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- EMDB-9066: CryoEM structure of AcrIIA2 in complex with CRISPR-Cas9 -

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Basic information

Entry
Database: EMDB / ID: EMD-9066
TitleCryoEM structure of AcrIIA2 in complex with CRISPR-Cas9
Map dataSpyCas9-A2a complex
Sample
  • Complex: AcrIIA2 interacting with sgRNA-bound Streptococcus pyogenes Cas9
    • Complex: sgRNA-bound Streptococcus pyogenes Cas9
      • RNA: Single guide RNA (116-MER)
      • Protein or peptide: CRISPR-associated endonuclease Cas9
    • Complex: AcrIIA2
      • Protein or peptide: Anti-CRISPR protein AcrIIA2
KeywordsCRISPR-Cas / Cas9 / Cas9 inhibitors / anti-CRISPR / AcrIIA2 / bacteriophage / gene editing / HYDROLASE-RNA-VIRAL PROTEIN complex
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
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 / CRISPR-associated endonuclease Cas9 / HNH endonuclease / Cas9-type HNH domain / Cas9-type HNH domain profile. ...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 / CRISPR-associated endonuclease Cas9 / HNH endonuclease / Cas9-type HNH domain / Cas9-type HNH domain profile. / HNH nuclease / Ribonuclease H superfamily
Similarity search - Domain/homology
CRISPR-associated endonuclease Cas9
Similarity search - Component
Biological speciesStreptococcus pyogenes M1 GAS (bacteria) / Listeria phage LP-101 (virus)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.4 Å
AuthorsJiang F / Liu JJ
Funding support United States, 1 items
OrganizationGrant numberCountry
Howard Hughes Medical Institute (HHMI) United States
CitationJournal: Mol Cell / Year: 2019
Title: Temperature-Responsive Competitive Inhibition of CRISPR-Cas9.
Authors: Fuguo Jiang / Jun-Jie Liu / Beatriz A Osuna / Michael Xu / Joel D Berry / Benjamin J Rauch / Eva Nogales / Joseph Bondy-Denomy / Jennifer A Doudna /
Abstract: CRISPR-Cas immune systems utilize RNA-guided nucleases to protect bacteria from bacteriophage infection. Bacteriophages have in turn evolved inhibitory "anti-CRISPR" (Acr) proteins, including six ...CRISPR-Cas immune systems utilize RNA-guided nucleases to protect bacteria from bacteriophage infection. Bacteriophages have in turn evolved inhibitory "anti-CRISPR" (Acr) proteins, including six inhibitors (AcrIIA1-AcrIIA6) that can block DNA cutting and genome editing by type II-A CRISPR-Cas9 enzymes. We show here that AcrIIA2 and its more potent homolog, AcrIIA2b, prevent Cas9 binding to DNA by occluding protein residues required for DNA binding. Cryo-EM-determined structures of AcrIIA2 or AcrIIA2b bound to S. pyogenes Cas9 reveal a mode of competitive inhibition of DNA binding that is distinct from other known Acrs. Differences in the temperature dependence of Cas9 inhibition by AcrIIA2 and AcrIIA2b arise from differences in both inhibitor structure and the local inhibitor-binding environment on Cas9. These findings expand the natural toolbox for regulating CRISPR-Cas9 genome editing temporally, spatially, and conditionally.
History
DepositionAug 31, 2018-
Header (metadata) releaseSep 26, 2018-
Map releaseJan 16, 2019-
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: 0.28
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.28
  • Imaged by UCSF Chimera
  • Download
  • Surface view with fitted model
  • Atomic models: PDB-6mcb
  • Surface level: 0.28
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_9066.png

Downloads & links

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Map

FileDownload / File: emd_9066.map.gz / Format: CCP4 / Size: 125 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationSpyCas9-A2a complex
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.83 Å/pix.
x 320 pix.
= 265.6 Å		0.83 Å/pix.
x 320 pix.
= 265.6 Å		0.83 Å/pix.
x 320 pix.
= 265.6 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.83 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.28 / Movie #1: 0.28
Minimum - Maximum-1.2549226 - 1.886544
Average (Standard dev.)0.00041811747 (±0.049322553)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions320320320
Spacing320320320
CellA=B=C: 265.6 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z0.830.830.83
M x/y/z320320320
origin x/y/z0.0000.0000.000
length x/y/z265.600265.600265.600
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS320320320
D min/max/mean-1.2551.8870.000

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Supplemental data

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Sample components

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Entire : AcrIIA2 interacting with sgRNA-bound Streptococcus pyogenes Cas9

EntireName: AcrIIA2 interacting with sgRNA-bound Streptococcus pyogenes Cas9
Components
  • Complex: AcrIIA2 interacting with sgRNA-bound Streptococcus pyogenes Cas9
    • Complex: sgRNA-bound Streptococcus pyogenes Cas9
      • RNA: Single guide RNA (116-MER)
      • Protein or peptide: CRISPR-associated endonuclease Cas9
    • Complex: AcrIIA2
      • Protein or peptide: Anti-CRISPR protein AcrIIA2

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Supramolecule #1: AcrIIA2 interacting with sgRNA-bound Streptococcus pyogenes Cas9

SupramoleculeName: AcrIIA2 interacting with sgRNA-bound Streptococcus pyogenes Cas9
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all

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Supramolecule #2: sgRNA-bound Streptococcus pyogenes Cas9

SupramoleculeName: sgRNA-bound Streptococcus pyogenes Cas9 / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1-#2
Source (natural)Organism: Streptococcus pyogenes M1 GAS (bacteria)

