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- EMDB-4900: Cryo-EM structure of St1Cas9-sgRNA-tDNA20-AcrIIA6 monomeric assembly. -
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Open data
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Basic information
Entry | Database: EMDB / ID: EMD-4900 | |||||||||
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Title | Cryo-EM structure of St1Cas9-sgRNA-tDNA20-AcrIIA6 monomeric assembly. | |||||||||
![]() | Sharpened 3D reconstruction of St1Cas9-sgRNA-tDNA20-AcrIIA6 monomeric assembly. | |||||||||
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![]() | CRISPR-Cas9 / anti-CRISPR protein / bacteriophages / Streptococcus thermophilus Cas9 / St1Cas9 / hydrolase | |||||||||
Function / homology | ![]() symbiont-mediated suppression of host CRISPR-cas system / 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 | |||||||||
Biological species | ![]() ![]() ![]() ![]() | |||||||||
Method | single particle reconstruction / cryo EM / Resolution: 3.2 Å | |||||||||
![]() | Swuec P | |||||||||
Funding support | ![]()
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![]() | ![]() 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 |
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Structure visualization
Movie |
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Structure viewer | EM map: ![]() ![]() ![]() |
Supplemental images |
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Downloads & links
-EMDB archive
Map data | ![]() | 92.8 MB | ![]() | |
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Header (meta data) | ![]() ![]() | 19.5 KB 19.5 KB | Display Display | ![]() |
FSC (resolution estimation) | ![]() | 10.6 KB | Display | ![]() |
Images | ![]() | 112.7 KB | ||
Masks | ![]() | 98.9 MB | ![]() | |
Filedesc metadata | ![]() | 7.3 KB | ||
Others | ![]() | 77.5 MB | ||
Archive directory | ![]() ![]() | HTTPS FTP |
-Validation report
Summary document | ![]() | 593.9 KB | Display | ![]() |
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Full document | ![]() | 593.5 KB | Display | |
Data in XML | ![]() | 11.4 KB | Display | |
Data in CIF | ![]() | 15.1 KB | Display | |
Arichive directory | ![]() ![]() | HTTPS FTP |
-Related structure data
Related structure data | ![]() 6rj9MC ![]() 4901C ![]() 4902C ![]() 4904C ![]() 6rjaC ![]() 6rjdC ![]() 6rjgC C: citing same article ( M: atomic model generated by this map |
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Similar structure data |
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Links
EMDB pages | ![]() ![]() |
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Related items in Molecule of the Month |
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Map
File | ![]() | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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Annotation | Sharpened 3D reconstruction of St1Cas9-sgRNA-tDNA20-AcrIIA6 monomeric assembly. | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Projections & slices | Image control
Images are generated by Spider. | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 0.889 Å | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Details | EMDB XML:
CCP4 map header:
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-Supplemental data
-Mask #1
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Projections & Slices |
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Density Histograms |
-Additional map: Unsharpened 3D reconstruction of St1Cas9-sgRNA-tDNA20-AcrIIA6 monomeric assembly....
File | emd_4900_additional.map | ||||||||||||
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Annotation | Unsharpened 3D reconstruction of St1Cas9-sgRNA-tDNA20-AcrIIA6 monomeric assembly. | ||||||||||||
Projections & Slices |
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Density Histograms |
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Sample components
-Entire : Cryo-EM structure of St1Cas9-sgRNA-tDNA20-AcrIIA6 monomeric assembly.
Entire | Name: Cryo-EM structure of St1Cas9-sgRNA-tDNA20-AcrIIA6 monomeric assembly. |
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Components |
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-Supramolecule #1: Cryo-EM structure of St1Cas9-sgRNA-tDNA20-AcrIIA6 monomeric assembly.
Supramolecule | Name: Cryo-EM structure of St1Cas9-sgRNA-tDNA20-AcrIIA6 monomeric assembly. type: complex / ID: 1 / Parent: 0 / Macromolecule list: all |
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-Supramolecule #2: AcrIIA6
Supramolecule | Name: AcrIIA6 / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1 |
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Source (natural) | Organism: ![]() |
-Supramolecule #3: CRISPR-associated endonuclease Cas9
Supramolecule | Name: CRISPR-associated endonuclease Cas9 / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #2 |
