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- PDB-7x7a: Cryo-EM structure of SbCas7-11 in complex with crRNA and target RNA -

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Basic information

Entry
Database: PDB / ID: 7x7a
TitleCryo-EM structure of SbCas7-11 in complex with crRNA and target RNA
Components
  • RAMP superfamily protein
  • RNA (33-MER)
KeywordsRNA BINDING PROTEIN/RNA / Complex / RNA-binding / RNA BINDING PROTEIN / RNA BINDING PROTEIN-RNA complex
Function / homologyCRISPR type III-associated protein / RAMP superfamily / defense response to virus / RNA / RNA (> 10) / RAMP superfamily protein
Function and homology information
Biological speciesCandidatus Scalindua brodae (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.2 Å
AuthorsYu, G. / Wang, X. / Deng, Z. / Zhang, H.
Funding support China, 1items
OrganizationGrant numberCountry
National Natural Science Foundation of China (NSFC)32071218 China
CitationJournal: Nat Microbiol / Year: 2022
Title: Structure and function of a bacterial type III-E CRISPR-Cas7-11 complex.
Authors: Guimei Yu / Xiaoshen Wang / Yi Zhang / Qiyin An / Yanan Wen / Xuzichao Li / Hang Yin / Zengqin Deng / Heng Zhang /
Abstract: The type III-E CRISPR-Cas system uses a single multidomain effector called Cas7-11 (also named gRAMP) to cleave RNA and associate with a caspase-like protease Csx29, showing promising potential for ...The type III-E CRISPR-Cas system uses a single multidomain effector called Cas7-11 (also named gRAMP) to cleave RNA and associate with a caspase-like protease Csx29, showing promising potential for RNA-targeting applications. The structural and molecular mechanisms of the type III-E CRISPR-Cas system remain poorly understood. Here we report four cryo-electron microscopy structures of Cas7-11 at different functional states. Cas7-11 has four Cas7-like domains, which assemble into a helical filament to accommodate CRISPR RNA (crRNA), and a Cas11-like domain facilitating crRNA-target RNA duplex formation. The Cas7.1 domain is critical for crRNA maturation, whereas Cas7.2 and Cas7.3 are responsible for target RNA cleavage. Target RNA binding induces the structural arrangements of Csx29, potentially exposing the catalytic site of Csx29. These results delineate the molecular mechanisms underlying pre-crRNA processing, target RNA recognition and cleavage for Cas7-11, and provide a structural framework to understand the role of Csx29 in type III-E CRISPR system.
History
DepositionMar 9, 2022Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Nov 16, 2022Provider: repository / Type: Initial release
Revision 1.1Dec 14, 2022Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation_author.identifier_ORCID

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: RAMP superfamily protein
C: RNA (33-MER)
hetero molecules


Theoretical massNumber of molelcules
Total (without water)208,4906
Polymers208,2282
Non-polymers2624
Water0
1


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

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Components

#1: Protein RAMP superfamily protein


Mass: 197823.797 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 BL21(DE3) (bacteria) / References: UniProt: A0A0B0EGF3
#2: RNA chain RNA (33-MER)


Mass: 10404.171 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Candidatus Scalindua brodae (bacteria) / Production host: Escherichia coli BL21(DE3) (bacteria)
#3: Chemical
ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: Zn / Feature type: SUBJECT OF INVESTIGATION
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

ComponentName: binary complex / Type: COMPLEX / Entity ID: #1-#2 / Source: RECOMBINANT
Source (natural)Organism: Candidatus Scalindua brodae (bacteria)
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria)
Buffer solutionpH: 7.5
SpecimenConc.: 0.6 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: No further treatment.
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 %

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Electron microscopy imaging

MicroscopyModel: JEOL CRYO ARM 300
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 2500 nm / Nominal defocus min: 500 nm
Image recordingElectron dose: 40 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

SoftwareName: PHENIX / Version: 1.19.2_4158: / Classification: refinement
CTF correctionType: PHASE FLIPPING ONLY
Particle selectionNum. of particles selected: 2817147
3D reconstructionResolution: 3.2 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 225876 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00210577
ELECTRON MICROSCOPYf_angle_d0.4714474
ELECTRON MICROSCOPYf_dihedral_angle_d12.3511731
ELECTRON MICROSCOPYf_chiral_restr0.041608
ELECTRON MICROSCOPYf_plane_restr0.0041748

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