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- EMDB-42838: Cryo-EM structure of iGeoCas9 in complex with sgRNA and target DNA -
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Open data
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Basic information
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Title | Cryo-EM structure of iGeoCas9 in complex with sgRNA and target DNA | |||||||||
![]() | Sharp map of deactivated iGeoCas9 with sgRNA and target DNA | |||||||||
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![]() | RNA-guided DNA endonuclease / ternary complex / CRISPR / HYDROLASE / HYDROLASE-RNA-DNA 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: 2.63 Å | |||||||||
![]() | Eggers AR / Soczek KM / Tuck OT / Doudna JA | |||||||||
Funding support | ![]()
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![]() | ![]() Title: Rapid DNA unwinding accelerates genome editing by engineered CRISPR-Cas9. Authors: Amy R Eggers / Kai Chen / Katarzyna M Soczek / Owen T Tuck / Erin E Doherty / Bryant Xu / Marena I Trinidad / Brittney W Thornton / Peter H Yoon / Jennifer A Doudna / ![]() Abstract: Thermostable clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas9) enzymes could improve genome-editing efficiency and delivery due to extended protein ...Thermostable clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas9) enzymes could improve genome-editing efficiency and delivery due to extended protein lifetimes. However, initial experimentation demonstrated Geobacillus stearothermophilus Cas9 (GeoCas9) to be virtually inactive when used in cultured human cells. Laboratory-evolved variants of GeoCas9 overcome this natural limitation by acquiring mutations in the wedge (WED) domain that produce >100-fold-higher genome-editing levels. Cryoelectron microscopy (cryo-EM) structures of the wild-type and improved GeoCas9 (iGeoCas9) enzymes reveal extended contacts between the WED domain of iGeoCas9 and DNA substrates. Biochemical analysis shows that iGeoCas9 accelerates DNA unwinding to capture substrates under the magnesium-restricted conditions typical of mammalian but not bacterial cells. These findings enabled rational engineering of other Cas9 orthologs to enhance genome-editing levels, pointing to a general strategy for editing enzyme improvement. Together, these results uncover a new role for the Cas9 WED domain in DNA unwinding and demonstrate how accelerated target unwinding dramatically improves Cas9-induced genome-editing activity. | |||||||||
History |
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Structure visualization
Supplemental images |
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Downloads & links
-EMDB archive
Map data | ![]() | 57.4 MB | ![]() | |
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Header (meta data) | ![]() ![]() | 23.7 KB 23.7 KB | Display Display | ![]() |
FSC (resolution estimation) | ![]() | 8.4 KB | Display | ![]() |
Images | ![]() | 52.2 KB | ||
Masks | ![]() | 64 MB | ![]() | |
Filedesc metadata | ![]() | 7.5 KB | ||
Others | ![]() ![]() ![]() | 31.2 MB 56.4 MB 56.4 MB | ||
Archive directory | ![]() ![]() | HTTPS FTP |
-Validation report
Summary document | ![]() | 882.1 KB | Display | ![]() |
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Full document | ![]() | 881.7 KB | Display | |
Data in XML | ![]() | 16.4 KB | Display | |
Data in CIF | ![]() | 20.9 KB | Display | |
Arichive directory | ![]() ![]() | HTTPS FTP |
-Related structure data
Related structure data | ![]() 8uzbMC ![]() 8uzaC 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
File | ![]() | ||||||||||||||||||||
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Annotation | Sharp map of deactivated iGeoCas9 with sgRNA and target DNA | ||||||||||||||||||||
Voxel size | X=Y=Z: 0.93 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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Projections & Slices |
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Density Histograms |
-Additional map: Map of deactivated iGeoCas9 with sgRNA and target DNA
File | emd_42838_additional_1.map | ||||||||||||
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Annotation | Map of deactivated iGeoCas9 with sgRNA and target DNA | ||||||||||||
Projections & Slices |
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Density Histograms |
-Half map: Half map of deactivated iGeoCas9 with sgRNA and target DNA
File | emd_42838_half_map_1.map | ||||||||||||
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Annotation | Half map of deactivated iGeoCas9 with sgRNA and target DNA | ||||||||||||
Projections & Slices |
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Density Histograms |
-Half map: Half map of deactivated iGeoCas9 with sgRNA and target DNA
File | emd_42838_half_map_2.map | ||||||||||||
