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- EMDB-43769: CryoEM structure of ThermoCas9 in post-cleavage state bound with ... -

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Basic information

Entry
Database: EMDB / ID: EMD-43769
TitleCryoEM structure of ThermoCas9 in post-cleavage state bound with the DNA containing NNNNCCA PAM
Map data
Sample
  • Complex: ThermoCas9 Post-cleavage complex
    • RNA: Single guide RNA
    • DNA: Cleaved 3' target DNA strand
    • DNA: Cleaved 3' non-target DNA strand
    • DNA: Cleaved 5' target DNA strand
    • DNA: Cleaved 5' non-target DNA strand
    • Protein or peptide: CRISPR-associated endonuclease Cas9
  • Ligand: MAGNESIUM ION
  • Ligand: GUANOSINE-5'-TRIPHOSPHATE
  • Ligand: water
KeywordsCRISPR-Cas9 / Cas9 / Epigenetics / endonuclease / HNH / RuvC / metal binding / RNA / DNA BINDING PROTEIN-DNA-RNA complex
Function / homology:
Function and homology information
Biological speciesGeobacillus thermodenitrificans (bacteria) / synthetic construct (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.2 Å
AuthorsShu Y / Zhao Y / Li H
Funding support United States, 1 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM099604 United States
CitationJournal: To Be Published
Title: Molecular Basis for Epigenetic-sensitive Editing by Cas9
Authors: Roth M / Shu Y / Trasanidis D / Zhao Y / Hoffman R / van der Oost J / Li H
History
DepositionFeb 22, 2024-
Header (metadata) releaseFeb 26, 2025-
Map releaseFeb 26, 2025-
UpdateFeb 26, 2025-
Current statusFeb 26, 2025Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_43769.png

Downloads & links

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Map

FileDownload / File: emd_43769.map.gz / Format: CCP4 / Size: 103 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.82 Å/pix.
x 300 pix.
= 246. Å		0.82 Å/pix.
x 300 pix.
= 246. Å		0.82 Å/pix.
x 300 pix.
= 246. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.82 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.36
Minimum - Maximum-4.5290713 - 7.8119097
Average (Standard dev.)0.0031039466 (±0.1424203)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions300300300
Spacing300300300
CellA=B=C: 246.0 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #2

Fileemd_43769_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_43769_half_map_2.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : ThermoCas9 Post-cleavage complex

EntireName: ThermoCas9 Post-cleavage complex
Components
  • Complex: ThermoCas9 Post-cleavage complex
    • RNA: Single guide RNA
    • DNA: Cleaved 3' target DNA strand
    • DNA: Cleaved 3' non-target DNA strand
    • DNA: Cleaved 5' target DNA strand
    • DNA: Cleaved 5' non-target DNA strand
    • Protein or peptide: CRISPR-associated endonuclease Cas9
  • Ligand: MAGNESIUM ION
  • Ligand: GUANOSINE-5'-TRIPHOSPHATE
  • Ligand: water

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Supramolecule #1: ThermoCas9 Post-cleavage complex

SupramoleculeName: ThermoCas9 Post-cleavage complex / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#6
Source (natural)Organism: Geobacillus thermodenitrificans (bacteria)

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Macromolecule #1: Single guide RNA

MacromoleculeName: Single guide RNA / type: rna / ID: 1 / Number of copies: 1
Source (natural)Organism: Geobacillus thermodenitrificans (bacteria)
Molecular weightTheoretical: 47.798094 KDa
SequenceString:
GGUAGGAUGG CAAGAUCCUG GUAGUCAUAG UUCCCCUGGA AACAGGGUUA CUAUGAUAAG GGCUUUCUGC CUAUAGGCAG ACUGACCCG UGGCGUUGGG GAUCGCCUAU CGCCCGCUUU CUUCGGGCAU UCCCCACUCU UAGGCGUUUU

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Macromolecule #2: Cleaved 3' target DNA strand

MacromoleculeName: Cleaved 3' target DNA strand / type: dna / ID: 2 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 7.883092 KDa
SequenceString:
(DC)(DA)(DG)(DG)(DA)(DT)(DC)(DT)(DT)(DG) (DC)(DC)(DA)(DT)(DC)(DC)(DT)(DA)(DC)(DC) (DT)(DC)(DT)(DA)(DG)(DA)

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Macromolecule #3: Cleaved 3' non-target DNA strand

MacromoleculeName: Cleaved 3' non-target DNA strand / type: dna / ID: 3 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 4.248793 KDa
SequenceString:
(DG)(DT)(DA)(DT)(DA)(DC)(DA)(DC)(DC)(DA) (DA)(DG)(DC)(DT)

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Macromolecule #4: Cleaved 5' target DNA strand

MacromoleculeName: Cleaved 5' target DNA strand / type: dna / ID: 4 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 4.310813 KDa
SequenceString:
(DA)(DG)(DC)(DT)(DT)(DG)(DG)(DT)(DG)(DT) (DA)(DT)(DA)(DC)

