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- EMDB-42837: Cryo-EM structure of GeoCas9 in complex with sgRNA and target DNA -

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Basic information

Entry
Database: EMDB / ID: EMD-42837
TitleCryo-EM structure of GeoCas9 in complex with sgRNA and target DNA
Map dataSharp map of deactivated WT-GeoCas9 with sgRNA and target DNA.
Sample
  • Complex: Ternary complex of deactivated wild type GeoCas9 with sgRNA and target DNA
    • Protein or peptide: CRISPR-associated endonuclease Cas9
    • RNA: sgRNA (107-MER)
    • DNA: Target strand DNA
    • DNA: Non-target strand DNA
KeywordsRNA-guided DNA endonuclease / ternary complex / CRISPR / hydrolase / HYDROLASE-RNA-DNA complex
Function / homology
Function and homology information


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
Cas9, alpha-helical lobe domain / Cas9 alpha-helical lobe domain / RuvC endonuclease subdomain 3 / RuvC endonuclease subdomain 3 / 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 speciesGeobacillus stearothermophilus (bacteria) / Mus musculus (house mouse)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.17 Å
AuthorsEggers AR / Soczek KM / Tuck OT / Doudna JA
Funding support United States, 1 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)AI171110 United States
CitationJournal: Cell / Year: 2024
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
DepositionNov 14, 2023-
Header (metadata) releaseMay 29, 2024-
Map releaseMay 29, 2024-
UpdateJul 3, 2024-
Current statusJul 3, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_42837.png

Downloads & links

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Map

FileDownload / File: emd_42837.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationSharp map of deactivated WT-GeoCas9 with sgRNA and target DNA.
Voxel sizeX=Y=Z: 0.93 Å
Density
Contour LevelBy AUTHOR: 0.27
Minimum - Maximum-1.0659535 - 1.9135756
Average (Standard dev.)-0.00069664506 (±0.05231394)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 238.08 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_42837_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Map of deactivated WT-GeoCas9 with sgRNA and target DNA.

Fileemd_42837_additional_1.map
AnnotationMap of deactivated WT-GeoCas9 with sgRNA and target DNA.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map of deactivated WT-GeoCas9 with sgRNA and target DNA.

Fileemd_42837_half_map_1.map
AnnotationHalf map of deactivated WT-GeoCas9 with sgRNA and target DNA.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map of deactivated WT-GeoCas9 with sgRNA and target DNA.

Fileemd_42837_half_map_2.map
AnnotationHalf map of deactivated WT-GeoCas9 with sgRNA and target DNA.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Ternary complex of deactivated wild type GeoCas9 with sgRNA and t...

EntireName: Ternary complex of deactivated wild type GeoCas9 with sgRNA and target DNA
Components
  • Complex: Ternary complex of deactivated wild type GeoCas9 with sgRNA and target DNA
    • Protein or peptide: CRISPR-associated endonuclease Cas9
    • RNA: sgRNA (107-MER)
    • DNA: Target strand DNA
    • DNA: Non-target strand DNA

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Supramolecule #1: Ternary complex of deactivated wild type GeoCas9 with sgRNA and t...

SupramoleculeName: Ternary complex of deactivated wild type GeoCas9 with sgRNA and target DNA
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Geobacillus stearothermophilus (bacteria)
Molecular weightTheoretical: 203 KDa

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Macromolecule #1: CRISPR-associated endonuclease Cas9

MacromoleculeName: CRISPR-associated endonuclease Cas9 / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Geobacillus stearothermophilus (bacteria)
Molecular weightTheoretical: 127.043883 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MRYKIGLAIG ITSVGWAVMN LDIPRIEDLG VRIFDRAENP QTGESLALPR RLARSARRRL RRRKHRLERI RRLVIREGIL TKEELDKLF EEKHEIDVWQ LRVEALDRKL NNDELARVLL HLAKRRGFKS NRKSERSNKE NSTMLKHIEE NRAILSSYRT V GEMIVKDP ...String:
MRYKIGLAIG ITSVGWAVMN LDIPRIEDLG VRIFDRAENP QTGESLALPR RLARSARRRL RRRKHRLERI RRLVIREGIL TKEELDKLF EEKHEIDVWQ LRVEALDRKL NNDELARVLL HLAKRRGFKS NRKSERSNKE NSTMLKHIEE NRAILSSYRT V GEMIVKDP KFALHKRNKG ENYTNTIARD DLEREIRLIF SKQREFGNMS 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 PPIANPVVMR 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 VTGAAHQETL RRYVGIDERS GKIQTVVKTK LSEIKLDASG HFPMYGKESD PRTYEAIRQR LLEHNNDPKK AFQEPLYKP KKNGEPGPVI RTVKIIDTKN 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

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Macromolecule #2: sgRNA (107-MER)

MacromoleculeName: sgRNA (107-MER) / type: rna / ID: 2 / Number of copies: 1
Source (natural)Organism: Geobacillus stearothermophilus (bacteria)
Molecular weightTheoretical: 44.488117 KDa
SequenceString:
CACUGCAUUC UAGUUGUGGU UGUCAUAGUU CCCCUGAGAA AUCAGGGUUA CUAUGAUAAG GGCUUUCUGC CUAAGGCAGA CUGACCCGC GGCGUUGGGG AUCGCCUGUC GCCCGCUUUU GGCGGGCAUU CCCCAUCCUU

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Macromolecule #3: Target strand DNA

MacromoleculeName: Target strand DNA / type: dna / ID: 3 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: Mus musculus (house mouse)
Molecular weightTheoretical: 15.797221 KDa
SequenceString:
(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)

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Macromolecule #4: Non-target strand DNA

MacromoleculeName: Non-target strand DNA / type: dna / ID: 4 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: Mus musculus (house mouse)
Molecular weightTheoretical: 15.618021 KDa
SequenceString:
(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)

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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: 7.5
Component:
ConcentrationFormulaName
20.0 mMC4H11NO3tris
100.0 mMKClpotassium chloride
1.0 mMC9H15O6PTCEP
0.25 %C3H8O3glycerol
GridModel: Quantifoil R1.2/1.3 / Material: GOLD / Mesh: 300 / Support film - Material: GOLD / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 281 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TALOS ARCTICA
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 50.0 e/Å2
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: OTHER / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.0 µm / Nominal defocus min: 0.8 µm
Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company

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Image processing

Startup modelType of model: OTHER / Details: cryoSPARC ab initio model
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.17 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 4.2.1) / Number images used: 117726
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 4.2.1)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 4.2.1)
Final 3D classificationSoftware - Name: cryoSPARC (ver. 4.2.1)
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial model
ChainDetails
source_name: AlphaFold, initial_model_type: in silico modelv1.4.0
source_name: Other, initial_model_type: in silico modelModelAngelo v1.0.1
RefinementSpace: REAL / Protocol: OTHER
Output model

PDB-8uza:
Cryo-EM structure of GeoCas9 in complex with sgRNA and target DNA

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