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- PDB-7y83: CryoEM structure of type III-E CRISPR Craspase gRAMP-crRNA in com... -

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Basic information

Entry
Database: PDB / ID: 7y83
TitleCryoEM structure of type III-E CRISPR Craspase gRAMP-crRNA in complex with TPR-CHAT protease bound to non-self RNA target
Components
  • CHAT domain protein
  • RAMP superfamily protein
  • crRNA
  • non-self RNA
KeywordsSTRUCTURAL PROTEIN/RNA / Nuclease / STRUCTURAL PROTEIN-RNA COMPLEX
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) / RNA (> 100) / RAMP superfamily protein / CHAT domain protein
Similarity search - Component
Biological speciesCandidatus Scalindua brodae (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.93 Å
AuthorsZhang, J.T. / Cui, N. / Huang, H.D. / Jia, N.
Funding support1items
OrganizationGrant numberCountry
Not funded
CitationJournal: Nat Commun / Year: 2022
Title: Structural basis for the non-self RNA-activated protease activity of the type III-E CRISPR nuclease-protease Craspase.
Authors: Ning Cui / Jun-Tao Zhang / Zhuolin Li / Xiao-Yu Liu / Chongyuan Wang / Hongda Huang / Ning Jia /
Abstract: The RNA-targeting type III-E CRISPR-gRAMP effector interacts with a caspase-like protease TPR-CHAT to form the CRISPR-guided caspase complex (Craspase), but their functional mechanism is unknown. ...The RNA-targeting type III-E CRISPR-gRAMP effector interacts with a caspase-like protease TPR-CHAT to form the CRISPR-guided caspase complex (Craspase), but their functional mechanism is unknown. Here, we report cryo-EM structures of the type III-E gRAMP and gRAMP-TPR-CHAT complexes, before and after either self or non-self RNA target binding, and elucidate the mechanisms underlying RNA-targeting and non-self RNA-induced protease activation. The associated TPR-CHAT adopted a distinct conformation upon self versus non-self RNA target binding, with nucleotides at positions -1 and -2 of the CRISPR-derived RNA (crRNA) serving as a sensor. Only binding of the non-self RNA target activated the TPR-CHAT protease, leading to cleavage of Csx30 protein. Furthermore, TPR-CHAT structurally resembled eukaryotic separase, but with a distinct mechanism for protease regulation. Our findings should facilitate the development of gRAMP-based RNA manipulation tools, and advance our understanding of the virus-host discrimination process governed by a nuclease-protease Craspase during type III-E CRISPR-Cas immunity.
History
DepositionJun 22, 2022Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Dec 14, 2022Provider: repository / Type: Initial release
Revision 1.1May 8, 2024Group: Data collection / Database references / Category: chem_comp_atom / chem_comp_bond / citation
Item: _citation.page_last / _citation.pdbx_database_id_PubMed / _citation.title

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: RAMP superfamily protein
B: crRNA
C: non-self RNA
D: CHAT domain protein
hetero molecules


Theoretical massNumber of molelcules
Total (without water)337,9929
Polymers337,7064
Non-polymers2865
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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Protein , 2 types, 2 molecules AD

#1: Protein RAMP superfamily protein


Mass: 198652.688 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Candidatus Scalindua brodae (bacteria) / Gene: SCABRO_02597 / Production host: Escherichia coli (E. coli) / References: UniProt: A0A0B0EGF3
#4: Protein CHAT domain protein


Mass: 85523.141 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Candidatus Scalindua brodae (bacteria) / Gene: SCABRO_02601 / Production host: Escherichia coli (E. coli) / References: UniProt: A0A0B0EKL4

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RNA chain , 2 types, 2 molecules BC

#2: RNA chain crRNA


Mass: 35432.090 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Candidatus Scalindua brodae (bacteria) / Production host: Escherichia coli (E. coli)
#3: RNA chain non-self RNA


Mass: 18097.797 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Candidatus Scalindua brodae (bacteria)

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Non-polymers , 2 types, 5 molecules

#5: Chemical
ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: Zn / Feature type: SUBJECT OF INVESTIGATION
#6: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Mg / Feature type: SUBJECT OF INVESTIGATION

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Details

Has ligand of interestY

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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

Component
IDNameTypeEntity IDParent-IDSource
1Type III-E CRISPR gRAMP-crRNA binary complexCOMPLEX#1-#40MULTIPLE SOURCES
2gRAMPCOMPLEX#11RECOMBINANT
3crRNACOMPLEX#21RECOMBINANT
4Non-self RNA targetCOMPLEX#31SYNTHETIC
5TPR-CHATCOMPLEX#41RECOMBINANT
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
21Candidatus Scalindua brodae (bacteria)237368
32Candidatus Scalindua brodae (bacteria)237368
44Candidatus Scalindua brodae (bacteria)237368
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
21Escherichia coli (E. coli)562
32Escherichia coli (E. coli)562
44Escherichia coli (E. coli)562
Buffer solutionpH: 8
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: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: SPOT SCAN
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 2500 nm / Nominal defocus min: 1500 nm
Image recordingElectron dose: 50 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

CTF correctionType: PHASE FLIPPING ONLY
3D reconstructionResolution: 2.93 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 53439 / Symmetry type: POINT

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