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- PDB-6o7i: Cryo-EM structure of Csm-crRNA-target RNA ternary bigger complex ... -

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Basic information

Entry
Database: PDB / ID: 6o7i
TitleCryo-EM structure of Csm-crRNA-target RNA ternary bigger complex in complex with cA4 in type III-A CRISPR-Cas system
Components
  • CRISPR system single-strand-specific deoxyribonuclease Cas10/Csm1 (subtype III-A)
  • Csm2
  • Csm3
  • Csm4
  • Csm5Saint-Michel-des-Saints Aerodrome
  • RNA (38-MER)
  • RNA (40-MER)
  • RNA (5'-R(P*AP*AP*AP*A)-3')
Keywordsimmune system/rna / cryo-EM structure / Csm-crRNA-target RNA ternary bigger complex in complex with CA4 / Type III CRISPR-Cas systerm / IMMUNE SYSTEM / immune system-rna-dna complex / immune system-rna complex
Function / homology
Function and homology information


exonuclease activity / Transferases; Transferring phosphorus-containing groups; Nucleotidyltransferases / transferase activity / defense response to virus / endonuclease activity / Hydrolases; Acting on ester bonds / RNA binding / ATP binding / identical protein binding
Similarity search - Function
CRISPR-associated protein Csm5 / CRISPR-associated protein, Csm2 Type III-A / Csm2 Type III-A / CRISPR-associated RAMP Csm3 / CRISPR type III-associated RAMP protein Csm4 / CRISPR system single-strand-specific deoxyribonuclease Cas10/Csm1 / Csm1, subunit domain B / Csm1 subunit domain B / CRISPR type III-associated protein / RAMP superfamily ...CRISPR-associated protein Csm5 / CRISPR-associated protein, Csm2 Type III-A / Csm2 Type III-A / CRISPR-associated RAMP Csm3 / CRISPR type III-associated RAMP protein Csm4 / CRISPR system single-strand-specific deoxyribonuclease Cas10/Csm1 / Csm1, subunit domain B / Csm1 subunit domain B / CRISPR type III-associated protein / RAMP superfamily / HD domain profile. / GGDEF domain profile. / GGDEF domain / HD domain / HD domain / Reverse transcriptase/Diguanylate cyclase domain
Similarity search - Domain/homology
RNA / RNA (> 10) / CRISPR system single-strand-specific deoxyribonuclease Cas10/Csm1 (subtype III-A) / CRISPR system Cms protein Csm2 / CRISPR system Cms endoribonuclease Csm3 / CRISPR system Cms protein Csm4 / CRISPR system Cms protein Csm5
Similarity search - Component
Biological speciesThermococcus onnurineus (archaea)
synthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.2 Å
AuthorsJia, N. / Patel, D.J.
CitationJournal: Mol Cell / Year: 2019
Title: Second Messenger cA Formation within the Composite Csm1 Palm Pocket of Type III-A CRISPR-Cas Csm Complex and Its Release Path.
Authors: Ning Jia / Roger Jones / George Sukenick / Dinshaw J Patel /
Abstract: Target RNA binding to crRNA-bound type III-A CRISPR-Cas multi-subunit Csm surveillance complexes activates cyclic-oligoadenylate (cA) formation from ATP subunits positioned within the composite pair ...Target RNA binding to crRNA-bound type III-A CRISPR-Cas multi-subunit Csm surveillance complexes activates cyclic-oligoadenylate (cA) formation from ATP subunits positioned within the composite pair of Palm domain pockets of the Csm1 subunit. The generated cA second messenger in turn targets the CARF domain of trans-acting RNase Csm6, triggering its HEPN domain-based RNase activity. We have undertaken cryo-EM studies on multi-subunit Thermococcus onnurineus Csm effector ternary complexes, as well as X-ray studies on Csm1-Csm4 cassette, both bound to substrate (AMPPNP), intermediates (pppA), and products (cA), to decipher mechanistic aspects of cA formation and release. A network of intermolecular hydrogen bond alignments accounts for the observed adenosine specificity, with ligand positioning dictating formation of linear pppA intermediates and subsequent cA formation by cyclization. We combine our structural results with published functional studies to highlight mechanistic insights into the role of the Csm effector complex in mediating the cA signaling pathway.
History
DepositionMar 7, 2019Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jul 31, 2019Provider: repository / Type: Initial release
Revision 1.1Sep 18, 2019Group: Data collection / Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first / _citation.page_last
Revision 1.2Mar 20, 2024Group: Data collection / Database references / Category: chem_comp_atom / chem_comp_bond / database_2
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession

