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- PDB-9k4h: SuperFi Cas9 - mismatch 22nt sgRNA - DNA ternary complex class3 -

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Basic information

Entry
Database: PDB / ID: 9k4h
TitleSuperFi Cas9 - mismatch 22nt sgRNA - DNA ternary complex class3
Components
  • (DNA (50-MER)) x 2
  • CRISPR-associated endonuclease Cas9/Csn1
  • RNA (102-MER)
KeywordsIMMUNE SYSTEM/DNA/RNA / Complex / cas / SuperFi cas9 / IMMUNE SYSTEM / IMMUNE SYSTEM-DNA-RNA complex
Function / homology
Function and homology information


maintenance of CRISPR repeat elements / 3'-5' exonuclease activity / DNA 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, bridge helix / Bridge helix of CRISPR-associated endonuclease Cas9 / CRISPR-associated endonuclease Cas9, PAM-interacting domain / CRISPR-associated endonuclease Cas9, REC lobe / REC lobe of CRISPR-associated endonuclease Cas9 / PAM-interacting domain of CRISPR-associated endonuclease Cas9 / : / Cas9 RuvC domain / HNH endonuclease / CRISPR-associated endonuclease Cas9 ...CRISPR-associated endonuclease Cas9, bridge helix / Bridge helix of CRISPR-associated endonuclease Cas9 / CRISPR-associated endonuclease Cas9, PAM-interacting domain / CRISPR-associated endonuclease Cas9, REC lobe / REC lobe of CRISPR-associated endonuclease Cas9 / PAM-interacting domain of CRISPR-associated endonuclease Cas9 / : / 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) / RNA (> 100) / CRISPR-associated endonuclease Cas9/Csn1
Similarity search - Component
Biological speciesStreptococcus pyogenes (bacteria)
synthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.36 Å
AuthorsZheng, R. / Ma, L.J.
Funding support1items
OrganizationGrant numberCountry
Not funded
CitationJournal: To Be Published
Title: 5' extended gRNA increases the cleavage activity of SuperFi-Cas9
Authors: Zheng, R. / Ma, L.J.
History
DepositionOct 21, 2024Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Oct 22, 2025Provider: repository / Type: Initial release
Revision 1.0Oct 22, 2025Data content type: EM metadata / Data content type: EM metadata / Provider: repository / Type: Initial release
Revision 1.0Oct 22, 2025Data content type: FSC / Data content type: FSC / Provider: repository / Type: Initial release
Revision 1.0Oct 22, 2025Data content type: Half map / Part number: 1 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0Oct 22, 2025Data content type: Half map / Part number: 2 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0Oct 22, 2025Data content type: Image / Data content type: Image / Provider: repository / Type: Initial release
Revision 1.0Oct 22, 2025Data content type: Mask / Part number: 1 / Data content type: Mask / Provider: repository / Type: Initial release
Revision 1.0Oct 22, 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/Csn1
B: DNA (50-MER)
C: DNA (50-MER)
D: RNA (102-MER)


Theoretical massNumber of molelcules
Total (without water)222,0894
Polymers222,0894
Non-polymers00
Water00
1


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

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Components

#1: Protein CRISPR-associated endonuclease Cas9/Csn1 / SpCas9 / SpyCas9


Mass: 158502.375 Da / Num. of mol.: 1
Mutation: Y1010D, Y1013D, Y1016D, V1018D, R1019D, Q1027D, K1031D
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Streptococcus pyogenes (bacteria) / Gene: cas9, csn1, SPy_1046 / Production host: Escherichia coli (E. coli)
References: UniProt: Q99ZW2, Hydrolases; Acting on ester bonds
#2: DNA chain DNA (50-MER)


Mass: 15438.878 Da / Num. of mol.: 1 / Source method: obtained synthetically / Details: Target Strand / Source: (synth.) synthetic construct (others)
#3: DNA chain DNA (50-MER)


Mass: 15371.812 Da / Num. of mol.: 1 / Source method: obtained synthetically / Details: None-Target Strand / Source: (synth.) synthetic construct (others)
#4: RNA chain RNA (102-MER)


Mass: 32775.438 Da / Num. of mol.: 1 / Source method: obtained synthetically / Details: sgRNA / Source: (synth.) synthetic construct (others)
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: SuperFi Cas9 - mismatch 22nt sgRNA - DNA ternary complex classC
Type: COMPLEX / Entity ID: all / Source: MULTIPLE SOURCES
Source (natural)Organism: Streptococcus pyogenes (bacteria)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7.4
Buffer component
IDConc.NameFormulaBuffer-ID
1150 mMNaClNaCl1
220 mMHEPES pH7.4C8H18N2O4S1
310 mMMgCl2MgCl21
40.5 mMTCEPC9H15O6P1
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: OTHER / Nominal defocus max: 2500 nm / Nominal defocus min: 1500 nm
Image recording
IDImaging-IDElectron dose (e/Å2)Film or detector model
111.572FEI FALCON IV (4k x 4k)
211.571FEI FALCON IV (4k x 4k)

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Processing

EM software
IDNameCategory
1cryoSPARCparticle selection
13EPU3D reconstruction
CTF correctionType: NONE
3D reconstructionResolution: 3.36 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 211388 / Symmetry type: POINT
RefinementHighest resolution: 3.36 Å
Stereochemistry target values: REAL-SPACE (WEIGHTED MAP SUM AT ATOM CENTERS)
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00312548
ELECTRON MICROSCOPYf_angle_d0.52717537
ELECTRON MICROSCOPYf_dihedral_angle_d18.3553000
ELECTRON MICROSCOPYf_chiral_restr0.0362023
ELECTRON MICROSCOPYf_plane_restr0.0031743

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