
Revision 1.3Dec 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 1.4Jan 1, 2020Group: Database references / Category: citation / Item: _citation.journal_volume / _citation.page_first
Revision 1.5Jun 30, 2021Group: Database references / Source and taxonomy / Structure summary
Category: em_entity_assembly_naturalsource / entity ...em_entity_assembly_naturalsource / entity / entity_name_com / entity_src_gen / struct_ref / struct_ref_seq / struct_ref_seq_dif
Item: _em_entity_assembly_naturalsource.ncbi_tax_id / _em_entity_assembly_naturalsource.organism ..._em_entity_assembly_naturalsource.ncbi_tax_id / _em_entity_assembly_naturalsource.organism / _entity.pdbx_description / _entity_src_gen.gene_src_strain / _entity_src_gen.pdbx_gene_src_gene / _entity_src_gen.pdbx_gene_src_ncbi_taxonomy_id / _entity_src_gen.pdbx_gene_src_scientific_name / _struct_ref.db_code / _struct_ref.db_name / _struct_ref.pdbx_db_accession / _struct_ref.pdbx_seq_one_letter_code / _struct_ref_seq.pdbx_db_accession
Revision 1.6May 22, 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
A: AcrIIA6
B: AcrIIA6
C: CRISPR-associated endonuclease Cas9 1
D: sgRNA
E: tDNA20


Theoretical massNumber of molelcules
Total (without water)216,1695
Polymers216,1695
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: microscopy
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area22820 Å2
ΔGint-133 kcal/mol
Surface area68640 Å2
MethodPISA

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Components

#1: Protein AcrIIA6


Mass: 21464.391 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Streptococcus phage D1811 (virus) / Gene: D1811_026 / Production host: Escherichia coli (E. coli) / References: UniProt: A0A2U7VKE8
#2: Protein CRISPR-associated endonuclease Cas9 1 / CRISPR-associated endonuclease Cas9


Mass: 129700.961 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Streptococcus thermophilus (strain ATCC BAA-491 / LMD-9) (bacteria)
Strain: ATCC BAA-491 / LMD-9 / Gene: cas9-1, csn1, STER_0709 / Production host: Escherichia coli (E. coli)
References: UniProt: Q03LF7, Hydrolases; Acting on ester bonds
#3: RNA chain sgRNA


Mass: 37438.039 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Streptococcus thermophilus (bacteria)
Production host: in vitro transcription vector pT7-Fluc(deltai) (others)
#4: DNA chain tDNA20


Mass: 6100.993 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Streptococcus virus 2972

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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
1Cryo-EM structure of St1Cas9-sgRNA-tDNA20-AcrIIA6 monomeric assembly.COMPLEXall0MULTIPLE SOURCES
2AcrIIA6COMPLEX#11RECOMBINANT
3CRISPR-associated endonuclease Cas9COMPLEX#21RECOMBINANT
4sgRNACOMPLEX#31RECOMBINANT
5ntPAMCOMPLEX#41RECOMBINANT
Molecular weightUnits: MEGADALTONS / Experimental value: NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
12Streptococcus phage D1811 (virus)2108111
23Streptococcus thermophilus (strain ATCC BAA-491 / LMD-9) (bacteria)322159
34Streptococcus thermophilus (bacteria)1308
45Streptococcus virus 2972306323
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
12Escherichia coli (E. coli)562
23Escherichia coli (E. coli)562
34in vitro transcription vector pT7-Fluc(deltai) (others)905932
45synthetic construct (others)32630
Buffer solutionpH: 7.5
SpecimenConc.: 0.7 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company
MicroscopyModel: FEI TALOS ARCTICA
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: SPOT SCAN
Electron lensMode: BRIGHT FIELD
Image recordingElectron dose: 40 e/Å2 / Detector mode: COUNTING / Film or detector model: FEI FALCON III (4k x 4k)

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Processing

Software
NameVersionClassification
PHENIX1.14_3260refinement
PHENIX1.14_3260refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.2 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 118189 / Symmetry type: POINT
Atomic model buildingProtocol: OTHER / Space: REAL
RefinementHighest resolution: 3.2 Å / Stereochemistry target values: CDL v1.2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.005812293
ELECTRON MICROSCOPYf_angle_d0.776216966
ELECTRON MICROSCOPYf_chiral_restr0.04631892
ELECTRON MICROSCOPYf_plane_restr0.00571823
ELECTRON MICROSCOPYf_dihedral_angle_d18.07517209

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