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- PDB-8gu6: Structure of the SbCas7-11-crRNA-NTR-Csx29 complex -

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Basic information

Entry
Database: PDB / ID: 8gu6
TitleStructure of the SbCas7-11-crRNA-NTR-Csx29 complex
Components
  • CHAT domain protein
  • RAMP superfamily protein
  • RNA (33-MER)
  • RNA (5'-R(P*GP*GP*GP*GP*CP*AP*GP*AP*AP*AP*AP*UP*UP*GP*GP*GP*U)-3')
KeywordsRNA BINDING PROTEIN / Complex / RNA-binding
Function / homologyCHAT domain / CHAT domain / RNA / RNA (> 10) / CHAT domain protein
Function and homology information
Biological speciesCandidatus Scalindua brodae (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.1 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: Nucleic Acids Res / Year: 2022
Title: Target RNA-guided protease activity in type III-E CRISPR-Cas system.
Authors: Xiaoshen Wang / Guimei Yu / Yanan Wen / Qiyin An / Xuzichao Li / Fumeng Liao / Chengwei Lian / Kai Zhang / Hang Yin / Yong Wei / Zengqin Deng / Heng Zhang /
Abstract: The type III-E CRISPR-Cas systems are newly identified adaptive immune systems in prokaryotes that use a single Cas7-11 protein to specifically cleave target RNA. Cas7-11 could associate with Csx29, ...The type III-E CRISPR-Cas systems are newly identified adaptive immune systems in prokaryotes that use a single Cas7-11 protein to specifically cleave target RNA. Cas7-11 could associate with Csx29, a putative caspase-like protein encoded by the gene frequently found in the type III-E loci, suggesting a functional linkage between the RNase and protease activities in type III-E systems. Here, we demonstrated that target RNA recognition would stimulate the proteolytic activity of Csx29, and protein Csx30 is the endogenous substrate. More interestingly, while the cognate target RNA recognition would activate Csx29, non-cognate target RNA with the complementary 3' anti-tag sequence inhibits the enzymatic activity. Csx30 could bind to the sigma factor RpoE, which may initiate the stress response after proteolytic cleavage. Combined with biochemical and structural studies, we have elucidated the mechanisms underlying the target RNA-guided proteolytic activity of Csx29. Our work will guide further developments leveraging this simple RNA targeting system for RNA and protein-related applications.
History
DepositionSep 10, 2022Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Jan 18, 2023Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
D: CHAT domain protein
A: RAMP superfamily protein
C: RNA (33-MER)
J: RNA (5'-R(P*GP*GP*GP*GP*CP*AP*GP*AP*AP*AP*AP*UP*UP*GP*GP*GP*U)-3')
hetero molecules


Theoretical massNumber of molelcules
Total (without water)296,6268
Polymers296,3644
Non-polymers2624
Water00
1


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

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Components

#1: Protein CHAT domain protein


Mass: 82549.992 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 BL21(DE3) (bacteria) / References: UniProt: A0A0B0EKL4
#2: 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)
#3: 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)
#4: RNA chain RNA (5'-R(P*GP*GP*GP*GP*CP*AP*GP*AP*AP*AP*AP*UP*UP*GP*GP*GP*U)-3')


Mass: 5586.398 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Candidatus Scalindua brodae (bacteria)
#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
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: ternary complex / Type: COMPLEX / Entity ID: #1-#3 / 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 FIELD / 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: 1.20.1_4487: / Classification: refinement
CTF correctionType: PHASE FLIPPING ONLY
3D reconstructionResolution: 3.1 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 70000 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00215702
ELECTRON MICROSCOPYf_angle_d0.51421506
ELECTRON MICROSCOPYf_dihedral_angle_d11.1562594
ELECTRON MICROSCOPYf_chiral_restr0.042432
ELECTRON MICROSCOPYf_plane_restr0.0042558

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