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- PDB-8dfs: type I-C Cascade bound to AcrIF2 -

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Basic information

Entry
Database: PDB / ID: 8dfs
Titletype I-C Cascade bound to AcrIF2
Components
  • (CRISPR-associated protein, CT1133 ...) x 2
  • Anti-CRISPR protein 30
  • CRISPR-associated protein, TM1801 family
  • RNA (48-MER)
  • pre-crRNA processing endonuclease
KeywordsRNA BINDING PROTEIN/RNA / CRISPR / Cascade / Type I-C / anti-CRISPR / IF2 / DNA BINDING PROTEIN / RNA BINDING PROTEIN-RNA complex
Function / homology
Function and homology information


symbiont-mediated suppression of host adaptive immune response / symbiont-mediated suppression of host CRISPR-cas system / maintenance of CRISPR repeat elements / defense response to virus / endonuclease activity / Hydrolases; Acting on ester bonds / virus-mediated perturbation of host defense response / RNA binding
Similarity search - Function
CRISPR-associated protein Csd2 / CRISPR-associated protein Cas7, subtype I-B/I-C / CRISPR-associated protein Cas7 / CRISPR-associated protein, Csd1-type / CRISPR-associated protein (Cas_Csd1) / CRISPR pre-crRNA endoribonuclease Cas5d / CRISPR-associated protein, Cas5 / CRISPR-associated protein (Cas_Cas5) / CRISPR-associated protein Cas5, N-terminal
Similarity search - Domain/homology
RNA / RNA (> 10) / Anti-CRISPR protein 30 / CRISPR-associated protein, TM1801 family / CRISPR-associated protein, CT1133 family / pre-crRNA processing endonuclease
Similarity search - Component
Biological speciesDesulfovibrio vulgaris (bacteria)
Desulfovibrio vulgaris str. Hildenborough (bacteria)
Casadabanvirus D3112
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3 Å
AuthorsO'Brien, R.E. / Bravo, J.P.K. / Ramos, D. / Hibshman, G.N. / Wright, J.T. / Taylor, D.W.
Funding support United States, 2items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R35GM138348 United States
Cancer Prevention and Research Institute of Texas (CPRIT)RR160088 United States
CitationJournal: Mol Cell / Year: 2023
Title: Structural snapshots of R-loop formation by a type I-C CRISPR Cascade.
Authors: Roisin E O'Brien / Jack P K Bravo / Delisa Ramos / Grace N Hibshman / Jacquelyn T Wright / David W Taylor /
Abstract: Type I CRISPR-Cas systems employ multi-subunit Cascade effector complexes to target foreign nucleic acids for destruction. Here, we present structures of D. vulgaris type I-C Cascade at various ...Type I CRISPR-Cas systems employ multi-subunit Cascade effector complexes to target foreign nucleic acids for destruction. Here, we present structures of D. vulgaris type I-C Cascade at various stages of double-stranded (ds)DNA target capture, revealing mechanisms that underpin PAM recognition and Cascade allosteric activation. We uncover an interesting mechanism of non-target strand (NTS) DNA stabilization via stacking interactions with the "belly" subunits, securing the NTS in place. This "molecular seatbelt" mechanism facilitates efficient R-loop formation and prevents dsDNA reannealing. Additionally, we provide structural insights into how two anti-CRISPR (Acr) proteins utilize distinct strategies to achieve a shared mechanism of type I-C Cascade inhibition by blocking PAM scanning. These observations form a structural basis for directional R-loop formation and reveal how different Acr proteins have converged upon common molecular mechanisms to efficiently shut down CRISPR immunity.
History
DepositionJun 22, 2022Deposition site: RCSB / Processing site: RCSB
Revision 1.0Feb 22, 2023Provider: repository / Type: Initial release
Revision 1.1Mar 1, 2023Group: Database references / Category: citation
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_ASTM / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.pdbx_database_id_DOI / _citation.title / _citation.year
Revision 1.2Mar 8, 2023Group: Database references / Category: citation / Item: _citation.pdbx_database_id_PubMed / _citation.title
Revision 1.3Mar 15, 2023Group: Database references / Category: citation / Item: _citation.journal_volume / _citation.page_first
Revision 1.4Jun 12, 2024Group: Data collection / Refinement description
Category: chem_comp_atom / chem_comp_bond / em_3d_fitting_list
Item: _em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id ..._em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id / _em_3d_fitting_list.source_name / _em_3d_fitting_list.type

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: pre-crRNA processing endonuclease
B: CRISPR-associated protein, TM1801 family
C: CRISPR-associated protein, TM1801 family
D: CRISPR-associated protein, TM1801 family
E: CRISPR-associated protein, TM1801 family
F: CRISPR-associated protein, TM1801 family
G: CRISPR-associated protein, TM1801 family
H: CRISPR-associated protein, TM1801 family
I: CRISPR-associated protein, CT1133 family
J: CRISPR-associated protein, CT1133 family
K: CRISPR-associated protein, CT1133 family
L: RNA (48-MER)
M: Anti-CRISPR protein 30


Theoretical massNumber of molelcules
Total (without water)374,05713
Polymers374,05713
Non-polymers00
Water00
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 , 3 types, 9 molecules ABCDEFGHM

#1: Protein pre-crRNA processing endonuclease / Cas5c


Mass: 25977.857 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Desulfovibrio vulgaris (bacteria)
Strain: ATCC 29579 / DSM 644 / NCIMB 8303 / VKM B-1760 / Hildenborough
Gene: DVUA0130 / Production host: Escherichia coli (E. coli)
References: UniProt: Q72WF9, Hydrolases; Acting on ester bonds
#2: Protein
CRISPR-associated protein, TM1801 family / Cas7c


Mass: 32358.912 Da / Num. of mol.: 7
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Desulfovibrio vulgaris (bacteria) / Strain: Hildenborough / ATCC 29579 / DSM 644 / NCIMB 8303 / Gene: DVUA0132 / Production host: Escherichia coli (E. coli) / References: UniProt: Q72WF7
#6: Protein Anti-CRISPR protein 30 / Gene product 30 / gp30 / AcrIF2


Mass: 9931.600 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Casadabanvirus D3112 / Gene: orf30 / Production host: Escherichia coli (E. coli) / References: UniProt: Q6TM72

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CRISPR-associated protein, CT1133 ... , 2 types, 3 molecules IJK

#3: Protein CRISPR-associated protein, CT1133 family / Cas8c


Mass: 68123.219 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Desulfovibrio vulgaris str. Hildenborough (bacteria)
Strain: ATCC 29579 / DSM 644 / NCIMB 8303 / VKM B-1760 / Hildenborough
Gene: DVUA0131 / Production host: Escherichia coli (E. coli) / References: UniProt: Q72WF8
#4: Protein CRISPR-associated protein, CT1133 family / Cas11c


Mass: 14017.981 Da / