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- EMDB-8749: cryo-EM structure of the Cas9-sgRNA-AcrIIA4 anti-CRISPR complex -

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Basic information

Entry
Database: EMDB / ID: EMD-8749
Titlecryo-EM structure of the Cas9-sgRNA-AcrIIA4 anti-CRISPR complex
Map dataCas9- sgRNA-AcrIIA4 anti-CRISPR complex
Sample
  • Complex: the complex of Streptococcus progenies Cas9 with sgRNA and AcrIIA4
    • Complex: Streptococcus pyogenes M1 GAS
      • RNA: single guide RNA (116-MER)
      • Protein or peptide: CRISPR-associated endonuclease Cas9
    • Complex: unidentified phage
      • Protein or peptide: phage anti-CRISPR AcrIIA4
Keywordsanti-CRISPR / Cas9 / CRISPR / gene editing / on-target / off-target / cryo-EM / Immune System-RNA complex
Function / homology
Function and homology information


symbiont-mediated suppression of host CRISPR-cas system / 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, PAM-interacting domain / CRISPR-associated endonuclease Cas9, bridge helix / CRISPR-associated endonuclease Cas9, REC lobe / REC lobe of CRISPR-associated endonuclease Cas9 / Bridge helix of CRISPR-associated endonuclease Cas9 / PAM-interacting domain of CRISPR-associated endonuclease Cas9 / Cas9 RuvC domain / CRISPR-associated endonuclease Cas9 / HNH endonuclease / Cas9-type HNH domain ...CRISPR-associated endonuclease Cas9, PAM-interacting domain / CRISPR-associated endonuclease Cas9, bridge helix / CRISPR-associated endonuclease Cas9, REC lobe / REC lobe of CRISPR-associated endonuclease Cas9 / Bridge helix of CRISPR-associated endonuclease Cas9 / PAM-interacting domain of CRISPR-associated endonuclease Cas9 / Cas9 RuvC domain / CRISPR-associated endonuclease Cas9 / HNH endonuclease / Cas9-type HNH domain / Cas9-type HNH domain profile. / HNH nuclease / Ribonuclease H superfamily
Similarity search - Domain/homology
: / phage anti-CRISPR AcrIIA4 / CRISPR-associated endonuclease Cas9/Csn1
Similarity search - Component
Biological speciesStreptococcus pyogenes M1 GAS (bacteria) / unidentified phage (virus)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.9 Å
AuthorsJiang F / Liu JJ
Funding support United States, 1 items
OrganizationGrant numberCountry
Howard Hughes Medical Institute (HHMI) United States
CitationJournal: Sci Adv / Year: 2017
Title: Disabling Cas9 by an anti-CRISPR DNA mimic.
Authors: Jiyung Shin / Fuguo Jiang / Jun-Jie Liu / Nicolas L Bray / Benjamin J Rauch / Seung Hyun Baik / Eva Nogales / Joseph Bondy-Denomy / Jacob E Corn / Jennifer A Doudna /
Abstract: CRISPR (clustered regularly interspaced short palindromic repeats)-Cas9 gene editing technology is derived from a microbial adaptive immune system, where bacteriophages are often the intended target. ...CRISPR (clustered regularly interspaced short palindromic repeats)-Cas9 gene editing technology is derived from a microbial adaptive immune system, where bacteriophages are often the intended target. Natural inhibitors of CRISPR-Cas9 enable phages to evade immunity and show promise in controlling Cas9-mediated gene editing in human cells. However, the mechanism of CRISPR-Cas9 inhibition is not known, and the potential applications for Cas9 inhibitor proteins in mammalian cells have not been fully established. We show that the anti-CRISPR protein AcrIIA4 binds only to assembled Cas9-single-guide RNA (sgRNA) complexes and not to Cas9 protein alone. A 3.9 Å resolution cryo-electron microscopy structure of the Cas9-sgRNA-AcrIIA4 complex revealed that the surface of AcrIIA4 is highly acidic and binds with a 1:1 stoichiometry to a region of Cas9 that normally engages the DNA protospacer adjacent motif. Consistent with this binding mode, order-of-addition experiments showed that AcrIIA4 interferes with DNA recognition but has no effect on preformed Cas9-sgRNA-DNA complexes. Timed delivery of AcrIIA4 into human cells as either protein or expression plasmid allows on-target Cas9-mediated gene editing while reducing off-target edits. These results provide a mechanistic understanding of AcrIIA4 function and demonstrate that inhibitors can modulate the extent and outcomes of Cas9-mediated gene editing.
History
DepositionMay 29, 2017-
Header (metadata) releaseJul 26, 2017-
Map releaseJul 26, 2017-
UpdateMar 13, 2024-
Current statusMar 13, 2024Processing site: RCSB / 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
  • Download
  • 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-5vzl
  • Surface level: 0.05
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_8749.png