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Structure visualization

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  • Deposited structure unit
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Assembly

Deposited unit
A: CRISPR system single-strand-specific deoxyribonuclease Cas10/Csm1 (subtype III-A)
B: Csm2
C: Csm3
D: Csm3
E: Csm4
J: Csm2
G: RNA (38-MER)
H: RNA (40-MER)
K: Csm3
I: RNA (5'-R(P*AP*AP*AP*A)-3')
F: Csm5
hetero molecules


Theoretical massNumber of molelcules
Total (without water)330,23515
Polymers329,97411
Non-polymers2624
Water0
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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Protein , 5 types, 8 molecules ABJCDKEF

#1: Protein CRISPR system single-strand-specific deoxyribonuclease Cas10/Csm1 (subtype III-A) / ssDNase Cas10 / Cyclic oligoadenylate synthase / ToCsm1


Mass: 89706.594 Da / Num. of mol.: 1 / Mutation: D589A
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Thermococcus onnurineus (strain NA1) (archaea)
Strain: NA1 / Gene: csm1, cas10, TON_0893 / Production host: Escherichia coli (E. coli)
References: UniProt: B6YWB8, Hydrolases; Acting on ester bonds, Transferases; Transferring phosphorus-containing groups; Nucleotidyltransferases
#2: Protein Csm2


Mass: 21210.293 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Thermococcus onnurineus (strain NA1) (archaea)
Strain: NA1 / Gene: TON_0894 / Production host: Escherichia coli (E. coli) / References: UniProt: B6YWB9
#3: Protein Csm3


Mass: 32809.012 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Thermococcus onnurineus (strain NA1) (archaea)
Strain: NA1 / Production host: Escherichia coli (E. coli) / References: UniProt: B6YWC0
#4: Protein Csm4


Mass: 32345.061 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Thermococcus onnurineus (strain NA1) (archaea)
Strain: NA1 / Gene: TON_0896 / Production host: Escherichia coli (E. coli) / References: UniProt: B6YWC1
#8: Protein Csm5 / Saint-Michel-des-Saints Aerodrome


Mass: 40588.410 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Thermococcus onnurineus (strain NA1) (archaea)
Strain: NA1 / Gene: TON_0897 / Production host: Escherichia coli (E. coli) / References: UniProt: B6YWC2*PLUS

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RNA chain , 3 types, 3 molecules GHI

#5: RNA chain RNA (38-MER)


Mass: 12463.438 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Thermococcus onnurineus (archaea)
#6: RNA chain RNA (40-MER)


Mass: 12750.658 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Thermococcus onnurineus (archaea)
#7: RNA chain RNA (5'-R(P*AP*AP*AP*A)-3')


Mass: 1271.866 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)

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Non-polymers , 1 types, 4 molecules

#9: Chemical
ChemComp-ZN / ZINC ION


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

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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: Csm-crRNA-target RNA complex / Type: COMPLEX / Entity ID: #1-#7 / Source: RECOMBINANT
Molecular weightValue: 0.3 MDa / Experimental value: YES
Source (natural)Organism: Thermococcus onnurineus (archaea)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 8.8 / Details: 20 mM Tris-HCl, pH 8.8, 250 mM NaCl, 2 mM DTT
Buffer componentFormula: Tris
SpecimenConc.: 0.5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 %

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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
Image recordingElectron dose: 1.35 e/Å2 / Film or detector model: GATAN K2 SUMMIT (4k x 4k)

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Processing

EM softwareName: RELION / Version: 2.1 / Category: 3D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.2 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 41472 / Symmetry type: POINT

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