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- PDB-9wac: Cryo-EM structure of AtCas9-sgRNA-underwound DNA (TTGA) ternary c... -

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Basic information

Entry
Database: PDB / ID: 9wac
TitleCryo-EM structure of AtCas9-sgRNA-underwound DNA (TTGA) ternary complex
Components
  • CRISPR-associated endonuclease Cas9
  • NTS DNA
  • TS DNA
  • sgRNA
KeywordsRNA BINDING PROTEIN/RNA/DNA / Complex / Endonuclease / Immunity / RNA BINDING PROTEIN-RNA-DNA complex
Function / homologyDNA / DNA (> 10) / RNA / RNA (> 10) / RNA (> 100)
Function and homology information
Biological speciesAlicyclobacillus tolerans (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.89 Å
AuthorsMeng, B. / Duan, M. / Wu, L.J. / Liu, Z.J. / Zhang, Y.
Funding support China, 1items
OrganizationGrant numberCountry
National Natural Science Foundation of China (NSFC) China
CitationJournal: Nat.Struct.Mol.Biol. / Year: 2026
Title: Structural basis of AtCas9 recognition of PAM mutants in underwound DNA topology
Authors: Duan, M. / Meng, B. / Zhou, L. / Wu, L.J. / Tong, X. / Huang, D. / Yin, H. / Liu, Z.J. / Zhang, Y.
History
DepositionAug 11, 2025Deposition site: PDBJ / Processing site: PDBC
Revision 1.0Jul 1, 2026Provider: repository / Type: Initial release
Revision 1.0Jul 1, 2026Data content type: EM metadata / Data content type: EM metadata / Provider: repository / Type: Initial release
Revision 1.0Jul 1, 2026Data content type: FSC / Data content type: FSC / Provider: repository / Type: Initial release
Revision 1.0Jul 1, 2026Data content type: Half map / Part number: 1 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0Jul 1, 2026Data content type: Half map / Part number: 2 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0Jul 1, 2026Data content type: Image / Data content type: Image / Provider: repository / Type: Initial release
Revision 1.0Jul 1, 2026Data 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: sgRNA
C: TS DNA
D: NTS DNA


Theoretical massNumber of molelcules
Total (without water)189,3114
Polymers189,3114
Non-polymers00
Water00
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

#1: Protein CRISPR-associated endonuclease Cas9


Mass: 132207.750 Da / Num. of mol.: 1 / Mutation: D8A, H617A, N640A
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Alicyclobacillus tolerans (bacteria) / Production host: Escherichia coli (E. coli)
#2: RNA chain sgRNA


Mass: 37424.258 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Alicyclobacillus tolerans (bacteria)
#3: DNA chain TS DNA


Mass: 9941.408 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Alicyclobacillus tolerans (bacteria)
#4: DNA chain NTS DNA


Mass: 9737.320 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Alicyclobacillus tolerans (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: AtCas9-sgRNA-underwound DNA (TTGA PAM) ternary complex
Type: COMPLEX / Entity ID: all / Source: MULTIPLE SOURCES
Source (natural)Organism: Alicyclobacillus tengchongensis (bacteria)
Source (recombinant)Organism: Escherichia coli (E. coli)
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: 1800 nm / Nominal defocus min: 1400 nm
Image recordingElectron dose: 50 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)

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Processing

EM software
IDNameVersionCategory
1cryoSPARC4.7particle selection
12cryoSPARC4.73D reconstruction
13PHENIX1.20.1_4487model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 2.89 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 1023398 / Symmetry type: POINT
RefinementStereochemistry target values: REAL-SPACE (WEIGHTED MAP SUM AT ATOM CENTERS)
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0049952
ELECTRON MICROSCOPYf_angle_d0.60814057
ELECTRON MICROSCOPYf_dihedral_angle_d15.5982480
ELECTRON MICROSCOPYf_chiral_restr0.0361657
ELECTRON MICROSCOPYf_plane_restr0.0041298

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