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Open data
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Basic information
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Title | Cas9:sgRNA:DNA (S. pyogenes) forming a 3-base-pair R-loop | |||||||||
![]() | LocScale-sharpened map | |||||||||
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Function / homology | ![]() 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 | |||||||||
Biological species | ![]() ![]() | |||||||||
Method | single particle reconstruction / cryo EM / Resolution: 3.3 Å | |||||||||
![]() | Cofsky JC / Soczek KM / Knott GJ / Nogales E / Doudna JA | |||||||||
Funding support | ![]()
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![]() | ![]() Title: CRISPR-Cas9 bends and twists DNA to read its sequence. Authors: Joshua C Cofsky / Katarzyna M Soczek / Gavin J Knott / Eva Nogales / Jennifer A Doudna / ![]() ![]() Abstract: In bacterial defense and genome editing applications, the CRISPR-associated protein Cas9 searches millions of DNA base pairs to locate a 20-nucleotide, guide RNA-complementary target sequence that ...In bacterial defense and genome editing applications, the CRISPR-associated protein Cas9 searches millions of DNA base pairs to locate a 20-nucleotide, guide RNA-complementary target sequence that abuts a protospacer-adjacent motif (PAM). Target capture requires Cas9 to unwind DNA at candidate sequences using an unknown ATP-independent mechanism. Here we show that Cas9 sharply bends and undertwists DNA on PAM binding, thereby flipping DNA nucleotides out of the duplex and toward the guide RNA for sequence interrogation. Cryogenic-electron microscopy (cryo-EM) structures of Cas9-RNA-DNA complexes trapped at different states of the interrogation pathway, together with solution conformational probing, reveal that global protein rearrangement accompanies formation of an unstacked DNA hinge. Bend-induced base flipping explains how Cas9 'reads' snippets of DNA to locate target sites within a vast excess of nontarget DNA, a process crucial to both bacterial antiviral immunity and genome editing. This mechanism establishes a physical solution to the problem of complementarity-guided DNA search and shows how interrogation speed and local DNA geometry may influence genome editing efficiency. | |||||||||
History |
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Structure visualization
Supplemental images |
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Downloads & links
-EMDB archive
Map data | ![]() | 1.8 MB | ![]() | |
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Header (meta data) | ![]() ![]() | 25.5 KB 25.5 KB | Display Display | ![]() |
FSC (resolution estimation) | ![]() | 9.1 KB | Display | ![]() |
Images | ![]() | 126.4 KB | ||
Masks | ![]() | 64 MB | ![]() | |
Others | ![]() ![]() ![]() ![]() | 49.3 MB 4.3 MB 49.7 MB 49.7 MB | ||
Archive directory | ![]() ![]() | HTTPS FTP |
-Related structure data
Related structure data | ![]() 7s38MC ![]() 7s36C ![]() 7s37C ![]() 7s3hC C: citing same article ( M: atomic model generated by this map |
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Similar structure data | Similarity search - Function & homology ![]() |
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Links
EMDB pages | ![]() ![]() |
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Related items in Molecule of the Month |
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Map
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Annotation | LocScale-sharpened map | ||||||||||||||||||||
Voxel size | X=Y=Z: 1.05 Å | ||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||
Details | EMDB XML:
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-Supplemental data
-Mask #1
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-Additional map: unsharpened map from Relion
File | emd_24819_additional_1.map | ||||||||||||
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Annotation | unsharpened map from Relion | ||||||||||||
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-Additional map: post-processed map from RELION
File | emd_24819_additional_2.map | ||||||||||||
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Annotation | post-processed map from RELION | ||||||||||||
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-Half map: half map 1 from RELION
File | emd_24819_half_map_1.map | ||||||||||||
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Annotation | half map 1 from RELION | ||||||||||||
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Density Histograms |
-Half map: half map 2 from RELION
File | emd_24819_half_map_2.map | ||||||||||||
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Annotation | half map 2 from RELION | ||||||||||||
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Sample components
-Entire : Cas9 bound to sgRNA and DNA with 3bp complementarity between RNA ...
Entire | Name: Cas9 bound to sgRNA and DNA with 3bp complementarity between RNA and DNA |
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Components |
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-Supramolecule #1: Cas9 bound to sgRNA and DNA with 3bp complementarity between RNA ...
