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- PDB-6xn4: Structure of the Lactococcus lactis Csm CTR_3:2 CRISPR-Cas Complex -

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Basic information

Entry
Database: PDB / ID: 6xn4
TitleStructure of the Lactococcus lactis Csm CTR_3:2 CRISPR-Cas Complex
Components
  • (CRISPR-associated protein ...) x 5
  • Crispr RNA
  • target RNA
KeywordsRNA BINDING PROTEIN/RNA / Type III-A CRISPR-Cas / Csm / RNA BINDING PROTEIN-RNA complex
Function / homology
Function and homology information


exonuclease activity / defense response to virus / endonuclease activity / RNA binding / ATP binding
Similarity search - Function
: / CRISPR RNA silencing complex Cmr2 subunit, second helical domain / Csm4, C-terminal / CRISPR Csm4 C-terminal 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 ...: / CRISPR RNA silencing complex Cmr2 subunit, second helical domain / Csm4, C-terminal / CRISPR Csm4 C-terminal 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 / CRISPR type III-associated protein / RAMP superfamily / GGDEF domain profile. / GGDEF domain / HD domain / HD domain / Reverse transcriptase/Diguanylate cyclase domain
Similarity search - Domain/homology
RNA / RNA (> 10) / CRISPR system Cms endoribonuclease Csm3 / CRISPR system single-strand-specific deoxyribonuclease Cas10/Csm1 (subtype III-A) / CRISPR system Cms protein Csm4 / CRISPR system Cms protein Csm2 / CRISPR system Cms protein Csm5
Similarity search - Component
Biological speciesLactococcus lactis subsp. lactis (lactic acid bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.35 Å
AuthorsRai, J. / Sridhara, S. / Li, H.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01 GM099604 United States
CitationJournal: Commun Biol / Year: 2022
Title: Structural and biochemical characterization of in vivo assembled Lactococcus lactis CRISPR-Csm complex.
Authors: Sagar Sridhara / Jay Rai / Charlisa Whyms / Hemant Goswami / Huan He / Walter Woodside / Michael P Terns / Hong Li /
Abstract: The small RNA-mediated immunity in bacteria depends on foreign RNA-activated and self RNA-inhibited enzymatic activities. The multi-subunit Type III-A CRISPR-Cas effector complex (Csm) exemplifies ...The small RNA-mediated immunity in bacteria depends on foreign RNA-activated and self RNA-inhibited enzymatic activities. The multi-subunit Type III-A CRISPR-Cas effector complex (Csm) exemplifies this principle and is in addition regulated by cellular metabolites such as divalent metals and ATP. Recognition of the foreign or cognate target RNA (CTR) triggers its single-stranded deoxyribonuclease (DNase) and cyclic oligoadenylate (cOA) synthesis activities. The same activities remain dormant in the presence of the self or non-cognate target RNA (NTR) that differs from CTR only in its 3'-protospacer flanking sequence (3'-PFS). Here we employ electron cryomicroscopy (cryoEM), functional assays, and comparative cross-linking to study in vivo assembled mesophilic Lactococcus lactis Csm (LlCsm) at the three functional states: apo, the CTR- and the NTR-bound. Unlike previously studied Csm complexes, we observed binding of 3'-PFS to Csm in absence of bound ATP and analyzed the structures of the four RNA cleavage sites. Interestingly, comparative crosslinking results indicate a tightening of the Csm3-Csm4 interface as a result of CTR but not NTR binding, reflecting a possible role of protein dynamics change during activation.
History
DepositionJul 2, 2020Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jan 12, 2022Provider: repository / Type: Initial release
Revision 1.1Apr 13, 2022Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year

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Structure visualization

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Assembly

Deposited unit
B: CRISPR-associated protein Csm4
F: CRISPR-associated protein Csm3
H: CRISPR-associated protein Csm3
G: CRISPR-associated protein Csm3
R: Crispr RNA
C: CRISPR-associated protein Csm2
D: CRISPR-associated protein Csm2
T: target RNA
A: CRISPR-associated protein Cas10
J: CRISPR-associated protein Csm5


Theoretical massNumber of molelcules
Total (without water)285,34910
Polymers285,34910
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: microscopy
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area42080 Å2
ΔGint-206 kcal/mol
Surface area87100 Å2

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Components

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CRISPR-associated protein ... , 5 types, 8 molecules BFHGCDAJ

#1: Protein CRISPR-associated protein Csm4


Mass: 33844.148 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Lactococcus lactis subsp. lactis (lactic acid bacteria)
Gene: csm4 / Production host: Escherichia coli (E. coli) / References: UniProt: L0CFH1
#2: Protein CRISPR-associated protein Csm3


Mass: 23825.160 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Lactococcus lactis subsp. lactis (lactic acid bacteria)
Gene: csm3 / Production host: Escherichia coli (E. coli) / References: UniProt: L0CEA3
#4: Protein CRISPR-associated protein Csm2


Mass: 15796.016 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Lactococcus lactis subsp. lactis (lactic acid bacteria)
Gene: csm2 / Production host: Escherichia coli (E. coli) / References: UniProt: L0CFW2
#6: Protein CRISPR-associated protein Cas10


Mass: 87132.586 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Lactococcus lactis subsp. lactis (lactic acid bacteria)
Gene: cas10 / Production host: Escherichia coli (E. coli) / References: UniProt: L0CEJ3
#7: Protein CRISPR-associated protein Csm5


Mass: 40492.617 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Lactococcus lactis subsp. lactis (lactic acid bacteria)
Gene: csm5 / Production host: Escherichia coli (E. coli) / References: UniProt: L0CG31

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RNA chain , 2 types, 2 molecules RT

#3: RNA chain Crispr RNA /


Mass: 11133.669 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Lactococcus lactis subsp. lactis (lactic acid bacteria)
Production host: Escherichia coli (E. coli)
#5: RNA chain target RNA


Mass: 9678.801 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Lactococcus lactis subsp. lactis (lactic acid bacteria)
Production host: Escherichia coli (E. coli)

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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: Lactococcus lactis Csm CRISPR-Cas CTR_3:2 Complex / Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Source (natural)Organism: Lactococcus lactis subsp. lactis (lactic acid bacteria)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7.5
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: unspecified
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: OTHER / Cs: 2.7 mm / C2 aperture diameter: 70 µm
Image recordingElectron dose: 60.14 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)

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Processing

SoftwareName: PHENIX / Version: 1.18.2_3874: / Classification: refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.35 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 62413 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00919742
ELECTRON MICROSCOPYf_angle_d1.18426888
ELECTRON MICROSCOPYf_dihedral_angle_d20.5583194
ELECTRON MICROSCOPYf_chiral_restr0.0673050
ELECTRON MICROSCOPYf_plane_restr0.0063212

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