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- PDB-7zol: Cryo-EM structure of a CRISPR effector in complex with regulator -

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Basic information

Entry
Database: PDB / ID: 7zol
TitleCryo-EM structure of a CRISPR effector in complex with regulator
Components
  • Cas7-11
  • TPR-CHAT
  • gRNAGuide RNA
KeywordsANTIVIRAL PROTEIN / CRISPR-cas / effector / regulator / CrRNA / guide RNA / antiphage
Function / homologyRNA / RNA (> 10)
Function and homology information
Biological speciesDesulfonema magnum (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.03 Å
AuthorsBabatunde, E.E. / Davide, T. / Bertrand, B. / Sergey, N. / Alexander, M. / Henning, S. / Dong, C.N.
Funding support Switzerland, 2items
OrganizationGrant numberCountry
Swiss National Science FoundationSNF Grants CRSII5_177195 Switzerland
Swiss National Science FoundationNCCR TransCure (185544) Switzerland
CitationJournal: Nat Struct Mol Biol / Year: 2023
Title: Structural insights into the regulation of Cas7-11 by TPR-CHAT.
Authors: Babatunde Ekundayo / Davide Torre / Bertrand Beckert / Sergey Nazarov / Alexander Myasnikov / Henning Stahlberg / Dongchun Ni /
Abstract: The CRISPR-guided caspase (Craspase) complex is an assembly of the target-specific RNA nuclease known as Cas7-11 bound to CRISPR RNA (crRNA) and an ancillary protein known as TPR-CHAT ...The CRISPR-guided caspase (Craspase) complex is an assembly of the target-specific RNA nuclease known as Cas7-11 bound to CRISPR RNA (crRNA) and an ancillary protein known as TPR-CHAT (tetratricopeptide repeats (TPR) fused with a CHAT domain). The Craspase complex holds promise as a tool for gene therapy and biomedical research, but its regulation is poorly understood. TPR-CHAT regulates Cas7-11 nuclease activity via an unknown mechanism. In the present study, we use cryoelectron microscopy to determine structures of the Desulfonema magnum (Dm) Craspase complex to gain mechanistic insights into its regulation. We show that DmTPR-CHAT stabilizes crRNA-bound DmCas7-11 in a closed conformation via a network of interactions mediated by the DmTPR-CHAT N-terminal domain, the DmCas7-11 insertion finger and Cas11-like domain, resulting in reduced target RNA accessibility and cleavage.
History
DepositionApr 26, 2022Deposition site: PDBE / Processing site: PDBE
Revision 1.0Nov 30, 2022Provider: repository / Type: Initial release
Revision 1.1Mar 1, 2023Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _citation_author.identifier_ORCID / _citation_author.name

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
B: Cas7-11
C: gRNA
A: TPR-CHAT
hetero molecules


Theoretical massNumber of molelcules
Total (without water)237,3707
Polymers237,1083
Non-polymers2624
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein Cas7-11


Mass: 207402.859 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Desulfonema magnum (bacteria) / Gene: cas7-11/gRAMP / Production host: Escherichia coli (E. coli)
#2: RNA chain gRNA / Guide RNA


Mass: 12344.251 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Desulfonema magnum (bacteria) / Production host: Escherichia coli (E. coli)
#3: Protein TPR-CHAT


Mass: 17361.045 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Desulfonema magnum (bacteria) / Gene: TPR-CHAT/ / 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
Has ligand of interestN

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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: Cas7-11 with TRP-CHAT-NTD / Type: COMPLEX / Entity ID: #1-#3 / Source: RECOMBINANT
Molecular weightValue: 0.22 MDa / Experimental value: YES
Source (natural)Organism: Desulfonema magnum (bacteria)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7.5
SpecimenConc.: 0.9 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid mesh size: 400 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 298 K

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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 FIELDBright-field microscopy / Nominal magnification: 96000 X / Nominal defocus max: 2500 nm / Nominal defocus min: 800 nm / Cs: 2.7 mm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 60 e/Å2 / Film or detector model: FEI FALCON IV (4k x 4k)

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Processing

SoftwareName: PHENIX / Version: 1.19.2_4158: / Classification: refinement
EM software
IDNameCategory
2EPUimage acquisition
4cryoSPARCCTF correction
7Cootmodel fitting
9cryoSPARCinitial Euler assignment
10cryoSPARCfinal Euler assignment
11cryoSPARCclassification
12cryoSPARC3D reconstruction
13PHENIXmodel refinement
CTF correctionType: NONE
Particle selectionNum. of particles selected: 1719283
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.03 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 53969 / Algorithm: BACK PROJECTION / Symmetry type: POINT
Atomic model buildingB value: 43 / Protocol: AB INITIO MODEL / Space: REAL
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00215828
ELECTRON MICROSCOPYf_angle_d0.52321533
ELECTRON MICROSCOPYf_dihedral_angle_d12.6412464
ELECTRON MICROSCOPYf_chiral_restr0.042390
ELECTRON MICROSCOPYf_plane_restr0.0052643

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