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- PDB-8zda: Cryo-EM structure of eSaCas9_NNG-guide RNA-target DNA complex in ... -

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Basic information

Entry
Database: PDB / ID: 8zda
TitleCryo-EM structure of eSaCas9_NNG-guide RNA-target DNA complex in a catalytically active state
Components
  • CRISPR-associated endonuclease Cas9
  • Non-target DNA strand
  • Target DNA strand1
  • Target DNA strand2
  • sgRNA
KeywordsDNA BINDING PROTEIN / CRISPR-CAS9 / GENOME ENGINEERING / 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
: / CRISPR-associated endonuclease Cas9, C-terminal domain / Cas9, PI domain / Cas9, WED domain / CRISPR-Cas9 WED domain / CRISPR-Cas9 PI domain / : / Cas9 RuvC domain / HNH endonuclease / CRISPR-associated endonuclease Cas9 ...: / CRISPR-associated endonuclease Cas9, C-terminal domain / Cas9, PI domain / Cas9, WED domain / CRISPR-Cas9 WED domain / CRISPR-Cas9 PI domain / : / Cas9 RuvC domain / HNH endonuclease / CRISPR-associated endonuclease Cas9 / Cas9-type HNH domain / Cas9-type HNH domain profile. / HNH nuclease / Ribonuclease H superfamily
Similarity search - Domain/homology
DNA / DNA (> 10) / RNA / RNA (> 10) / CRISPR-associated endonuclease Cas9
Similarity search - Component
Biological speciesStaphylococcus aureus (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.14 Å
AuthorsOmura, S.N. / Nakagawa, R. / Yamashita, K. / Nishimasu, H. / Nureki, O.
Funding support Japan, 2items
OrganizationGrant numberCountry
Japan Agency for Medical Research and Development (AMED) Japan
Japan Society for the Promotion of Science (JSPS) Japan
CitationJournal: To Be Published
Title: Molecular engineering and structural mechanism of the compact CRISPR-Cas9 nuclease
Authors: Omura, S.N. / Nakagawa, R. / Kajimoto, S. / Okazaki, S. / Ishiguro, S. / Mori, H. / Kashiwakura, Y. / Hiramoto, T. / Hirano, H. / Yamashita, K. / Jividen, K. / Tsai, S.Q. / Yachie, N. / ...Authors: Omura, S.N. / Nakagawa, R. / Kajimoto, S. / Okazaki, S. / Ishiguro, S. / Mori, H. / Kashiwakura, Y. / Hiramoto, T. / Hirano, H. / Yamashita, K. / Jividen, K. / Tsai, S.Q. / Yachie, N. / Ohmori, T. / Nishimasu, H. / Nureki, O.
History
DepositionMay 1, 2024Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Nov 5, 2025Provider: repository / Type: Initial release
Revision 1.0Nov 5, 2025Data content type: EM metadata / Data content type: EM metadata / Provider: repository / Type: Initial release
Revision 1.0Nov 5, 2025Data content type: FSC / Data content type: FSC / Provider: repository / Type: Initial release
Revision 1.0Nov 5, 2025Data content type: Half map / Part number: 1 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0Nov 5, 2025Data content type: Half map / Part number: 2 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0Nov 5, 2025Data content type: Image / Data content type: Image / Provider: repository / Type: Initial release
Revision 1.0Nov 5, 2025Data content type: Mask / Part number: 1 / Data content type: Mask / Provider: repository / Type: Initial release
Revision 1.0Nov 5, 2025Data content type: Primary map / Data content type: Primary map / Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: CRISPR-associated endonuclease Cas9
B: sgRNA
C: Target DNA strand1
D: Non-target DNA strand
E: Target DNA strand2
hetero molecules


Theoretical massNumber of molelcules
Total (without water)182,9288
Polymers182,8555
Non-polymers733
Water181
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

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DNA chain , 3 types, 3 molecules CDE

#3: DNA chain Target DNA strand1


Mass: 5267.556 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Staphylococcus aureus (bacteria) / Production host: Escherichia coli (E. coli)
#4: DNA chain Non-target DNA strand


Mass: 13386.006 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Staphylococcus aureus (bacteria) / Production host: Escherichia coli (E. coli)
#5: DNA chain Target DNA strand2


Mass: 8002.456 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Staphylococcus aureus (bacteria) / Production host: Escherichia coli (E. coli)

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Protein / RNA chain , 2 types, 2 molecules AB

#1: Protein CRISPR-associated endonuclease Cas9 / SaCas9


Mass: 124305.898 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Staphylococcus aureus (bacteria) / Gene: cas9 / Production host: Escherichia coli (E. coli)
References: UniProt: J7RUA5, Hydrolases; Acting on ester bonds
#2: RNA chain sgRNA


Mass: 31892.889 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Staphylococcus aureus (bacteria) / Production host: Escherichia coli (E. coli)

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Non-polymers , 2 types, 4 molecules

#6: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: Mg
#7: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Formula: H2O

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Details

Has protein modificationN

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: eSaCas9-NNG-sgRNA-target DNA ternary complex in a catalytically active state
Type: COMPLEX / Entity ID: #1-#5 / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Staphylococcus aureus (bacteria)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7.6
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: TFS KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: OTHER
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 1600 nm / Nominal defocus min: 800 nm
Image recordingElectron dose: 50 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

EM softwareName: Servalcat / Version: 0.4.68 / Category: model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.14 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 59818 / Symmetry type: POINT

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