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- PDB-9ko9: Cryo-EM structure of PsCas9-sgRNA binary complex -

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Basic information

Entry
Database: PDB / ID: 9ko9
TitleCryo-EM structure of PsCas9-sgRNA binary complex
Components
  • CRISPR-associated endonuclease Cas9
  • RNA (131-MER)
KeywordsRNA BINDING PROTEIN/RNA / CRISPR-Cas9 / RNA / binary complex / RNA BINDING PROTEIN-RNA complex
Function / homologyRNA / RNA (> 10) / RNA (> 100)
Function and homology information
Biological speciesParasutterella secunda (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.7 Å
AuthorsShen, P.P. / Lu, X.Y. / Huang, J.-W. / Chen, C.-C. / Gut, 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 20, 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)


Theoretical massNumber of molelcules
Total (without water)207,3212
Polymers207,3212
Non-polymers00
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: 165211.922 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)
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: all / 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: 2.7 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 270781 / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT / Space: REAL
RefinementHighest resolution: 2.7 Å
Stereochemistry target values: REAL-SPACE (WEIGHTED MAP SUM AT ATOM CENTERS)
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00314343
ELECTRON MICROSCOPYf_angle_d0.59319896
ELECTRON MICROSCOPYf_dihedral_angle_d17.8763167
ELECTRON MICROSCOPYf_chiral_restr0.0372303
ELECTRON MICROSCOPYf_plane_restr0.0042127

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