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- PDB-9kor: Cryo-EM structure of PsCas9-sgRNA-dsDNA ternary complex -

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Basic information

Entry
Database: PDB / ID: 9kor
TitleCryo-EM structure of PsCas9-sgRNA-dsDNA ternary complex
Components
  • CRISPR-associated endonuclease Cas9
  • DNA (25-MER)
  • DNA (25-mer)
  • RNA (131-MER)
KeywordsRNA BINDING PROTEIN/DNA/RNA / CRISPR-Cas9 / RNA / DNA / complex / RNA BINDING PROTEIN-DNA-RNA complex
Function / homologyDNA / DNA (> 10) / RNA / RNA (> 10) / RNA (> 100)
Function and homology information
Biological speciesParasutterella secunda (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.8 Å
AuthorsShen, P.P. / Lu, X.Y. / Huang, J.-W. / Chen, C.-C. / Guo, R.-T.
Funding support China, 1items
OrganizationGrant numberCountry
National Natural Science Foundation of China (NSFC) China
CitationJournal: Acs Catalysis / Year: 2025
Title: Structural and Mechanistic Investigations of a High-Fidelity Cas9 from Parasutterella secunda
Authors: Shen, P. / Lu, X. / Wang, C. / Huang, J.W. / Min, J. / Luo, J. / Sun, Y. / Chen, C.C. / Guo, R.T.
History
DepositionNov 21, 2024Deposition site: PDBJ / Processing site: PDBC
Revision 1.0Jul 30, 2025Provider: repository / Type: Initial release
Revision 1.0Jul 30, 2025Data content type: EM metadata / Data content type: EM metadata / Provider: repository / Type: Initial release
Revision 1.0Jul 30, 2025Data content type: FSC / Data content type: FSC / Provider: repository / Type: Initial release
Revision 1.0Jul 30, 2025Data content type: Half map / Part number: 1 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0Jul 30, 2025Data content type: Half map / Part number: 2 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0Jul 30, 2025Data content type: Image / Data content type: Image / Provider: repository / Type: Initial release
Revision 1.0Jul 30, 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

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Assembly

Deposited unit
A: CRISPR-associated endonuclease Cas9
B: RNA (131-MER)
C: DNA (25-mer)
D: DNA (25-MER)
hetero molecules


Theoretical massNumber of molelcules
Total (without water)222,7368
Polymers222,6394
Non-polymers974
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: not applicable
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein CRISPR-associated endonuclease Cas9


Mass: 165168.891 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Parasutterella secunda (bacteria) / Production host: Escherichia coli BL21(DE3) (bacteria)
#2: RNA chain RNA (131-MER)


Mass: 42108.891 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Parasutterella secunda (bacteria) / Production host: Escherichia coli BL21(DE3) (bacteria)
#3: DNA chain DNA (25-mer)


Mass: 7845.045 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Parasutterella secunda (bacteria) / Production host: Escherichia coli BL21(DE3) (bacteria)
#4: DNA chain DNA (25-MER)


Mass: 7515.838 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Parasutterella secunda (bacteria) / Production host: Escherichia coli BL21(DE3) (bacteria)
#5: Chemical
ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: Mg / Feature type: SUBJECT OF INVESTIGATION
Has ligand of interestY
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: type II CRISPR RNA-guided endonuclease Cas9 / Type: COMPLEX / Entity ID: #1-#4 / Source: RECOMBINANT
Molecular weightValue: 0.16 MDa / Experimental value: NO
Source (natural)Organism: Parasutterella secunda (bacteria)
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria)
Buffer solutionpH: 7.5
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: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 2000 nm / Nominal defocus min: 1000 nm
Image recordingElectron dose: 52 e/Å2 / Film or detector model: FEI FALCON IV (4k x 4k)

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Processing

EM softwareName: PHENIX / Category: model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 2.8 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 349205 / Symmetry type: POINT
RefinementHighest resolution: 2.8 Å
Stereochemistry target values: REAL-SPACE (WEIGHTED MAP SUM AT ATOM CENTERS)
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00313733
ELECTRON MICROSCOPYf_angle_d0.60119241
ELECTRON MICROSCOPYf_dihedral_angle_d22.5523396
ELECTRON MICROSCOPYf_chiral_restr0.0392236
ELECTRON MICROSCOPYf_plane_restr0.0051913

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