Downloads & links

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Map

FileDownload / File: emd_8749.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationCas9- sgRNA-AcrIIA4 anti-CRISPR complex
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.07 Å/pix.
x 256 pix.
= 273.92 Å		1.07 Å/pix.
x 256 pix.
= 273.92 Å		1.07 Å/pix.
x 256 pix.
= 273.92 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.07 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.05 / Movie #1: 0.05
Minimum - Maximum-0.1356536 - 0.21605033
Average (Standard dev.)0.00034119125 (±0.004950442)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 273.92 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.071.071.07
M x/y/z256256256
origin x/y/z0.0000.0000.000
length x/y/z273.920273.920273.920
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS256256256
D min/max/mean-0.1360.2160.000

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Supplemental data

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Sample components

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Entire : the complex of Streptococcus progenies Cas9 with sgRNA and AcrIIA4

EntireName: the complex of Streptococcus progenies Cas9 with sgRNA and AcrIIA4
Components
  • Complex: the complex of Streptococcus progenies Cas9 with sgRNA and AcrIIA4
    • Complex: Streptococcus pyogenes M1 GAS
      • RNA: single guide RNA (116-MER)
      • Protein or peptide: CRISPR-associated endonuclease Cas9
    • Complex: unidentified phage
      • Protein or peptide: phage anti-CRISPR AcrIIA4

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Supramolecule #1: the complex of Streptococcus progenies Cas9 with sgRNA and AcrIIA4

SupramoleculeName: the complex of Streptococcus progenies Cas9 with sgRNA and AcrIIA4
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Streptococcus pyogenes M1 GAS (bacteria)

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Supramolecule #2: Streptococcus pyogenes M1 GAS

SupramoleculeName: Streptococcus pyogenes M1 GAS / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1-#2
Source (natural)Organism: unidentified phage (virus)

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Supramolecule #3: unidentified phage

SupramoleculeName: unidentified phage / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #3

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Macromolecule #1: single guide RNA (116-MER)

MacromoleculeName: single guide RNA (116-MER) / type: rna / ID: 1 / Number of copies: 1
Source (natural)Organism: Streptococcus pyogenes M1 GAS (bacteria)
Molecular weightTheoretical: 38.292645 KDa
SequenceString:
(GTP)GCGCAUAAA GAUGAGACGC GUUUUAGAGC UAUGCUGUUU UGAAAAAAAC AGCAUAGCAA GUUAAAAUAA GGCUAG UCC GUUAUCAACU UGAAAAAGUG GCACCGAGUC GGUGCUUCG

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Macromolecule #2: CRISPR-associated endonuclease Cas9

