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- PDB-8gs2: Structure of the Cas7-11-Csx29-guide RNA-target RNA (non-matching... -

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Basic information

Entry
Database: PDB / ID: 8gs2
TitleStructure of the Cas7-11-Csx29-guide RNA-target RNA (non-matching PFS) complex
Components
  • CHAT domain-containing protein
  • CRISPR-associated RAMP family protein
  • crRNA
  • target RNA with non-matching PFS
KeywordsRNA BINDING PROTEIN/RNA / CRISPR / RNase / 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
ADENOSINE MONOPHOSPHATE / CYTIDINE-5'-MONOPHOSPHATE / RNA / RNA (> 10) / CRISPR-associated RAMP family protein / CHAT domain-containing protein
Similarity search - Component
Biological speciesDesulfonema ishimotonii (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.84 Å
AuthorsKato, K. / Okazaki, S. / Ishikawa, J. / Isayama, Y. / Nishizawa, T. / Nishimasu, H.
Funding support Japan, 1items
OrganizationGrant numberCountry
Japan Agency for Medical Research and Development (AMED) Japan
CitationJournal: Science / Year: 2022
Title: RNA-triggered protein cleavage and cell growth arrest by the type III-E CRISPR nuclease-protease.
Authors: Kazuki Kato / Sae Okazaki / Cian Schmitt-Ulms / Kaiyi Jiang / Wenyuan Zhou / Junichiro Ishikawa / Yukari Isayama / Shungo Adachi / Tomohiro Nishizawa / Kira S Makarova / Eugene V Koonin / ...Authors: Kazuki Kato / Sae Okazaki / Cian Schmitt-Ulms / Kaiyi Jiang / Wenyuan Zhou / Junichiro Ishikawa / Yukari Isayama / Shungo Adachi / Tomohiro Nishizawa / Kira S Makarova / Eugene V Koonin / Omar O Abudayyeh / Jonathan S Gootenberg / Hiroshi Nishimasu /
Abstract: The type III-E CRISPR-Cas7-11 effector binds a CRISPR RNA (crRNA) and the putative protease Csx29 and catalyzes crRNA-guided RNA cleavage. We report cryo-electron microscopy structures of the Cas7-11- ...The type III-E CRISPR-Cas7-11 effector binds a CRISPR RNA (crRNA) and the putative protease Csx29 and catalyzes crRNA-guided RNA cleavage. We report cryo-electron microscopy structures of the Cas7-11-crRNA-Csx29 complex with and without target RNA (tgRNA), and demonstrate that tgRNA binding induces conformational changes in Csx29. Biochemical experiments revealed tgRNA-dependent cleavage of the accessory protein Csx30 by Csx29. Reconstitution of the system in bacteria showed that Csx30 cleavage yields toxic protein fragments that cause growth arrest, which is regulated by Csx31. Csx30 binds Csx31 and the associated sigma factor RpoE (RNA polymerase, extracytoplasmic E), suggesting that Csx30-mediated RpoE inhibition modulates the cellular response to infection. We engineered the Cas7-11-Csx29-Csx30 system for programmable RNA sensing in mammalian cells. Overall, the Cas7-11-Csx29 effector is an RNA-dependent nuclease-protease.
History
DepositionSep 4, 2022Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Nov 9, 2022Provider: repository / Type: Initial release
Revision 1.1Dec 7, 2022Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_PubMed / _citation.title
Revision 2.0May 29, 2024Group: Advisory / Atomic model ...Advisory / Atomic model / Author supporting evidence / Data collection / Derived calculations / Non-polymer description / Structure summary
Category: atom_site / chem_comp ...atom_site / chem_comp / chem_comp_atom / chem_comp_bond / entity / pdbx_entity_instance_feature / pdbx_entity_nonpoly / pdbx_nonpoly_scheme / pdbx_unobs_or_zero_occ_atoms / pdbx_validate_close_contact
Item: _atom_site.B_iso_or_equiv / _atom_site.Cartn_x ..._atom_site.B_iso_or_equiv / _atom_site.Cartn_x / _atom_site.Cartn_y / _atom_site.Cartn_z / _atom_site.auth_atom_id / _atom_site.auth_comp_id / _atom_site.label_atom_id / _atom_site.label_comp_id / _atom_site.type_symbol / _chem_comp.formula / _chem_comp.formula_weight / _chem_comp.id / _chem_comp.mon_nstd_flag / _chem_comp.name / _chem_comp.type / _entity.pdbx_description / _pdbx_entity_instance_feature.auth_comp_id / _pdbx_entity_instance_feature.comp_id / _pdbx_entity_nonpoly.comp_id / _pdbx_entity_nonpoly.name / _pdbx_nonpoly_scheme.mon_id / _pdbx_nonpoly_scheme.pdb_mon_id / _pdbx_unobs_or_zero_occ_atoms.auth_atom_id / _pdbx_unobs_or_zero_occ_atoms.auth_comp_id / _pdbx_unobs_or_zero_occ_atoms.label_atom_id / _pdbx_unobs_or_zero_occ_atoms.label_comp_id / _pdbx_validate_close_contact.auth_comp_id_1 / _pdbx_validate_close_contact.auth_comp_id_2

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
R: crRNA
D: target RNA with non-matching PFS
A: CRISPR-associated RAMP family protein
B: CHAT domain-containing protein
hetero molecules


Theoretical massNumber of molelcules
Total (without water)316,39011
Polymers315,1114
Non-polymers1,2797
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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RNA chain , 2 types, 2 molecules RD

#1: RNA chain crRNA


Mass: 30532.891 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)
#2: RNA chain target RNA with non-matching PFS


Mass: 10662.481 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Desulfonema ishimotonii (bacteria)

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Protein , 2 types, 2 molecules AB

#3: Protein CRISPR-associated RAMP family protein


Mass: 185603.750 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
#4: 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

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Non-polymers , 3 types, 7 molecules

#5: Chemical ChemComp-C5P / CYTIDINE-5'-MONOPHOSPHATE


Mass: 323.197 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C9H14N3O8P
#6: Chemical ChemComp-AMP / ADENOSINE MONOPHOSPHATE


Mass: 347.221 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C10H14N5O7P / Feature type: SUBJECT OF INVESTIGATION / Comment: AMP*YM
#7: 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 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
1Cas7-11 in complex with a crRNA and its target RNACOMPLEX#1-#40MULTIPLE SOURCES
2crRNACOMPLEX#11RECOMBINANT
3target RNA with 3'-tagCOMPLEX#21SYNTHETIC
4Cas7-11, Csx29COMPLEX#3-#41RECOMBINANT
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
11Desulfonema ishimotonii (bacteria)45657
23Desulfonema ishimotonii (bacteria)45657
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
11Escherichia coli (E. coli)562
23Escherichia coli (E. coli)562
Buffer solutionpH: 7
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: 1500 nm / Nominal defocus min: 600 nm
Image recordingElectron dose: 76.5 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 2.84 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 71543 / Symmetry type: POINT

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