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- EMDB-4904: Cryo-EM structure of St1Cas9-sgRNA-AcrIIA6-tDNA59-ntPAM complex. -

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Basic information

Entry
Database: EMDB / ID: EMD-4904
TitleCryo-EM structure of St1Cas9-sgRNA-AcrIIA6-tDNA59-ntPAM complex.
Map dataPostprocessed 3D reconstruction of St1Cas9%u2022sgRNA%u2022AcrIIA6%u2022tDNA59-ntPAM complex.
Sample
  • Complex: Cryo-EM structure of St1Cas9-sgRNA-AcrIIA6-tDNA59-ntPAM complex.
    • Complex: AcrIIA6
      • Protein or peptide: AcrIIA6
    • Complex: Cas 9
      • Protein or peptide: Cas 9
    • Complex: sgRNASubgenomic mRNA
      • RNA: sgRNASubgenomic mRNA
    • Complex: tDNA59
      • DNA: tDNA59
    • Complex: ntPAM
      • DNA: ntPAM
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 / 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
AcrIIA6 / CRISPR-associated endonuclease Cas9 1
Similarity search - Component
Biological speciesStreptococcus phage D1811 (virus) / Streptococcus thermophilus DGCC 7710 (bacteria) / Streptococcus thermophilus (bacteria) / Streptococcus virus 2972
Methodsingle particle reconstruction / cryo EM / Resolution: 3.2 Å
AuthorsSwuec P
Funding support France, 1 items
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, 2019-
Header (metadata) releaseMay 22, 2019-
Map releaseOct 2, 2019-
UpdateDec 2, 2020-
Current statusDec 2, 2020Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.05
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.05
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6rjg
  • Surface level: 0.05
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_4904.png

Downloads & links

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Map

FileDownload / File: emd_4904.map.gz / Format: CCP4 / Size: 98.9 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationPostprocessed 3D reconstruction of St1Cas9%u2022sgRNA%u2022AcrIIA6%u2022tDNA59-ntPAM complex.
Voxel sizeX=Y=Z: 0.889 Å
Density
Contour LevelBy AUTHOR: 0.05 / Movie #1: 0.05
Minimum - Maximum-0.20779811 - 0.3956438
Average (Standard dev.)0.00001331418 (±0.010677476)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions296296296
Spacing296296296
CellA=B=C: 263.144 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z0.8890.8890.889
M x/y/z296296296
origin x/y/z0.0000.0000.000
length x/y/z263.144263.144263.144
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ320320320
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS296296296
D min/max/mean-0.2080.3960.000

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Supplemental data

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Mask #1

Fileemd_4904_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Unsharpened 3D reconstruction of St1Cas9%u2022sgRNA%u2022AcrIIA6%u2022tDNA59-ntPAM complex....

Fileemd_4904_additional.map
AnnotationUnsharpened 3D reconstruction of St1Cas9%u2022sgRNA%u2022AcrIIA6%u2022tDNA59-ntPAM complex.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Cryo-EM structure of St1Cas9-sgRNA-AcrIIA6-tDNA59-ntPAM complex.

EntireName: Cryo-EM structure of St1Cas9-sgRNA-AcrIIA6-tDNA59-ntPAM complex.
Components
  • Complex: Cryo-EM structure of St1Cas9-sgRNA-AcrIIA6-tDNA59-ntPAM complex.
    • Complex: AcrIIA6
      • Protein or peptide: AcrIIA6
    • Complex: Cas 9
      • Protein or peptide: Cas 9
    • Complex: sgRNASubgenomic mRNA
      • RNA: sgRNASubgenomic mRNA
    • Complex: tDNA59
      • DNA: tDNA59
    • Complex: ntPAM
      • DNA: ntPAM

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Supramolecule #1: Cryo-EM structure of St1Cas9-sgRNA-AcrIIA6-tDNA59-ntPAM complex.

SupramoleculeName: Cryo-EM structure of St1Cas9-sgRNA-AcrIIA6-tDNA59-ntPAM complex.
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all

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Supramolecule #2: AcrIIA6

SupramoleculeName: AcrIIA6 / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1
Source (natural)Organism: Streptococcus phage D1811 (virus)
Recombinant expressionOrganism: Escherichia coli (E. coli)

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Supramolecule #3: Cas 9

SupramoleculeName: Cas 9 / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #2
Source (natural)Organism: Streptococcus thermophilus DGCC 7710 (bacteria)
Recombinant expressionOrganism: Escherichia coli (E. coli)

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Supramolecule #4: sgRNA

SupramoleculeName: sgRNA / type: complex / ID: 4 / Parent: 1 / Macromolecule list: #3
Source (natural)Organism: Streptococcus thermophilus (bacteria)
Recombinant expressionOrganism: in vitro transcription vector pT7-Fluc(deltai (others)