MacromoleculeName: CRISPR-associated endonuclease Cas9 / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO / EC number: Hydrolases; Acting on ester bonds
Source (natural)Organism: Streptococcus pyogenes M1 GAS (bacteria)
Molecular weightTheoretical: 158.772891 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: SMDKKYSIGL DIGTNSVGWA VITDDYKVPS KKFKVLGNTD RHSIKKNLIG ALLFDSGETA EATRLKRTAR RRYTRRKNRI CYLQEIFSN EMAKVDDSFF HRLEESFLVE EDKKHERHPI FGNIVDEVAY HEKYPTIYHL RKKLVDSTDK ADLRLIYLAL A HMIKFRGH ...String:
SMDKKYSIGL DIGTNSVGWA VITDDYKVPS KKFKVLGNTD RHSIKKNLIG ALLFDSGETA EATRLKRTAR RRYTRRKNRI CYLQEIFSN EMAKVDDSFF HRLEESFLVE EDKKHERHPI FGNIVDEVAY HEKYPTIYHL RKKLVDSTDK ADLRLIYLAL A HMIKFRGH FLIEGDLNPD NSDVDKLFIQ LVQTYNQLFE ENPINASGVD AKAILSARLS KSRRLENLIA QLPGEKKNGL FG NLIALSL GLTPNFKSNF DLAEDAKLQL SKDTYDDDLD NLLAQIGDQY ADLFLAAKNL SDAILLSDIL RVNTEITKAP LSA SMIKRY DEHHQDLTLL KALVRQQLPE KYKEIFFDQS KNGYAGYIDG GASQEEFYKF IKPILEKMDG TEELLVKLNR EDLL RKQRT FDNGSIPHQI HLGELHAILR RQEDFYPFLK DNREKIEKIL TFRIPYYVGP LARGNSRFAW MTRKSEETIT PWNFE EVVD KGASAQSFIE RMTNFDKNLP NEKVLPKHSL LYEYFTVYNE LTKVKYVTEG MRKPAFLSGE QKKAIVDLLF KTNRKV TVK QLKEDYFKKI ECFDSVEISG VEDRFNASLG TYHDLLKIIK DKDFLDNEEN EDILEDIVLT LTLFEDREMI EERLKTY AH LFDDKVMKQL KRRRYTGWGR LSRKLINGIR DKQSGKTILD FLKSDGFANR NFMQLIHDDS LTFKEDIQKA QVSGQGDS L HEHIANLAGS PAIKKGILQT VKVVDELVKV MGRHKPENIV IEMARENQTT QKGQKNSRER MKRIEEGIKE LGSQILKEH PVENTQLQNE KLYLYYLQNG RDMYVDQELD INRLSDYDVD HIVPQSFLKD DSIDNKVLTR SDKNRGKSDN VPSEEVVKKM KNYWRQLLN AKLITQRKFD NLTKAERGGL SELDKAGFIK RQLVETRQIT KHVAQILDSR MNTKYDENDK LIREVKVITL K SKLVSDFR KDFQFYKVRE INNYHHAHDA YLNAVVGTAL IKKYPKLESE FVYGDYKVYD VRKMIAKSEQ EIGKATAKYF FY SNIMNFF KTEITLANGE IRKRPLIETN GETGEIVWDK GRDFATVRKV LSMPQVNIVK KTEVQTGGFS KESILPKRNS DKL IARKKD WDPKKYGGFD SPTVAYSVLV VAKVEKGKSK KLKSVKELLG ITIMERSSFE KNPIDFLEAK GYKEVKKDLI IKLP KYSLF ELENGRKRML ASAGELQKGN ELALPSKYVN FLYLASHYEK LKGSPEDNEQ KQLFVEQHKH YLDEIIEQIS EFSKR VILA DANLDKVLSA YNKHRDKPIR EQAENIIHLF TLTNLGAPAA FKYFDTTIDR KRYTSTKEVL DATLIHQSIT GLYETR IDL SQLGGD

UniProtKB: UNIPROTKB: A0A0C6FZC2

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Macromolecule #3: phage anti-CRISPR AcrIIA4

MacromoleculeName: phage anti-CRISPR AcrIIA4 / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: unidentified phage (virus)
Molecular weightTheoretical: 10.182073 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
MNINDLIREI KNKDYTVKLS GTDSNSITQL IIRVNNDGNE YVISESENES IVEKFISAFK NGWNQEYEDE EEFYNDMQTI TLKSELN

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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

Concentration1.25 mg/mL
BufferpH: 8
GridModel: C-flat-2/2 / Material: COPPER / Mesh: 400 / Support film - topology: HOLEY / Pretreatment - Type: PLASMA CLEANING / Pretreatment - Time: 12 sec.
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 281 K / Instrument: FEI VITROBOT MARK IV
Details: blot for 4.5 seconds with force of 12 before plunging.
DetailsspyCas9, sgRNA and AcrIIA4 complex

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: SUPER-RESOLUTION / Average electron dose: 46.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 100.0 µm / Calibrated defocus max: 4.0 µm / Calibrated defocus min: 0.9 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 0.01 mm / Nominal defocus max: 3.0 µm / Nominal defocus min: 1.0 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: EMDB MAP
EMDB ID:

3276

Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.9 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 185000
Initial angle assignmentType: ANGULAR RECONSTITUTION
Final angle assignmentType: ANGULAR RECONSTITUTION / Software - Name: RELION (ver. 2.0)

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Atomic model buiding 1

RefinementSpace: REAL / Protocol: AB INITIO MODEL
Output model

PDB-5vzl:
cryo-EM structure of the Cas9-sgRNA-AcrIIA4 anti-CRISPR complex

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Atomic model buiding 2

RefinementSpace: REAL / Protocol: RIGID BODY FIT
Output model

PDB-5vzl:
cryo-EM structure of the Cas9-sgRNA-AcrIIA4 anti-CRISPR complex

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