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- PDB-9wdx: Structure of SpCas9 with engineered disulfide bonds (R1210C/V1280... -

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Basic information

Entry
Database: PDB / ID: 9wdx
TitleStructure of SpCas9 with engineered disulfide bonds (R1210C/V1280C and L1119C/P1128C)
ComponentsCRISPR-associated endonuclease Cas9/Csn1
KeywordsHYDROLASE / endonuclease / disulfide engineering / thermostability
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
CRISPR-associated endonuclease Cas9/Csn1
Similarity search - Component
Biological speciesStreptococcus pyogenes serotype M1 (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.72 Å
AuthorsTeo, Z.T. / Ravi, L.R.V. / Jia, X. / Xue, B. / Qu, K. / Yew, W.S.
Funding support Singapore, 3items
OrganizationGrant numberCountry
Other privateI1901E0040 Singapore
Other privateI2301E0021 Singapore
Other governmentNUHSRO/2022/033/Startup/03 Singapore
CitationJournal: To Be Published
Title: Engineering Enhanced Thermostability and High-Temperature Activity in SpCas9 through Strategic C-Terminal Domain Disulfide Bond Incorporation
Authors: Teo, Z.T. / Hartono, Y.D. / Xue, B. / Jia, X. / Ravi, L.R.V. / Qu, K. / Carstens, C.-P. / Bruhn, L. / Yew, W.S.
History
DepositionAug 19, 2025Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Aug 26, 2026Provider: repository / Type: Initial release
Revision 1.0Aug 26, 2026Data content type: EM metadata / Data content type: EM metadata / Provider: repository / Type: Initial release
Revision 1.0Aug 26, 2026Data content type: FSC / Data content type: FSC / Provider: repository / Type: Initial release
Revision 1.0Aug 26, 2026Data content type: Half map / Part number: 1 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0Aug 26, 2026Data content type: Half map / Part number: 2 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0Aug 26, 2026Data content type: Image / Data content type: Image / Provider: repository / Type: Initial release
Revision 1.0Aug 26, 2026Data content type: Mask / Part number: 1 / Data content type: Mask / Provider: repository / Type: Initial release
Revision 1.0Aug 26, 2026Data content type: Primary map / Data content type: Primary map / Provider: repository / Type: Initial release

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Structure visualization

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Assembly

Deposited unit
P: CRISPR-associated endonuclease Cas9/Csn1


Theoretical massNumber of molelcules
Total (without water)159,9191
Polymers159,9191
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: 159919.156 Da / Num. of mol.: 1 / Mutation: R1210C/V1280C/L1119C/P1128C
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Streptococcus pyogenes serotype M1 (bacteria)
Gene: cas9, csn1, SPy_1046 / Production host: Escherichia coli K-12 (bacteria)
References: UniProt: Q99ZW2, Hydrolases; Acting on ester bonds
Has protein modificationY

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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: SpCas9 with engineered disulfide bonds (R1210C/V1280C and L1119C/P1128C)
Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Streptococcus pyogenes serotype M1 (bacteria)
Source (recombinant)Organism: Escherichia coli K-12 (bacteria)
Buffer solutionpH: 8
SpecimenConc.: 1 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: C-flat-2/1
VitrificationInstrument: LEICA EM GP / Cryogen name: ETHANE / Humidity: 90 % / Chamber temperature: 288 K

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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 magnification: 105000 X / Nominal defocus max: 2400 nm / Nominal defocus min: 1800 nm / Cs: 2.7 mm / C2 aperture diameter: 70 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 53 e/Å2 / Film or detector model: GATAN K3 BIOCONTINUUM (6k x 4k)

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Processing

EM software
IDNameVersionCategory
1cryoSPARC4.5.3particle selection
2SerialEM4.0.6image acquisition
4cryoSPARC4.5.3CTF correction
7UCSF Chimera1.17.3model fitting
9PHENIX1.21.1_5286model refinement
10cryoSPARC4.5.3initial Euler assignment
11cryoSPARC4.5.3final Euler assignment
12cryoSPARC4.5.3classification
13cryoSPARC4.5.33D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 3618234
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.72 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 610131 / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL
Atomic model buildingPDB-ID: 7S3H

7s3h


Accession code: 7S3H / Source name: PDB / Type: experimental model
RefinementHighest resolution: 3.72 Å
Stereochemistry target values: REAL-SPACE (WEIGHTED MAP SUM AT ATOM CENTERS)
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0028510
ELECTRON MICROSCOPYf_angle_d0.44211435
ELECTRON MICROSCOPYf_dihedral_angle_d12.0833299
ELECTRON MICROSCOPYf_chiral_restr0.0391273
ELECTRON MICROSCOPYf_plane_restr0.0031465

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