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Source (natural) | Organism: ![]() |
-Supramolecule #4: sgRNA
Supramolecule | Name: sgRNA / type: complex / ID: 4 / Parent: 1 / Macromolecule list: #3 |
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Source (natural) | Organism: ![]() |
-Supramolecule #5: ntPAM
Supramolecule | Name: ntPAM / type: complex / ID: 5 / Parent: 1 / Macromolecule list: #4 |
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Source (natural) | Organism: ![]() |
-Macromolecule #1: AcrIIA6
Macromolecule | Name: AcrIIA6 / type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO |
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Source (natural) | Organism: ![]() |
Molecular weight | Theoretical: 21.464391 KDa |
Recombinant expression | Organism: ![]() ![]() |
Sequence | String: MKINDDIKEL ILEYMSRYFK FENDFYKLPG IKFTDANWQK FKNGGTDIEK MGAARVNAML DCLFDDFELA MIGKAQTNYY NDNSLKMNM PFYTYYDMFK KQQLLKWLKN NRDDVIGGTG RMYTASGNYI ANAYLEVALE SSSLGSGSYM LQMRFKDYSK G QEPIPSGR QNRLEWIENN LENIR UniProtKB: AcrIIA6 |
-Macromolecule #2: CRISPR-associated endonuclease Cas9 1
Macromolecule | Name: CRISPR-associated endonuclease Cas9 1 / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO / EC number: Hydrolases; Acting on ester bonds |
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Source (natural) | Organism: ![]() Strain: ATCC BAA-491 / LMD-9 |
Molecular weight | Theoretical: 129.700961 KDa |
Recombinant expression | Organism: ![]() ![]() |
Sequence | String: MSDLVLGLDI GIGSVGVGIL NKVTGEIIHK NSRIFPAAQA ENNLVRRTNR QGRRLTRRKK HRRVRLNRLF EESGLITDFT KISINLNPY QLRVKGLTDE LSNEELFIAL KNMVKHRGIS YLDDASDDGN SSIGDYAQIV KENSKQLETK TPGQIQLERY Q TYGQLRGD ...String: MSDLVLGLDI GIGSVGVGIL NKVTGEIIHK NSRIFPAAQA ENNLVRRTNR QGRRLTRRKK HRRVRLNRLF EESGLITDFT KISINLNPY QLRVKGLTDE LSNEELFIAL KNMVKHRGIS YLDDASDDGN SSIGDYAQIV KENSKQLETK TPGQIQLERY Q TYGQLRGD FTVEKDGKKH RLINVFPTSA YRSEALRILQ TQQEFNPQIT DEFINRYLEI LTGKRKYYHG PGNEKSRTDY GR YRTSGET LDNIFGILIG KCTFYPDEFR AAKASYTAQE FNLLNDLNNL TVPTETKKLS KEQKNQIINY VKNEKAMGPA KLF KYIAKL LSCDVADIKG YRIDKSGKAE IHTFEAYRKM KTLETLDIEQ MDRETLDKLA YVLTLNTERE GIQEALEHEF ADGS FSQKQ VDELVQFRKA NSSIFGKGWH NFSVKLMMEL IPELYETSEE QMTILTRLGK QKTTSSSNKT KYIDEKLLTE EIYNP VVAK SVRQAIKIVN AAIKEYGDFD NIVIEMARET NEDDEKKAIQ KIQKANKDEK DAAMLKAANQ YNGKAELPHS VFHGHK QLA TKIRLWHQQG ERCLYTGKTI SIHDLINNSN QFEVDHILPL SITFDDSLAN KVLVYATANQ EKGQRTPYQA LDSMDDA WS FRELKAFVRE SKTLSNKKKE YLLTEEDISK FDVRKKFIER NLVDTRYASR VVLNALQEHF RAHKIDTKVS VVRGQFTS Q LRRHWGIEKT RDTYHHHAVD ALIIAASSQL NLWKKQKNTL VSYSEDQLLD IETGELISDD EYKESVFKAP YQHFVDTLK SKEFEDSILF SYQVDSKFNR KISDATIYAT RQAKVGKDKA DETYVLGKIK DIYTQDGYDA FMKIYKKDKS KFLMYRHDPQ TFEKVIEPI LENYPNKQIN EKGKEVPCNP FLKYKEEHGY IRKYSKKGNG PEIKSLKYYD SKLGNHIDIT PKDSNNKVVL Q SVSPWRAD VYFNKTTGKY EILGLKYADL QFEKGTGTYK ISQEKYNDIK KKEGVDSDSE FKFTLYKNDL LLVKDTETKE QQ LFRFLSR TMPKQKHYVE LKPYDKQKFE GGEALIKVLG NVANSGQCKK GLGKSNISIY KVRTDVLGNQ HIIKNEGDKP KLD F UniProtKB: CRISPR-associated endonuclease Cas9 1 |
-Macromolecule #3: sgRNA
Macromolecule | Name: sgRNA / type: rna / ID: 3 / Number of copies: 1 |
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Source (natural) | Organism: ![]() |
Molecular weight | Theoretical: 37.438039 KDa |
Sequence | String: GUUGCGUUGA UAAAAGUAUU GUUUUUGUAC UCUCAAGAUU CAAUAAUCUU GCAGAAGCUA CAAAGAUAAG GCUUCAUGCC GAAAUCAAC ACCCUGUCAU UUUAUGGCAG GGUGUUUU |
-Macromolecule #4: tDNA20
Macromolecule | Name: tDNA20 / type: dna / ID: 4 / Number of copies: 1 / Classification: DNA |
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Source (natural) | Organism: ![]() |
Molecular weight | Theoretical: 6.100993 KDa |
Sequence | String: (DA)(DA)(DT)(DA)(DC)(DT)(DT)(DT)(DT)(DA) (DT)(DC)(DA)(DA)(DC)(DG)(DC)(DA)(DA)(DG) |
-Experimental details
-Structure determination
Method | cryo EM |
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![]() | single particle reconstruction |
Aggregation state | particle |
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Sample preparation
Concentration | 0.7 mg/mL |
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Buffer | pH: 7.5 |
Vitrification | Cryogen name: ETHANE / Instrument: FEI VITROBOT MARK IV |
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Electron microscopy
Microscope | FEI TALOS ARCTICA |
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Image recording | Film or detector model: FEI FALCON III (4k x 4k) / Detector mode: COUNTING / Average electron dose: 40.0 e/Å2 |
Electron beam | Acceleration voltage: 200 kV / Electron source: ![]() |
Electron optics | Illumination mode: SPOT SCAN / Imaging mode: BRIGHT FIELD |
Experimental equipment | ![]() Model: Talos Arctica / Image courtesy: FEI Company |
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Image processing
-Atomic model buiding 1
Refinement | Space: REAL / Protocol: OTHER |
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Output model | ![]() PDB-6rj9: |