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- EMDB-0584: Conformational states of Cas9-sgRNA-DNA ternary complex in the pr... -
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Open data
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Basic information
Entry | Database: EMDB / ID: EMD-0584 | ||||||||||||
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Title | Conformational states of Cas9-sgRNA-DNA ternary complex in the presence of magnesium | ||||||||||||
![]() | primary map | ||||||||||||
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![]() | CRISPR / Cas9 / Nuclease / Genome editing / HYDROLASE / HYDROLASE-RNA-DNA complex | ||||||||||||
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: 3.37 Å | ||||||||||||
![]() | Zhu X / Clarke R | ||||||||||||
Funding support | ![]()
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![]() | ![]() Title: Cryo-EM structures reveal coordinated domain motions that govern DNA cleavage by Cas9. Authors: Xing Zhu / Ryan Clarke / Anupama K Puppala / Sagar Chittori / Alan Merk / Bradley J Merrill / Miljan Simonović / Sriram Subramaniam / ![]() ![]() Abstract: The RNA-guided Cas9 endonuclease from Streptococcus pyogenes is a single-turnover enzyme that displays a stable product state after double-stranded-DNA cleavage. Here, we present cryo-EM structures ...The RNA-guided Cas9 endonuclease from Streptococcus pyogenes is a single-turnover enzyme that displays a stable product state after double-stranded-DNA cleavage. Here, we present cryo-EM structures of precatalytic, postcatalytic and product states of the active Cas9-sgRNA-DNA complex in the presence of Mg. In the precatalytic state, Cas9 adopts the 'checkpoint' conformation with the HNH nuclease domain positioned far away from the DNA. Transition to the postcatalytic state involves a dramatic ~34-Å swing of the HNH domain and disorder of the REC2 recognition domain. The postcatalytic state captures the cleaved substrate bound to the catalytically competent HNH active site. In the product state, the HNH domain is disordered, REC2 returns to the precatalytic conformation, and additional interactions of REC3 and RuvC with nucleic acids are formed. The coupled domain motions and interactions between the enzyme and the RNA-DNA hybrid provide new insights into the mechanism of genome editing by Cas9. | ||||||||||||
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 | ![]() | 53.5 MB | ![]() | |
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Header (meta data) | ![]() ![]() | 14.5 KB 14.5 KB | Display Display | ![]() |
Images | ![]() | 135.7 KB | ||
Filedesc metadata | ![]() | 6.9 KB | ||
Archive directory | ![]() ![]() | HTTPS FTP |
-Related structure data
Related structure data | ![]() 6o0yMC ![]() 0583C ![]() 0585C ![]() 6o0xC ![]() 6o0zC M: atomic model generated by this map C: citing same article ( |
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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
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Annotation | primary map | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 0.836 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 : Cas9-sgRNA-DNA ternary complex
Entire | Name: Cas9-sgRNA-DNA ternary complex |
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Components |
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-Supramolecule #1: Cas9-sgRNA-DNA ternary complex
Supramolecule | Name: Cas9-sgRNA-DNA ternary complex / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all |
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Source (natural) | Organism: ![]() |
-Macromolecule #1: CRISPR-associated endonuclease Cas9/Csn1
Macromolecule | Name: CRISPR-associated endonuclease Cas9/Csn1 / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO / EC number: Hydrolases; Acting on ester bonds |
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Source (natural) | Organism: ![]() |
Molecular weight | Theoretical: 158.986109 KDa |
Recombinant expression | Organism: ![]() ![]() |