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Supramolecule #3: AcrIIA2

SupramoleculeName: AcrIIA2 / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #3
Source (natural)Organism: Listeria phage LP-101 (virus)

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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: 37.642258 KDa
SequenceString:
(GTP)GCGCAUAAA GAUGAGACGC GUUUUAGAGC UAUGCUGUUU UGAAAAAAAC AGCAUAGCAA GUUAAAAUAA GGCUAG UCC GUUAUCAACU UGAAAAAGUG GCACCGAGUC GGUGCUU

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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: 158.685828 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MDKKYSIGLD IGTNSVGWAV ITDDYKVPSK KFKVLGNTDR HSIKKNLIGA LLFDSGETAE ATRLKRTARR RYTRRKNRIC YLQEIFSNE MAKVDDSFFH RLEESFLVEE DKKHERHPIF GNIVDEVAYH EKYPTIYHLR KKLVDSTDKA DLRLIYLALA H MIKFRGHF ...String:
MDKKYSIGLD IGTNSVGWAV ITDDYKVPSK KFKVLGNTDR HSIKKNLIGA LLFDSGETAE ATRLKRTARR RYTRRKNRIC YLQEIFSNE MAKVDDSFFH RLEESFLVEE DKKHERHPIF GNIVDEVAYH EKYPTIYHLR KKLVDSTDKA DLRLIYLALA H MIKFRGHF LIEGDLNPDN SDVDKLFIQL VQTYNQLFEE NPINASGVDA KAILSARLSK SRRLENLIAQ LPGEKKNGLF GN LIALSLG LTPNFKSNFD LAEDAKLQLS KDTYDDDLDN LLAQIGDQYA DLFLAAKNLS DAILLSDILR VNTEITKAPL SAS MIKRYD EHHQDLTLLK ALVRQQLPEK YKEIFFDQSK NGYAGYIDGG ASQEEFYKFI KPILEKMDGT EELLVKLNRE DLLR KQRTF DNGSIPHQIH LGELHAILRR QEDFYPFLKD NREKIEKILT FRIPYYVGPL ARGNSRFAWM TRKSEETITP WNFEE VVDK GASAQSFIER MTNFDKNLPN EKVLPKHSLL YEYFTVYNEL TKVKYVTEGM RKPAFLSGEQ KKAIVDLLFK TNRKVT VKQ LKEDYFKKIE CFDSVEISGV EDRFNASLGT YHDLLKIIKD KDFLDNEENE DILEDIVLTL TLFEDREMIE ERLKTYA HL FDDKVMKQLK RRRYTGWGRL SRKLINGIRD KQSGKTILDF LKSDGFANRN FMQLIHDDSL TFKEDIQKAQ VSGQGDSL H EHIANLAGSP AIKKGILQTV KVVDELVKVM GRHKPENIVI EMARENQTTQ KGQKNSRERM KRIEEGIKEL GSQILKEHP VENTQLQNEK LYLYYLQNGR DMYVDQELDI NRLSDYDVDH IVPQSFLKDD SIDNKVLTRS DKNRGKSDNV PSEEVVKKMK NYWRQLLNA KLITQRKFDN LTKAERGGLS ELDKAGFIKR QLVETRQITK HVAQILDSRM NTKYDENDKL IREVKVITLK S KLVSDFRK DFQFYKVREI NNYHHAHDAY LNAVVGTALI KKYPKLESEF VYGDYKVYDV RKMIAKSEQE IGKATAKYFF YS NIMNFFK TEITLANGEI RKRPLIETNG ETGEIVWDKG RDFATVRKVL SMPQVNIVKK TEVQTGGFSK ESILPKRNSD KLI ARKKDW DPKKYGGFDS PTVAYSVLVV AKVEKGKSKK LKSVKELLGI TIMERSSFEK NPIDFLEAKG YKEVKKDLII KLPK YSLFE LENGRKRMLA SAGELQKGNE LALPSKYVNF LYLASHYEKL KGSPEDNEQK QLFVEQHKHY LDEIIEQISE FSKRV ILAD ANLDKVLSAY NKHRDKPIRE QAENIIHLFT LTNLGAPAAF KYFDTTIDRK RYTSTKEVLD ATLIHQSITG LYETRI DLS QLGGD

UniProtKB: CRISPR-associated endonuclease Cas9

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Macromolecule #3: Anti-CRISPR protein AcrIIA2

MacromoleculeName: Anti-CRISPR protein AcrIIA2 / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Listeria phage LP-101 (virus)
Molecular weightTheoretical: 14.167594 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
MTLTRAQKKY AEAMHEFINM VDDFEESTPD FAKEVLHDSD YVVITKNEKY AVALCSLSTD ECEYDTNLYL DEKLVDYSTV DVNGVTYYI NIVETNDIDD LEIATDEDEM KSGNQEIILK SELK

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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

BufferpH: 8
Details: 30 mM Tris-HCl, pH 8.0, 150 mM sodium chloride, 5 mM DTT, 0.1% glycerol
GridModel: C-flat-2/0.5 4C
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK II
DetailsSpyCas9-sgRNA-AcrIIA2 complex

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Average electron dose: 43.2 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: EMDB MAP
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.4 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 263270
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD

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Atomic model buiding 1

Initial modelPDB ID:

4zt0


Chain - Source name: PDB / Chain - Initial model type: experimental model
RefinementSpace: REAL / Protocol: OTHER
Output model

PDB-6mcb:
CryoEM structure of AcrIIA2 in complex with CRISPR-Cas9

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