Supramolecule | Name: Cas9 bound to sgRNA and DNA with 3bp complementarity between RNA and DNA type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#4 |
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Source (natural) | Organism: ![]() |
-Macromolecule #1: Non-target DNA strand
Macromolecule | Name: Non-target DNA strand / type: dna / ID: 1 / Number of copies: 1 / Classification: DNA |
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Source (natural) | Organism: synthetic construct (others) |
Molecular weight | Theoretical: 9.33604 KDa |
Sequence | String: (DG)(DA)(DC)(DG)(DC)(DA)(DT)(DA)(DA)(DA) (DG)(DA)(DT)(DG)(DA)(DG)(DA)(DG)(DT)(DT) (DT)(DG)(DG)(DC)(DG)(DA)(DT)(DT)(DA) (DC) |
-Macromolecule #4: Target DNA strand
Macromolecule | Name: Target DNA strand / type: dna / ID: 4 Details: The 5'-most deoxycytidine is actually N4-cystamine-2'-deoxycytidine Number of copies: 1 / Classification: DNA |
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Source (natural) | Organism: synthetic construct (others) |
Molecular weight | Theoretical: 9.190992 KDa |
Sequence | String: (DG)(DT)(DA)(DA)(DT)(2YR)(DG)(DC)(DC)(DA) (DT)(DT)(DG)(DT)(DC)(DT)(DC)(DA)(DT) (DC)(DT)(DT)(DT)(DA)(DT)(DG)(DC)(DG)(DT) (DC) |
-Macromolecule #2: CRISPR-associated endonuclease Cas9/Csn1
Macromolecule | Name: CRISPR-associated endonuclease Cas9/Csn1 / 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: 158.974125 KDa |
Recombinant expression | Organism: ![]() ![]() |
Sequence | String: SNAMDKKYSI GLDIGTNSVG WAVITDEYKV PSKKFKVLGN TDRHSIKKNL IGALLFDSGE TAEATRLKRT ARRRYTRRKN RICYLQEIF SNEMAKVDDS FFHRLEESFL VEEDKKHERH PIFGNIVDEV AYHEKYPTIY HLRKKLVDST DKADLRLIYL A LAHMIKFR ...String: SNAMDKKYSI GLDIGTNSVG WAVITDEYKV PSKKFKVLGN TDRHSIKKNL IGALLFDSGE TAEATRLKRT ARRRYTRRKN RICYLQEIF SNEMAKVDDS FFHRLEESFL VEEDKKHERH PIFGNIVDEV AYHEKYPTIY HLRKKLVDST DKADLRLIYL A LAHMIKFR GHFLIEGDLN PDNSDVDKLF IQLVQTYNQL FEENPINASG VDAKAILSAR LSKSRRLENL IAQLPGEKKN GL FGNLIAL SLGLTPNFKS NFDLAEDAKL QLSKDTYDDD LDNLLAQIGD QYADLFLAAK NLSDAILLSD ILRVNTEITK APL SASMIK RYDEHHQDLT LLKALVRQQL PEKYKEIFFD QSKNGYAGYI DGGASQEEFY KFIKPILEKM DGTEELLVKL NRED LLRKQ RTFDNGSIPH QIHLGELHAI LRRQEDFYPF LKDNREKIEK ILTFRIPYYV GPLARGNSRF AWMTRKSEET ITPWN FEEV VDKGASAQSF IERMTNFDKN LPNEKVLPKH SLLYEYFTVY NELTKVKYVT EGMRKPAFLS GEQKKAIVDL LFKTNR KVT VKQLKEDYFK KIECFDSVEI SGVEDRFNAS LGTYHDLLKI IKDKDFLDNE ENEDILEDIV LTLTLFEDRE MIEERLK TY AHLFDDKVMK QLKRRRYTGW GRLSRKLING IRDKQSGKTI LDFLKSDGFA NRNFMQLIHD DSLTFKEDIQ KAQVSGQG D SLHEHIANLA GSPAIKKGIL QTVKVVDELV KVMGRHKPEN IVIEMARENQ TTQKGQKNSR ERMKRIEEGI KELGSQILK EHPVENTQLQ NEKLYLYYLQ NGRDMYVDQE LDINRLSDYD VDHIVPQSFL KDDSIDNKVL TRSDKNRGKS DNVPSEEVVK KMKNYWRQL LNAKLITQRK FDNLTKAERG GLSELDKAGF IKRQLVETRQ ITKHVAQILD SRMNTKYDEN DKLIREVKVI T LKSKLVSD FRKDFQFYKV REINNYHHAH DAYLNAVVGT ALIKKYPKLE SEFVYGDYKV YDVRKMIAKS EQEIGKATAK YF FYSNIMN FFKTEITLAN GEIRKRPLIE TNGETGEIVW DKGRDFATVR KVLSMPQVNI VKKTEVQTGG FSKESILPKR NSD KLIARK KDWDPKKYGG FDSPTVAYSV LVVAKVEKGK SKKLKSVKEL LGITIMERSS FEKNPIDFLE AKGYKEVKKD LIIK LPKYS LFELENGRKR MLASAGELQK GNELALPSKY VNFLYLASHY EKLKGSPEDN EQKQLFVEQH KHYLDEIIEQ ISEFS KRVI LADANLDKVL SAYNKHRDKP IREQAENIIH LFTLTNLGAP AAFKYFDTTI DRKRYCSTKE VLDATLIHQS ITGLYE TRI DLSQLGGD |
-Macromolecule #3: Single-guide RNA
Macromolecule | Name: Single-guide RNA / type: rna / ID: 3 / Number of copies: 1 |
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Source (natural) | Organism: ![]() |
Molecular weight | Theoretical: 32.79341 KDa |
Sequence | String: GGCUGCGUAU UUCUACUCUC AAGUUUUAGA GCUAGAAAUA GCAAGUUAAA AUAAGGCUAG UCCGUUAUCA ACUUGAAAAA GUGGCACCG AGUCGGUGCU UCG |
-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: 7.5 Component:
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Grid | Model: UltrAuFoil R1.2/1.3 / Material: GOLD / Mesh: 300 / Pretreatment - Type: GLOW DISCHARGE / Details: 25mA | ||||||||||||||||||
Vitrification | Cryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 281 K |
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Electron microscopy
Microscope | FEI TITAN KRIOS |
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Image recording | Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average electron dose: 50.0 e/Å2 |
Electron beam | Acceleration voltage: 300 kV / Electron source: ![]() |
Electron optics | Illumination mode: OTHER / Imaging mode: BRIGHT FIELD |
Experimental equipment | ![]() Model: Titan Krios / Image courtesy: FEI Company |