Sequence | String: GAASMDKKYS IGLDIGTNSV GWAVITDEYK VPSKKFKVLG NTDRHSIKKN LIGALLFDSG ETAEATRLKR TARRRYTRRK NRICYLQEI FSNEMAKVDD SFFHRLEESF LVEEDKKHER HPIFGNIVDE VAYHEKYPTI YHLRKKLVDS TDKADLRLIY L ALAHMIKF ...String: GAASMDKKYS IGLDIGTNSV GWAVITDEYK VPSKKFKVLG NTDRHSIKKN LIGALLFDSG ETAEATRLKR TARRRYTRRK NRICYLQEI FSNEMAKVDD SFFHRLEESF LVEEDKKHER HPIFGNIVDE VAYHEKYPTI YHLRKKLVDS TDKADLRLIY L ALAHMIKF RGHFLIEGDL NPDNSDVDKL FIQLVQTYNQ LFEENPINAS GVDAKAILSA RLSKSRRLEN LIAQLPGEKK NG LFGNLIA LSLGLTPNFK SNFDLAEDAK LQLSKDTYDD DLDNLLAQIG DQYADLFLAA KNLSDAILLS DILRVNTEIT KAP LSASMI KRYDEHHQDL TLLKALVRQQ LPEKYKEIFF DQSKNGYAGY IDGGASQEEF YKFIKPILEK MDGTEELLVK LNRE DLLRK QRTFDNGSIP HQIHLGELHA ILRRQEDFYP FLKDNREKIE KILTFRIPYY VGPLARGNSR FAWMTRKSEE TITPW NFEE VVDKGASAQS FIERMTNFDK NLPNEKVLPK HSLLYEYFTV YNELTKVKYV TEGMRKPAFL SGEQKKAIVD LLFKTN RKV TVKQLKEDYF KKIECFDSVE ISGVEDRFNA SLGTYHDLLK IIKDKDFLDN EENEDILEDI VLTLTLFEDR EMIEERL KT YAHLFDDKVM KQLKRRRYTG WGRLSRKLIN GIRDKQSGKT ILDFLKSDGF ANRNFMQLIH DDSLTFKEDI QKAQVSGQ G DSLHEHIANL AGSPAIKKGI LQTVKVVDEL VKVMGRHKPE NIVIEMAREN QTTQKGQKNS RERMKRIEEG IKELGSQIL KEHPVENTQL QNEKLYLYYL QNGRDMYVDQ ELDINRLSDY DVDHIVPQSF LKDDSIDNKV LTRSDKNRGK SDNVPSEEVV KKMKNYWRQ LLNAKLITQR KFDNLTKAER GGLSELDKAG FIKRQLVETR QITKHVAQIL DSRMNTKYDE NDKLIREVKV I TLKSKLVS DFRKDFQFYK VREINNYHHA HDAYLNAVVG TALIKKYPKL ESEFVYGDYK VYDVRKMIAK SEQEIGKATA KY FFYSNIM NFFKTEITLA NGEIRKRPLI ETNGETGEIV WDKGRDFATV RKVLSMPQVN IVKKTEVQTG GFSKESILPK RNS DKLIAR KKDWDPKKYG GFDSPTVAYS VLVVAKVEKG KSKKLKSVKE LLGITIMERS SFEKNPIDFL EAKGYKEVKK DLII KLPKY SLFELENGRK RMLASAGELQ KGNELALPSK YVNFLYLASH YEKLKGSPED NEQKQLFVEQ HKHYLDEIIE QISEF SKRV ILADANLDKV LSAYNKHRDK PIREQAENII HLFTLTNLGA PAAFKYFDTT IDRKRYTSTK EVLDATLIHQ SITGLY ETR IDLSQLGGD UniProtKB: CRISPR-associated endonuclease Cas9/Csn1 |
-Macromolecule #2: single guide RNA
Macromolecule | Name: single guide RNA / type: rna / ID: 2 / Number of copies: 1 |
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Source (natural) | Organism: synthetic construct (others) |
Molecular weight | Theoretical: 33.059699 KDa |
Sequence | String: GGGGCGCAUA AAGAUGAGAC GCGUUUUAGA GCUAGAAAUA GCAAGUUAAA AUAAGGCUAG UCCGUUAUCA ACUUGAAAAA GUGGCACCG AGUCGGUGCU UGC |
-Macromolecule #3: 5' product of target strand DNA
Macromolecule | Name: 5' product of target strand DNA / type: dna / ID: 3 / Number of copies: 1 / Classification: DNA |
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Source (natural) | Organism: synthetic construct (others) |
Molecular weight | Theoretical: 3.936575 KDa |
Sequence | String: (DC)(DT)(DA)(DA)(DT)(DC)(DG)(DC)(DC)(DA) (DG)(DC)(DG) |
-Macromolecule #4: non-target strand DNA
Macromolecule | Name: non-target strand DNA / type: dna / ID: 4 / Number of copies: 1 / Classification: DNA |
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Source (natural) | Organism: synthetic construct (others) |
Molecular weight | Theoretical: 12.452001 KDa |
Sequence | String: (DC)(DC)(DA)(DG)(DT)(DG)(DC)(DG)(DT)(DA) (DG)(DG)(DC)(DG)(DC)(DA)(DT)(DA)(DA)(DA) (DG)(DA)(DT)(DG)(DA)(DG)(DA)(DC)(DG) (DC)(DT)(DG)(DG)(DC)(DG)(DA)(DT)(DT)(DA) (DG) |
-Macromolecule #5: 3' product of target strand DNA
Macromolecule | Name: 3' product of target strand DNA / type: dna / ID: 5 / Number of copies: 1 / Classification: DNA |
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Source (natural) | Organism: synthetic construct (others) |
Molecular weight | Theoretical: 8.194269 KDa |
Sequence | String: (DT)(DC)(DT)(DC)(DA)(DT)(DC)(DT)(DT)(DT) (DA)(DT)(DG)(DC)(DG)(DC)(DC)(DT)(DA)(DC) (DG)(DC)(DA)(DC)(DT)(DG)(DG) |
-Experimental details
-Structure determination
Method | cryo EM |
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![]() | single particle reconstruction |
Aggregation state | particle |
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Sample preparation
Concentration | 0.5 mg/mL |
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Buffer | pH: 7.5 |
Grid | Pretreatment - Type: PLASMA CLEANING / Details: unspecified |
Vitrification | Cryogen name: ETHANE / Instrument: LEICA EM GP |
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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: 73.0 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: NONE |
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Final reconstruction | Applied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.37 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 27433 |
Initial angle assignment | Type: RANDOM ASSIGNMENT |
Final angle assignment | Type: MAXIMUM LIKELIHOOD |