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

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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 / homologyCsm1 subunit domain B / CRISPR type III-associated protein / HD domain profile. / GGDEF domain profile. / RAMP superfamily / Csm2 Type III-A / HD domain / CRISPR-associated RAMP Csm3 / CRISPR system single-strand-specific deoxyribonuclease Cas10/Csm1 / CRISPR-associated protein Csm5 ...Csm1 subunit domain B / CRISPR type III-associated protein / HD domain profile. / GGDEF domain profile. / RAMP superfamily / Csm2 Type III-A / HD domain / CRISPR-associated RAMP Csm3 / CRISPR system single-strand-specific deoxyribonuclease Cas10/Csm1 / CRISPR-associated protein Csm5 / HD domain / CRISPR-associated protein, Csm2 Type III-A / CRISPR type III-associated RAMP protein Csm4 / GGDEF domain / HD/PDEase domain / exonuclease activity / defense response to virus / Transferases, Transferring phosphorus-containing groups, Nucleotidyltransferases / transferase activity / endonuclease activity / Hydrolases, Acting on ester bonds / RNA binding / ATP binding / identical protein binding / CRISPR system single-strand-specific deoxyribonuclease Cas10/Csm1 (subtype III-A) / Uncharacterized protein / Uncharacterized protein / CRISPR system Cms protein Csm4 / Uncharacterized protein
Function and homology information
Specimen sourceThermococcus onnurineus (archaea)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / 3.1 Å resolution
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.
Validation Report
SummaryFull reportAbout validation report
DateDeposition: Oct 23, 2018 / Release: Dec 12, 2018
RevisionDateData content typeGroupCategoryItemProviderType
1.0Dec 12, 2018Structure modelrepositoryInitial release
1.1Dec 19, 2018Structure modelData collection / Database referencescitation / citation_author_citation.journal_abbrev / _citation.journal_id_ISSN / _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID
1.2Jan 30, 2019Structure modelData collection / Database referencescitation_citation.journal_volume / _citation.page_first / _citation.year

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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
Polyers281,6148
Non-polymers1963
Water0
1


TypeNameSymmetry operationNumber
identity operation1_5551
Buried area (Å2)28910
ΔGint (kcal/M)-122
Surface area (Å2)85250

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Components

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

#1: Protein/peptide Uncharacterized protein Csm1


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


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


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


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


Mass: 46091.016 Da / Num. of mol.: 1 / 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: (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: (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 / Formula: Zn / Zinc

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 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: 1,2,3,4,5,6,7 / Source: RECOMBINANT
Molecular weightValue: 0.25 MDa / Experimental value: YES
Source (natural)Organism: Thermococcus onnurineus (archaea)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionDetails: 20 mM Tris-HCl, pH 8.8, 250 mM NaCl, 2 mM DTT / pH: 8.8
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
MicroscopyMicroscope model: 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
SymmetryPoint symmetry: C1
3D reconstructionResolution: 3.1 Å / Resolution method: FSC 0.143 CUT-OFF / Number of particles: 57170 / Symmetry type: POINT

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