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- PDB-21ea: Cryo-EM structure of AtCas9-sgRNA-underwound DNA (TATA PAM) terna... -

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Basic information

Entry
Database: PDB / ID: 21ea
TitleCryo-EM structure of AtCas9-sgRNA-underwound DNA (TATA PAM) 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: 2.65 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: Min Duan / Bing Meng / Lei Zhou / Lijie Wu / Xiaohan Tong / Dongchao Huang / Hao Yin / Zhi-Jie Liu / Ying Zhang /
Abstract: The CRISPR-Cas9 system locates targets through guide RNA pairing and recognition of a protospacer-adjacent motif (PAM). Although PAM specificity is sequence-determined, DNA topology can relax PAM ...The CRISPR-Cas9 system locates targets through guide RNA pairing and recognition of a protospacer-adjacent motif (PAM). Although PAM specificity is sequence-determined, DNA topology can relax PAM requirements and enable near-PAMless cleavage by the type II-C Alicyclobacillus tengchongensis Cas9 (AtCas9). However, the structural mechanism underlying this regulation remains unknown. Here we report cryogenic-electron microscopy (cryo-EM) structures of AtCas9 bound to B-form DNA or a 340 bp underwound minicircle DNA containing wild-type or mutant PAMs. Despite PAM sequences differences, all three underwound complexes adopt an almost identical architecture distinct from the B-form DNA-bound state. On B-form DNA, AtCas9 recognizes the PAM through base-specific hydrogen bonds and steric exclusion, conferring preference for NCNNN and NRNNA (R = A/G). By contrast, underwound DNA widens the PAM major groove and promotes sequence-independent backbone contacts, explaining the near-PAMless cleavage. These findings uncover a topology-dependent mechanism of PAM recognition and establish a cryo-EM platform using underwound minicircle DNA for structural studies under native-like topological states.
History
DepositionDec 9, 2025Deposition site: PDBJ / Processing site: PDBC
Revision 1.0Jul 1, 2026Provider: repository / Type: Initial release
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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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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)191,1654
Polymers191,1654
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: 10894.997 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Alicyclobacillus tolerans (bacteria)
#4: DNA chain NTS DNA


Mass: 10637.904 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 (TATA PAM) ternary complex
Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Source (natural)Organism: Alicyclobacillus tolerans (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
2PHENIX1.20.1_4487model refinement
13cryoSPARC4.73D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 2.65 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 457333 / Symmetry type: POINT
RefinementStereochemistry target values: REAL-SPACE (WEIGHTED MAP SUM AT ATOM CENTERS)
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00410220
ELECTRON MICROSCOPYf_angle_d0.60514449
ELECTRON MICROSCOPYf_dihedral_angle_d16.2732566
ELECTRON MICROSCOPYf_chiral_restr0.0391704
ELECTRON MICROSCOPYf_plane_restr0.0051319

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