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- PDB-8d9h: gRAMP-TPR-CHAT match PFS target RNA(Craspase) -

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Basic information

Entry
Database: PDB / ID: 8d9h
TitlegRAMP-TPR-CHAT match PFS target RNA(Craspase)
Components
  • CHAT domain protein
  • RAMP superfamily protein
  • RNA (36-MER)
  • RNA (5'-R(P*UP*CP*CP*GP*GP*GP*GP*CP*AP*GP*AP*AP*AP*AP*UP*UP*GP*GP*GP*U)-3')
KeywordsRNA BINDING PROTEIN/RNA / CRISPR / GRAMP / RNA BINDING PROTEIN / Craspase / 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
PHOSPHATE ION / RNA / RNA (> 10) / RAMP superfamily protein / CHAT domain protein
Similarity search - Component
Biological speciesCandidatus Scalindua brodae (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.6 Å
AuthorsHu, C. / Nam, K.H. / Schuler, G. / Ke, A.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM118174 United States
CitationJournal: Science / Year: 2022
Title: Craspase is a CRISPR RNA-guided, RNA-activated protease.
Authors: Chunyi Hu / Sam P B van Beljouw / Ki Hyun Nam / Gabriel Schuler / Fran Ding / Yanru Cui / Alicia Rodríguez-Molina / Anna C Haagsma / Menno Valk / Martin Pabst / Stan J J Brouns / Ailong Ke /
Abstract: The CRISPR-Cas type III-E RNA-targeting effector complex gRAMP/Cas7-11 is associated with a caspase-like protein (TPR-CHAT/Csx29) to form Craspase (CRISPR-guided caspase). Here, we use cryo-electron ...The CRISPR-Cas type III-E RNA-targeting effector complex gRAMP/Cas7-11 is associated with a caspase-like protein (TPR-CHAT/Csx29) to form Craspase (CRISPR-guided caspase). Here, we use cryo-electron microscopy snapshots of Craspase to explain its target RNA cleavage and protease activation mechanisms. Target-guide pairing extending into the 5' region of the guide RNA displaces a gating loop in gRAMP, which triggers an extensive conformational relay that allosterically aligns the protease catalytic dyad and opens an amino acid side-chain-binding pocket. We further define Csx30 as the endogenous protein substrate that is site-specifically proteolyzed by RNA-activated Craspase. This protease activity is switched off by target RNA cleavage by gRAMP and is not activated by RNA targets containing a matching protospacer flanking sequence. We thus conclude that Craspase is a target RNA-activated protease with self-regulatory capacity.
History
DepositionJun 9, 2022Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jun 14, 2023Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
B: RAMP superfamily protein
C: RNA (36-MER)
D: RNA (5'-R(P*UP*CP*CP*GP*GP*GP*GP*CP*AP*GP*AP*AP*AP*AP*UP*UP*GP*GP*GP*U)-3')
E: CHAT domain protein
hetero molecules


Theoretical massNumber of molelcules
Total (without water)240,57811
Polymers240,0314
Non-polymers5477
Water0
1


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

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Components

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Protein , 2 types, 2 molecules BE

#1: Protein RAMP superfamily protein / gRAMP


Mass: 145301.531 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-Gold(DE3)pLysS AG' (bacteria)
References: UniProt: A0A0B0EGF3
#4: Protein CHAT domain protein


Mass: 76802.992 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-Gold(DE3)pLysS AG' (bacteria)
References: UniProt: A0A0B0EKL4

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RNA chain , 2 types, 2 molecules CD

#2: RNA chain RNA (36-MER)


Mass: 11423.787 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-Gold(DE3)pLysS AG' (bacteria)
#3: RNA chain RNA (5'-R(P*UP*CP*CP*GP*GP*GP*GP*CP*AP*GP*AP*AP*AP*AP*UP*UP*GP*GP*GP*U)-3')


Mass: 6502.928 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-Gold(DE3)pLysS AG' (bacteria)

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Non-polymers , 2 types, 7 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-PO4 / PHOSPHATE ION / Phosphate


Mass: 94.971 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: PO4 / 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

ComponentName: gRAMP-TPR-CHAT with match PFS target RNA Craspase / Type: COMPLEX / Entity ID: #1-#4 / Source: RECOMBINANT
Source (natural)Organism: Candidatus Scalindua brodae (bacteria)
Source (recombinant)Organism: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
Buffer solutionpH: 7.5
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 FIELDBright-field microscopy / Nominal defocus max: 2500 nm / Nominal defocus min: 1000 nm
Image recordingElectron dose: 50 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)

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Processing

SoftwareName: PHENIX / Version: 1.17.1_3660: / Classification: refinement
CTF correctionType: NONE
3D reconstructionResolution: 3.6 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 126021 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00717343
ELECTRON MICROSCOPYf_angle_d0.67523649
ELECTRON MICROSCOPYf_dihedral_angle_d17.6872760
ELECTRON MICROSCOPYf_chiral_restr0.0452569
ELECTRON MICROSCOPYf_plane_restr0.0042832

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