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- PDB-8wmc: Cryo-EM structure of DiCas7-11-crRNA in complex with regulator -

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Basic information

Entry
Database: PDB / ID: 8wmc
TitleCryo-EM structure of DiCas7-11-crRNA in complex with regulator
Components
  • CHAT domain-containing protein
  • CRISPR-associated RAMP family protein
  • RNA (38-MER)
KeywordsIMMUNE SYSTEM / CRISPR-Cas / Gene editing / TPR-CHAT
Function / homology
Function and homology information


defense response to virus
Similarity search - Function
CHAT domain / CHAT domain / : / CRISPR type III-associated protein / RAMP superfamily
Similarity search - Domain/homology
RNA / RNA (> 10) / CRISPR-associated RAMP family protein / CHAT domain-containing protein
Similarity search - Component
Biological speciesDesulfonema ishimotonii (bacteria)
Escherichia coli (E. coli)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.9 Å
AuthorsMa, H.Y. / Tang, X.D.
Funding support China, 2items
OrganizationGrant numberCountry
Ministry of Science and Technology (MoST, China)81971974 China
Ministry of Science and Technology (MoST, China)32222040 China
CitationJournal: Signal Transduct Target Ther / Year: 2024
Title: Structural basis of negative regulation of CRISPR-Cas7-11 by TPR-CHAT.
Authors: Tian Hong / Qinghua Luo / Haiyun Ma / Xin Wang / Xinqiong Li / Chongrong Shen / Jie Pang / Yan Wang / Yuejia Chen / Changbin Zhang / Zhaoming Su / Haohao Dong / Xiaodi Tang /
Abstract: CRISPR‒Cas7-11 is a Type III-E CRISPR-associated nuclease that functions as a potent RNA editing tool. Tetratrico-peptide repeat fused with Cas/HEF1-associated signal transducer (TPR-CHAT) acts as ...CRISPR‒Cas7-11 is a Type III-E CRISPR-associated nuclease that functions as a potent RNA editing tool. Tetratrico-peptide repeat fused with Cas/HEF1-associated signal transducer (TPR-CHAT) acts as a regulatory protein that interacts with CRISPR RNA (crRNA)-bound Cas7-11 to form a CRISPR-guided caspase complex (Craspase). However, the precise modulation of Cas7-11's nuclease activity by TPR-CHAT to enhance its utility requires further study. Here, we report cryo-electron microscopy (cryo-EM) structures of Desulfonema ishimotonii (Di) Cas7-11-crRNA, complexed with or without the full length or the N-terminus of TPR-CHAT. These structures unveil the molecular features of the Craspase complex. Structural analysis, combined with in vitro nuclease assay and electrophoretic mobility shift assay, reveals that DiTPR-CHAT negatively regulates the activity of DiCas7-11 by preventing target RNA from binding through the N-terminal 65 amino acids of DiTPR-CHAT (DiTPR-CHAT). Our work demonstrates that DiTPR-CHAT can function as a small unit of DiCas7-11 regulator, potentially enabling safe applications to prevent overcutting and off-target effects of the CRISPR‒Cas7-11 system.
History
DepositionOct 3, 2023Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0May 29, 2024Provider: repository / Type: Initial release
Revision 1.0May 29, 2024Data content type: EM metadata / Data content type: EM metadata / Provider: repository / Type: Initial release
Revision 1.0May 29, 2024Data content type: FSC / Data content type: FSC / Provider: repository / Type: Initial release
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Revision 1.0May 29, 2024Data content type: Primary map / Data content type: Primary map / Provider: repository / Type: Initial release
Revision 1.1Jun 25, 2025Group: Data collection / Structure summary / Category: em_admin / em_software / pdbx_entry_details
Item: _em_admin.last_update / _em_software.name / _pdbx_entry_details.has_protein_modification
Revision 1.1Jun 25, 2025Data content type: EM metadata / Data content type: EM metadata / EM metadata / Group: Data processing / Experimental summary / Data content type: EM metadata / EM metadata / Category: em_admin / em_software / Data content type: EM metadata / EM metadata / Item: _em_admin.last_update / _em_software.name

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
B: CHAT domain-containing protein
A: CRISPR-associated RAMP family protein
R: RNA (38-MER)
hetero molecules


Theoretical massNumber of molelcules
Total (without water)284,6417
Polymers284,3793
Non-polymers2624
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 CHAT domain-containing protein


Mass: 88311.875 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Desulfonema ishimotonii (bacteria) / Gene: DENIS_1081 / Production host: Escherichia coli (E. coli) / References: UniProt: A0A401FT52
#2: Protein CRISPR-associated RAMP family protein


Mass: 183933.000 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Desulfonema ishimotonii (bacteria) / Gene: DENIS_1082 / Production host: Escherichia coli (E. coli) / References: UniProt: A0A401FT36
#3: RNA chain RNA (38-MER)


Mass: 12134.133 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (E. coli) / Production host: Escherichia coli (E. coli)
#4: Chemical
ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: Zn / Feature type: SUBJECT OF INVESTIGATION
Has ligand of interestY
Has protein modificationN

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: CELL / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: Ternary complex of Cas7-11_crRNA_TPR-CHAT / Type: COMPLEX / Entity ID: #2, #1, #3 / Source: RECOMBINANT
Source (natural)Organism: Escherichia coli (E. coli)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 8
SpecimenConc.: 0.8 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: NITROGEN

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 3500 nm / Nominal defocus min: 960 nm / Cs: 2.7 mm
Image recordingElectron dose: 53.7 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 QUANTUM (4k x 4k)

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Processing

EM softwareName: PHENIX / Category: model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 2.9 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 78984 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00317379
ELECTRON MICROSCOPYf_angle_d0.59223585
ELECTRON MICROSCOPYf_dihedral_angle_d7.1682625
ELECTRON MICROSCOPYf_chiral_restr0.042501
ELECTRON MICROSCOPYf_plane_restr0.0042936

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