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- PDB-8eey: Cas7-11 in complex with DR-mismatched target RNA, Csx29 and Csx30 -

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Basic information

Entry
Database: PDB / ID: 8eey
TitleCas7-11 in complex with DR-mismatched target RNA, Csx29 and Csx30
Components
  • Cas7-11
  • Csx29
  • Csx30
  • DR-mismatched target RNA
  • crRNA
KeywordsRNA BINDING PROTEIN/RNA / CRISPR / endonuclease / endopeptidase / RNA BINDING 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) / CRISPR-associated RAMP family protein / Uncharacterized protein / CHAT domain-containing protein
Similarity search - Component
Biological speciesDesulfonema ishimotonii (bacteria)
synthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.53 Å
AuthorsDemircioglu, F.E. / Wilkinson, M.E. / Strecker, J. / Li, D. / Faure, G. / Macrae, R.K. / Zhang, F.
Funding support United States, 2items
OrganizationGrant numberCountry
National Institutes of Health/National Heart, Lung, and Blood Institute (NIH/NHLBI)1DP1-HL141201 United States
National Institutes of Health/National Human Genome Research Institute (NIH/NHGRI)2R01HG009761-05 United States
CitationJournal: Science / Year: 2022
Title: RNA-activated protein cleavage with a CRISPR-associated endopeptidase.
Authors: Jonathan Strecker / F Esra Demircioglu / David Li / Guilhem Faure / Max E Wilkinson / Jonathan S Gootenberg / Omar O Abudayyeh / Hiroshi Nishimasu / Rhiannon K Macrae / Feng Zhang /
Abstract: In prokaryotes, CRISPR-Cas systems provide adaptive immune responses against foreign genetic elements through RNA-guided nuclease activity. Recently, additional genes with non-nuclease functions have ...In prokaryotes, CRISPR-Cas systems provide adaptive immune responses against foreign genetic elements through RNA-guided nuclease activity. Recently, additional genes with non-nuclease functions have been found in genetic association with CRISPR systems, suggesting that there may be other RNA-guided non-nucleolytic enzymes. One such gene from encodes the TPR-CHAT protease Csx29, which is associated with the CRISPR effector Cas7-11. Here, we demonstrate that this CRISPR-associated protease (CASP) exhibits programmable RNA-activated endopeptidase activity against a sigma factor inhibitor to regulate a transcriptional response. Cryo-electron microscopy of an active and substrate-bound CASP complex reveals an allosteric activation mechanism that reorganizes Csx29 catalytic residues upon target RNA binding. This work reveals an RNA-guided function in nature that can be leveraged for RNA-sensing applications in vitro and in human cells.
History
DepositionSep 7, 2022Deposition site: RCSB / Processing site: RCSB
Revision 1.0Nov 16, 2022Provider: repository / Type: Initial release
Revision 1.1Dec 7, 2022Group: Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_PubMed / _citation.title
Revision 1.2Jun 19, 2024Group: Data collection / Category: chem_comp_atom / chem_comp_bond

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
E: Csx30
A: Cas7-11
B: Csx29
C: crRNA
D: DR-mismatched target RNA
hetero molecules


Theoretical massNumber of molelcules
Total (without water)356,1729
Polymers355,9115
Non-polymers2624
Water00
1


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

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Components

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Protein , 3 types, 3 molecules EAB

#1: Protein Csx30


Mass: 62688.184 Da / Num. of mol.: 1
Mutation: 427-MKKDKKA-433 replaced with 427-GGS-429. A gap exists because low-resolution density in EM map permitted modeling of only a single residue of the insertion, G at position 427.
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Desulfonema ishimotonii (bacteria) / Gene: DENIS_1080 / Production host: Escherichia coli (E. coli) / References: UniProt: A0A401FT41
#2: Protein Cas7-11


Mass: 183844.984 Da / Num. of mol.: 1 / Mutation: D429A,D654A
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: Protein Csx29


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

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RNA chain , 2 types, 2 molecules CD

#4: RNA chain crRNA


Mass: 12134.133 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Desulfonema ishimotonii (bacteria) / Production host: Escherichia coli (E. coli)
#5: RNA chain DR-mismatched target RNA


Mass: 8931.381 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)

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Non-polymers , 1 types, 4 molecules

#6: Chemical
ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: Zn

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Details

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 in complex with DR-mismatched target RNA, Csx29 and Csx30
Type: COMPLEX / Entity ID: #1-#5 / Source: MULTIPLE SOURCES
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
21Desulfonema ishimotonii (bacteria)45657
31synthetic construct (others)32630
Source (recombinant)Organism: Escherichia coli (E. coli)
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: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 2000 nm / Nominal defocus min: 500 nm / Cs: 2.7 mm
Image recordingElectron dose: 40 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)

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Processing

CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 2.53 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 65733 / Symmetry type: POINT

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