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- PDB-7zoq: Cryo-EM structure of a CRISPR effector in complex with a caspase ... -

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Basic information

Entry
Database: PDB / ID: 7zoq
TitleCryo-EM structure of a CRISPR effector in complex with a caspase regulator
Components
  • RNA (39-MER)
  • TPR-CHAT
  • cas7-11
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: 3.2 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
Revision 1.2Jul 24, 2024Group: Data collection / Category: chem_comp_atom / chem_comp_bond / em_admin / Item: _em_admin.last_update

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: TPR-CHAT
B: cas7-11
C: RNA (39-MER)
hetero molecules


Theoretical massNumber of molelcules
Total (without water)315,3967
Polymers315,1343
Non-polymers2624
Water00
1


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

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Components

#1: Protein TPR-CHAT


Mass: 95386.820 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)
#2: 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) / Production host: Escherichia coli (E. coli)
#3: RNA chain RNA (39-MER)


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)
#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-full / Type: COMPLEX / Entity ID: #1-#3 / Source: RECOMBINANT
Molecular weightValue: 0.31 MDa / Experimental value: YES
Source (natural)Organism: Desulfonema magnum (bacteria)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7.5
SpecimenConc.: 1.2 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 FIELD / 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
8UCSF Chimeramodel fitting
10cryoSPARCinitial Euler assignment
11cryoSPARCfinal Euler assignment
12cryoSPARCclassification
13cryoSPARC3D reconstruction
14PHENIXmodel refinement
CTF correctionType: NONE
Particle selectionNum. of particles selected: 1719283
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.2 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 65755 / 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_d0.00221496
ELECTRON MICROSCOPYf_angle_d0.50229182
ELECTRON MICROSCOPYf_dihedral_angle_d10.5443241
ELECTRON MICROSCOPYf_chiral_restr0.0383191
ELECTRON MICROSCOPYf_plane_restr0.0043602

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