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- PDB-6rjd: Cryo-EM structure of St1Cas9-sgRNA-tDNA59-ntPAM complex. -

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Basic information

Entry
Database: PDB / ID: 6rjd
TitleCryo-EM structure of St1Cas9-sgRNA-tDNA59-ntPAM complex.
Components
  • Streptococcus Thermophilus 1 Cas9
  • ntPAM
  • sgRNA (78-MER)
  • tDNA59
KeywordsHYDROLASE / CRISPR-Cas9 / anti-CRISPR protein / bacteriophages / Streptococcus thermophilus Cas9 / St1Cas9
Function / homology
Function and homology information


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 / HNH endonuclease ...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) / CRISPR-associated endonuclease Cas9 1
Similarity search - Component
Biological speciesStreptococcus thermophilus DGCC 7710 (bacteria)
Streptococcus phage D1811 (virus)
Streptococcus Phage 2972 (virus)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.3 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 1.2Dec 18, 2019Group: Other / Category: atom_sites / cell
Item: _atom_sites.fract_transf_matrix[1][1] / _atom_sites.fract_transf_matrix[2][2] ..._atom_sites.fract_transf_matrix[1][1] / _atom_sites.fract_transf_matrix[2][2] / _atom_sites.fract_transf_matrix[3][3] / _cell.Z_PDB
Revision 1.3Jan 1, 2020Group: Database references / Category: citation / Item: _citation.journal_volume / _citation.page_first

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Structure visualization

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  • Deposited structure unit
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Structure viewerMolecule:
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Assembly

Deposited unit
C: Streptococcus Thermophilus 1 Cas9
D: sgRNA (78-MER)
E: tDNA59
G: ntPAM


Theoretical massNumber of molelcules
Total (without water)192,3844
Polymers192,3844
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: microscopy
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area19010 Å2
ΔGint-148 kcal/mol
Surface area57940 Å2
MethodPISA

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Components

#1: Protein Streptococcus Thermophilus 1 Cas9


Mass: 129700.961 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Streptococcus thermophilus DGCC 7710 (bacteria)
Gene: cas9, CDA68_00396 / Production host: Escherichia coli (E. coli)
References: UniProt: Q03LF7*PLUS, Hydrolases; Acting on ester bonds
#2: RNA chain sgRNA (78-MER)


Mass: 37438.039 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Streptococcus thermophilus DGCC 7710 (bacteria)
Production host: in vitro transcription vector pT7-Fluc(deltai) (others)
#3: DNA chain tDNA59


Mass: 18160.625 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Streptococcus phage D1811 (virus)
#4: DNA chain ntPAM


Mass: 7084.642 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Streptococcus Phage 2972 (virus)

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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-tDNA59-ntPAM complex.COMPLEXall0MULTIPLE SOURCES
2Streptococcus Thermophilus 1 Cas9COMPLEX#11RECOMBINANT
3sgRNA (78-MER)COMPLEX#21RECOMBINANT
4tDNA59COMPLEX#31RECOMBINANT
5ntPAMCOMPLEX#41RECOMBINANT
Molecular weightUnits: MEGADALTONS / Experimental value: NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
12Streptococcus thermophilus DGCC 7710 (bacteria)1268061
23Streptococcus thermophilus (bacteria)1268061
34Streptococcus phage D1811 (virus)2108111
45Streptococcus phage 2972 (virus)306323
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
12Escherichia coli (E. coli)562
23in vitro transcription vector pT7-Fluc(deltai) (others)905932
34synthetic construct (others)32630
45synthetic construct (others)32630
Buffer solutionpH: 7.5
SpecimenConc.: 0.75 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 FIELDBright-field microscopy
Image recordingElectron dose: 40 e/Å2 / Detector mode: COUNTING / Film or detector model: FEI FALCON III (4k x 4k)

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Processing

EM software
IDNameVersionCategory
1RELION3particle selection
4CTFFIND4.1.10CTF correction
13RELION33D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.3 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 68361 / Symmetry type: POINT
Atomic model buildingProtocol: OTHER / Space: REAL

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