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Annotation | Half map of deactivated iGeoCas9 with sgRNA and target DNA | ||||||||||||
Projections & Slices |
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Density Histograms |
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Sample components
-Entire : Ternary complex of deactivated iGeoCas9 with sgRNA and target DNA
Entire | Name: Ternary complex of deactivated iGeoCas9 with sgRNA and target DNA |
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Components |
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-Supramolecule #1: Ternary complex of deactivated iGeoCas9 with sgRNA and target DNA
Supramolecule | Name: Ternary complex of deactivated iGeoCas9 with sgRNA and target DNA type: complex / ID: 1 / Parent: 0 / Macromolecule list: all |
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Source (natural) | Organism: ![]() ![]() |
Molecular weight | Theoretical: 203 KDa |
-Macromolecule #1: CRISPR-associated endonuclease Cas9
Macromolecule | Name: CRISPR-associated endonuclease Cas9 / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO |
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Source (natural) | Organism: ![]() ![]() |
Molecular weight | Theoretical: 127.000977 KDa |
Recombinant expression | Organism: ![]() ![]() |
Sequence | String: MRYKIGLAIG ITSVGWAVMN LDIPRIEDLG VRIFDRAENP QTGESLALPR RLARSARRRL RRRKHRLERI RRLVIREGIL TKEELDKLF EEKHEIDVWQ LRVEALDRKL NNDELARVLL HLAKRRGFKS NRKSERSNKE NSTMLKHIEG NRAILSSYRT V GEMIVKDP ...String: MRYKIGLAIG ITSVGWAVMN LDIPRIEDLG VRIFDRAENP QTGESLALPR RLARSARRRL RRRKHRLERI RRLVIREGIL TKEELDKLF EEKHEIDVWQ LRVEALDRKL NNDELARVLL HLAKRRGFKS NRKSERSNKE NSTMLKHIEG NRAILSSYRT V GEMIVKDP KFALHKRNKG ENYINTIARD DLEREIRLIF SKQREFGDMS CTEEFENEYI TIWASQRPVA SKDDIEKKVG FC TFEPKEK RAPKATYTFQ SFIAWEHINK LRLISPSGAR GLTDEERRLL YEQAFQKNKI TYHDIRTLLH LPDDTYFKGI VYD RGESRK QNENIRFLEL DAYHQIRKAV DKVYGKGKSS SFLPIDFDTF GYALTLFKDD ADIHSYLRNE YEQNGKRMPN LANK VYDNE LIEELLNLSF TKFGHLSLKA LRSILPYMEQ GEVYSSACER AGYTFTGPKK KQKTMLLPNI QPIANPVVMR ALTQA RKVV NAIIKKYGSP VSIHIELARD LSQTFDERRK TKKEQDENRK KNETAIRQLM EYGLTLNPTG HDIVKFKLWS EQNGRC AYS LQPIEIERLL EPGYVEVDAV IPYSRSLDDS YTNKVLVLTR ENREKGNRIP AEYLGVGTER WQQFETFVLT NKQFSKK KR DRLLRLHYDE NEETEFKNRN LNDTRYISRF FANFIREHLK FAESDDKQKV YTVNGRVTAH LRSRWEFNKN REESDLHH A VDAVIVACTT PSDIAKVTAF YQRREQNKEL AKKTEPHFPQ PWPHFADELR ARLSKHPKES IKALNLGNYD DQKLESLQP VFVSRMPKRS VTGAAHRETL RRYVGIDERS GKIQTVVKTK LSKIKLDASG HFPMYGKESD PRTYEAIRQR LLEHNNDPKK AFQGPLYKP KKNGEPGPVI RTVKIIDTRN QVIPLNDGKT VAYNSNIVRV DVFEKDGKYY CVPVYTMDIM KGILPNKAIE P NKPYSEWK EMTEDYTFRF SLYPNDLIRI ELPREKTVKT AAGEEINVKD VFVYYKTIDS ANGGLELISH DHRFSLRGVG SR TLKRFEK YQVDVLGNIY KVRGEKRVGL ASSAHSKPGK TIRPLQSTRD UniProtKB: CRISPR-associated endonuclease Cas9 |
-Macromolecule #2: RNA (107-MER)
Macromolecule | Name: RNA (107-MER) / type: rna / ID: 2 / Number of copies: 1 |
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Source (natural) | Organism: ![]() ![]() |
Molecular weight | Theoretical: 44.488117 KDa |
Sequence | String: CACUGCAUUC UAGUUGUGGU UGUCAUAGUU CCCCUGAGAA AUCAGGGUUA CUAUGAUAAG GGCUUUCUGC CUAAGGCAGA CUGACCCGC GGCGUUGGGG AUCGCCUGUC GCCCGCUUUU GGCGGGCAUU CCCCAUCCUU |
-Macromolecule #3: Target strand DNA (39-MER)
Macromolecule | Name: Target strand DNA (39-MER) / type: dna / ID: 3 / Number of copies: 1 / Classification: DNA |
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Source (natural) | Organism: ![]() ![]() |
Molecular weight | Theoretical: 15.797221 KDa |
Sequence | String: (DT)(DA)(DC)(DA)(DT)(DT)(DG)(DA)(DT)(DG) (DA)(DG)(DT)(DT)(DT)(DG)(DG)(DA)(DC)(DA) (DA)(DA)(DC)(DC)(DA)(DC)(DA)(DA)(DC) (DT)(DA)(DG)(DA)(DA)(DT)(DG)(DC)(DA)(DG) (DT) (DG)(DA)(DA)(DA)(DA)(DA)(DA)(DA) (DT)(DG)(DC) |
-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: ![]() ![]() |
Molecular weight | Theoretical: 15.618021 KDa |
Sequence | String: (DG)(DC)(DA)(DT)(DT)(DT)(DT)(DT)(DT)(DT) (DC)(DA)(DC)(DT)(DG)(DC)(DA)(DT)(DT)(DC) (DT)(DA)(DG)(DT)(DT)(DG)(DT)(DG)(DG) (DT)(DT)(DT)(DG)(DT)(DC)(DC)(DA)(DA)(DA) (DC) (DT)(DC)(DA)(DT)(DC)(DA)(DA)(DT) (DG)(DT)(DA) |
-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 / Support film - Material: GOLD / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE | |||||||||||||||
Vitrification | Cryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 281 K / Instrument: FEI VITROBOT MARK IV |
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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 / Nominal defocus max: 1.8 µm / Nominal defocus min: 0.8 µm |
Experimental equipment | ![]() Model: Titan Krios / Image courtesy: FEI Company |
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Image processing
-Atomic model buiding 1
Initial model |
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Refinement | Space: REAL / Protocol: OTHER | ||||||
Output model | ![]() PDB-8uzb: |