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Macromolecule #5: Cleaved 5' non-target DNA strand

MacromoleculeName: Cleaved 5' non-target DNA strand / type: dna / ID: 5 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 1.504037 KDa
SequenceString:
(DC)(DT)(DA)(DG)(DA)

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Macromolecule #6: CRISPR-associated endonuclease Cas9

MacromoleculeName: CRISPR-associated endonuclease Cas9 / type: protein_or_peptide / ID: 6 / Number of copies: 1 / Enantiomer: LEVO / EC number: Hydrolases; Acting on ester bonds
Source (natural)Organism: Geobacillus thermodenitrificans (bacteria)
Molecular weightTheoretical: 126.563461 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MKYKIGLDIG ITSIGWAVIN LDIPRIEDLG VRIFDRAENP KTGESLALPR RLARSARRRL RRRKHRLERI RRLFVREGIL TKEELNKLF EKKHEIDVWQ LRVEALDRKL NNDELARILL HLAKRRGFRS NRKSERTNKE NSTMLKHIEE NQSILSSYRT V AEMVVKDP ...String:
MKYKIGLDIG ITSIGWAVIN LDIPRIEDLG VRIFDRAENP KTGESLALPR RLARSARRRL RRRKHRLERI RRLFVREGIL TKEELNKLF EKKHEIDVWQ LRVEALDRKL NNDELARILL HLAKRRGFRS NRKSERTNKE NSTMLKHIEE NQSILSSYRT V AEMVVKDP KFSLHKRNKE DNYTNTVARD DLEREIKLIF AKQREYGNIV CTEAFEHEYI SIWASQRPFA SKDDIEKKVG FC TFEPKEK RAPKATYTFQ SFTVWEHINK LRLVSPGGIR ALTDDERRLI YKQAFHKNKI TFHDVRTLLN LPDDTRFKGL LYD RNTTLK ENEKVRFLEL GAYHKIRKAI DSVYGKGAAK SFRPIDFDTF GYALTMFKDD TDIRSYLRNE YEQNGKRMEN LADK VYDEE LIEELLNLSF SKFGHLSLKA LRNILPYMEQ GEVYSTACER AGYTFTGPKK KQKTVLLPNI PPIANPVVMR ALTQA RKVV NAIIKKYGSP VSIHIELARE LSQSFDERRK MQKEQEGNRK KNETAIRQLV EYGLTLNPTG LDIVKFKLWS EQNGKC AYS LQPIEIERLL EPGYTEVDHV IPYSRSLDDS YTNKVLVLTK ENREKGNRTP AEYLGLGSER WQQFETFVLT NKQFSKK KR DRLLRLHYDE NEENEFKNRN LNDTRYISRF LANFIREHLK FADSDDKQKV YTVNGRITAH LRSRWNFNKN REESNLHH A VDAAIVACTT PSDIARVTAF YQRREQNKEL SKKTDPQFPQ PWPHFADELQ ARLSKNPKES IKALNLGNYD NEKLESLQP VFVSRMPKRS ITGAAHQETL RRYIGIDERS GKIQTVVKKK LSEIQLDKTG HFPMYGKESD PRTYEAIRQR LLEHNNDPKK AFQEPLYKP KKNGELGPII RTIKIIDTTN QVIPLNDGKT VAYNSNIVRV DVFEKDGKYY CVPIYTIDMM KGILPNKAIE P NKPYSEWK EMTEDYTFRF SLYPNDLIRI EFPREKTIKT AVGEEIKIKD LFAYYQTIDS SNGGLSLVSH DNNFSLRSIG SR TLKRFEK YQVDVLGNIY KVRGEKRVGV ASSSHSKAGE TIRPL

UniProtKB: UNIPROTKB: A0A1W6VMQ3

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Macromolecule #7: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 7 / Number of copies: 6 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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Macromolecule #8: GUANOSINE-5'-TRIPHOSPHATE

MacromoleculeName: GUANOSINE-5'-TRIPHOSPHATE / type: ligand / ID: 8 / Number of copies: 1 / Formula: GTP
Molecular weightTheoretical: 523.18 Da
Chemical component information

ChemComp-GTP:
GUANOSINE-5'-TRIPHOSPHATE / GTP, energy-carrying molecule*YM

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Macromolecule #9: water

MacromoleculeName: water / type: ligand / ID: 9 / Number of copies: 21 / Formula: HOH
Molecular weightTheoretical: 18.015 Da
Chemical component information

ChemComp-HOH:
WATER

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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
Sugar embeddingMaterial: ice
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average electron dose: 60.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.0 µm / Nominal defocus min: 1.0 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: NONE
Final reconstructionResolution.type: BY AUTHOR / Resolution: 2.2 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 353229
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD

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