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- PDB-6mut: Cryo-EM structure of ternary Csm-crRNA-target RNA with anti-tag s... -

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Basic information

Entry
Database: PDB / ID: 6mut
TitleCryo-EM structure of ternary Csm-crRNA-target RNA with anti-tag sequence complex in type III-A CRISPR-Cas system
Components
  • (Uncharacterized protein ...) x 5
  • RNA (5'-R(*GP*UP*GP*GP*AP*AP*AP*GP*GP*CP*GP*GP*GP*CP*AP*GP*AP*GP*GP*C)-3')
  • RNA (5'-R(P*CP*CP*UP*CP*UP*GP*CP*CP*CP*GP*CP*CP*UP*UP*UP*CP*CP*AP*C)-3')
KeywordsRNA BINDING PROTEIN/RNA / cryo-EM structure / ternary Csm-crRNA-target RNA with anti-tag sequence complex / Type III CRISPR-Cas system / RNA BINDING PROTEIN-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 / Cas10/Cmr2, second palm domain ...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 / Cas10/Cmr2, second palm domain / : / 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)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.1 Å
AuthorsJia, N. / Wang, C. / Eng, E.T. / Patel, D.J.
CitationJournal: Mol Cell / Year: 2019
Title: Type III-A CRISPR-Cas Csm Complexes: Assembly, Periodic RNA Cleavage, DNase Activity Regulation, and Autoimmunity.
Authors: Ning Jia / Charlie Y Mo / Chongyuan Wang / Edward T Eng / Luciano A Marraffini / Dinshaw J Patel /
Abstract: Type ΙΙΙ CRISPR-Cas systems provide robust immunity against foreign RNA and DNA by sequence-specific RNase and target RNA-activated sequence-nonspecific DNase and RNase activities. We report on ...Type ΙΙΙ CRISPR-Cas systems provide robust immunity against foreign RNA and DNA by sequence-specific RNase and target RNA-activated sequence-nonspecific DNase and RNase activities. We report on cryo-EM structures of Thermococcus onnurineus Csm binary, Csm-target RNA and Csm-target RNA ternary complexes in the 3.1 Å range. The topological features of the crRNA 5'-repeat tag explains the 5'-ruler mechanism for defining target cleavage sites, with accessibility of positions -2 to -5 within the 5'-repeat serving as sensors for avoidance of autoimmunity. The Csm3 thumb elements introduce periodic kinks in the crRNA-target RNA duplex, facilitating cleavage of the target RNA with 6-nt periodicity. Key Glu residues within a Csm1 loop segment of Csm adopt a proposed autoinhibitory conformation suggestive of DNase activity regulation. These structural findings, complemented by mutational studies of key intermolecular contacts, provide insights into Csm complex assembly, mechanisms underlying RNA targeting and site-specific periodic cleavage, regulation of DNase cleavage activity, and autoimmunity suppression.
History
DepositionOct 23, 2018Deposition site: RCSB / Processing site: RCSB
Revision 1.0Dec 12, 2018Provider: repository / Type: Initial release
Revision 1.1Dec 19, 2018Group: Data collection / Database references / Category: citation / citation_author
Item: _citation.journal_abbrev / _citation.journal_id_ISSN ..._citation.journal_abbrev / _citation.journal_id_ISSN / _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID
Revision 1.2Jan 30, 2019Group: Data collection / Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first / _citation.year
Revision 1.3Mar 13, 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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Assembly

Deposited unit
A: Uncharacterized protein Csm1
B: Uncharacterized protein Csm2
C: Uncharacterized protein Csm3
D: Uncharacterized protein Csm3
E: Uncharacterized protein Csm4
G: RNA (5'-R(*GP*UP*GP*GP*AP*AP*AP*GP*GP*CP*GP*GP*GP*CP*AP*GP*AP*GP*GP*C)-3')
H: RNA (5'-R(P*CP*CP*UP*CP*UP*GP*CP*CP*CP*GP*CP*CP*UP*UP*UP*CP*CP*AP*C)-3')
F: Uncharacterized protein Csm5
hetero molecules


Theoretical massNumber of molelcules
Total (without water)281,81011
Polymers281,6148
Non-polymers1963
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area28910 Å2
ΔGint-122 kcal/mol
Surface area85250 Å2

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Components

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Uncharacterized protein ... , 5 types, 6 molecules ABCDEF

#1: Protein Uncharacterized protein Csm1


Mass: 89750.602 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Thermococcus onnurineus (archaea) / Gene: TON_0893 / Production host: Escherichia coli (E. coli) / References: UniProt: B6YWB8
#2: Protein Uncharacterized protein Csm2


Mass: 21210.293 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Thermococcus onnurineus (archaea) / Gene: TON_0894 / Production host: Escherichia coli (E. coli) / References: UniProt: B6YWB9
#3: Protein Uncharacterized protein Csm3


Mass: 32765.002 Da / Num. of mol.: 2 / Mutation: D36A
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Thermococcus onnurineus (archaea) / Gene: TON_0895 / Production host: Escherichia coli (E. coli) / References: UniProt: B6YWC0
#4: Protein Uncharacterized protein Csm4


Mass: 32345.061 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Thermococcus onnurineus (archaea) / Gene: TON_0896 / Production host: Escherichia coli (E. coli) / References: UniProt: B6YWC1
#7: Protein Uncharacterized protein Csm5


Mass: 46091.016 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Thermococcus onnurineus (archaea) / Gene: TON_0897 / Production host: Escherichia coli (E. coli) / References: UniProt: B6YWC2

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RNA chain , 2 types, 2 molecules GH

#5: RNA chain RNA (5'-R(*GP*UP*GP*GP*AP*AP*AP*GP*GP*CP*GP*GP*GP*CP*AP*GP*AP*GP*GP*C)-3')


Mass: 12463.438 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Thermococcus onnurineus (archaea)
#6: RNA chain RNA (5'-R(P*CP*CP*UP*CP*UP*GP*CP*CP*CP*GP*CP*CP*UP*UP*UP*CP*CP*AP*C)-3')


Mass: 14223.495 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Thermococcus onnurineus (archaea)

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Non-polymers , 1 types, 3 molecules

#8: Chemical ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 3 / 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: ternary Csm-crRNA-target RNA with anti-tag sequence complex
Type: COMPLEX / Entity ID: #1-#7 / Source: RECOMBINANT
Molecular weightValue: 0.25 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
Specimen supportDetails: unspecified
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 FIELD
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
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.1 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 57170 / Symmetry type: POINT

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