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Supramolecule #5: tDNA59

SupramoleculeName: tDNA59 / type: complex / ID: 5 / Parent: 1 / Macromolecule list: #4
Source (natural)Organism: Streptococcus virus 2972
Recombinant expressionOrganism: synthetic construct (others)

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Supramolecule #6: ntPAM

SupramoleculeName: ntPAM / type: complex / ID: 6 / Parent: 1 / Macromolecule list: #5
Source (natural)Organism: Streptococcus virus 2972
Recombinant expressionOrganism: synthetic construct (others)

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Macromolecule #1: AcrIIA6

MacromoleculeName: AcrIIA6 / type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Streptococcus phage D1811 (virus)
Molecular weightTheoretical: 21.464391 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
MKINDDIKEL ILEYMSRYFK FENDFYKLPG IKFTDANWQK FKNGGTDIEK MGAARVNAML DCLFDDFELA MIGKAQTNYY NDNSLKMNM PFYTYYDMFK KQQLLKWLKN NRDDVIGGTG RMYTASGNYI ANAYLEVALE SSSLGSGSYM LQMRFKDYSK G QEPIPSGR QNRLEWIENN LENIR

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Macromolecule #2: Cas 9

MacromoleculeName: Cas 9 / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO / EC number: Hydrolases; Acting on ester bonds
Source (natural)Organism: Streptococcus thermophilus DGCC 7710 (bacteria)
Molecular weightTheoretical: 129.700961 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: 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

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Macromolecule #3: sgRNA

MacromoleculeName: sgRNA / type: rna / ID: 3 / Number of copies: 1
Source (natural)Organism: Streptococcus thermophilus (bacteria)
Molecular weightTheoretical: 37.438039 KDa
SequenceString:
GUUGCGUUGA UAAAAGUAUU GUUUUUGUAC UCUCAAGAUU CAAUAAUCUU GCAGAAGCUA CAAAGAUAAG GCUUCAUGCC GAAAUCAAC ACCCUGUCAU UUUAUGGCAG GGUGUUUU

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Macromolecule #4: tDNA59

MacromoleculeName: tDNA59 / type: dna / ID: 4 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: Streptococcus virus 2972
Molecular weightTheoretical: 18.160625 KDa
SequenceString: (DA)(DT)(DG)(DG)(DT)(DT)(DC)(DT)(DG)(DG) (DT)(DT)(DT)(DC)(DA)(DT)(DT)(DT)(DT)(DC) (DT)(DG)(DC)(DA)(DA)(DT)(DA)(DC)(DT) (DT)(DT)(DT)(DA)(DT)(DC)(DA)(DA)(DC)(DG) (DC) (DA)(DA)(DG)(DA)(DG)(DG) ...String:
(DA)(DT)(DG)(DG)(DT)(DT)(DC)(DT)(DG)(DG) (DT)(DT)(DT)(DC)(DA)(DT)(DT)(DT)(DT)(DC) (DT)(DG)(DC)(DA)(DA)(DT)(DA)(DC)(DT) (DT)(DT)(DT)(DA)(DT)(DC)(DA)(DA)(DC)(DG) (DC) (DA)(DA)(DG)(DA)(DG)(DG)(DT)(DG) (DC)(DT)(DT)(DC)(DT)(DG)(DT)(DT)(DA)(DT) (DG)

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Macromolecule #5: ntPAM

MacromoleculeName: ntPAM / type: dna / ID: 5 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: Streptococcus virus 2972
Molecular weightTheoretical: 7.084642 KDa
SequenceString:
(DG)(DC)(DA)(DG)(DA)(DA)(DA)(DA)(DT)(DG) (DA)(DA)(DA)(DC)(DC)(DA)(DG)(DA)(DA)(DC) (DC)(DA)(DT)

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration0.75 mg/mL
BufferpH: 7.5
VitrificationCryogen name: ETHANE / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TALOS ARCTICA
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: SPOT SCAN / Imaging mode: BRIGHT FIELDBright-field microscopy
Image recordingFilm or detector model: FEI FALCON III (4k x 4k) / Detector mode: COUNTING / Average electron dose: 40.0 e/Å2
Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company

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Image processing

CTF correctionSoftware - Name: CTFFIND (ver. 4.1.10)
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.2 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.0) / Number images used: 50728
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementSpace: REAL / Protocol: OTHER
Output model

PDB-6rjg:
Cryo-EM structure of St1Cas9-sgRNA-AcrIIA6-tDNA59-ntPAM complex.

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