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Open data
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Basic information
Entry | Database: EMDB / ID: EMD-9066 | |||||||||
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Title | CryoEM structure of AcrIIA2 in complex with CRISPR-Cas9 | |||||||||
![]() | SpyCas9-A2a complex | |||||||||
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![]() | CRISPR-Cas / Cas9 / Cas9 inhibitors / anti-CRISPR / AcrIIA2 / bacteriophage / gene editing / HYDROLASE-RNA-VIRAL PROTEIN complex | |||||||||
Function / homology | ![]() 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: 3.4 Å | |||||||||
![]() | Jiang F / Liu JJ | |||||||||
Funding support | ![]()
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![]() | ![]() 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 |
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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 | ![]() | 117.3 MB | ![]() | |
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Header (meta data) | ![]() ![]() | 14.6 KB 14.6 KB | Display Display | ![]() |
Images | ![]() | 45.9 KB | ||
Filedesc metadata | ![]() | 6.8 KB | ||
Archive directory | ![]() ![]() | HTTPS FTP |
-Related structure data
Related structure data | ![]() 6mcbMC ![]() 9067C ![]() 6mccC 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 | SpyCas9-A2a complex | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 0.83 Å | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Details | EMDB XML:
CCP4 map header:
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-Supplemental data
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Sample components
-Entire : AcrIIA2 interacting with sgRNA-bound Streptococcus pyogenes Cas9
Entire | Name: AcrIIA2 interacting with sgRNA-bound Streptococcus pyogenes Cas9 |
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Components |
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-Supramolecule #1: AcrIIA2 interacting with sgRNA-bound Streptococcus pyogenes Cas9
Supramolecule | Name: 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
Supramolecule | Name: sgRNA-bound Streptococcus pyogenes Cas9 / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1-#2 |
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Source (natural) | Organism: ![]() |
-Supramolecule #3: AcrIIA2
Supramolecule | Name: AcrIIA2 / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #3 |
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Source (natural) | Organism: ![]() |
-Macromolecule #1: Single guide RNA (116-MER)
Macromolecule | Name: Single guide RNA (116-MER) / type: rna / ID: 1 / Number of copies: 1 |
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Source (natural) | Organism: ![]() |
Molecular weight | Theoretical: 37.642258 KDa |
Sequence | String: (GTP)GCGCAUAAA GAUGAGACGC GUUUUAGAGC UAUGCUGUUU UGAAAAAAAC AGCAUAGCAA GUUAAAAUAA GGCUAG UCC GUUAUCAACU UGAAAAAGUG GCACCGAGUC GGUGCUU |
-Macromolecule #2: CRISPR-associated endonuclease Cas9
Macromolecule | Name: CRISPR-associated endonuclease Cas9 / 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: ![]() |
Molecular weight | Theoretical: 158.685828 KDa |
Recombinant expression | Organism: ![]() ![]() |
Sequence | String: 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 |
-Macromolecule #3: Anti-CRISPR protein AcrIIA2
Macromolecule | Name: Anti-CRISPR protein AcrIIA2 / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO |
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Source (natural) | Organism: ![]() |
Molecular weight | Theoretical: 14.167594 KDa |
Recombinant expression | Organism: ![]() ![]() |
Sequence | String: MTLTRAQKKY AEAMHEFINM VDDFEESTPD FAKEVLHDSD YVVITKNEKY AVALCSLSTD ECEYDTNLYL DEKLVDYSTV DVNGVTYYI NIVETNDIDD LEIATDEDEM KSGNQEIILK SELK |
-Experimental details
-Structure determination
Method | cryo EM |
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![]() | single particle reconstruction |
Aggregation state | particle |
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Sample preparation
Buffer | pH: 8 Details: 30 mM Tris-HCl, pH 8.0, 150 mM sodium chloride, 5 mM DTT, 0.1% glycerol |
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Grid | Model: C-flat-2/0.5 4C |
Vitrification | Cryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK II |
Details | SpyCas9-sgRNA-AcrIIA2 complex |
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Electron microscopy
Microscope | FEI TITAN KRIOS |
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Image recording | Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Average electron dose: 43.2 e/Å2 |
Electron beam | Acceleration voltage: 300 kV / Electron source: ![]() |
Electron optics | Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD |
Experimental equipment | ![]() Model: Titan Krios / Image courtesy: FEI Company |
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Image processing
Startup model | Type of model: EMDB MAP |
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Final reconstruction | Resolution.type: BY AUTHOR / Resolution: 3.4 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 263270 |
Initial angle assignment | Type: MAXIMUM LIKELIHOOD |
Final angle assignment | Type: MAXIMUM LIKELIHOOD |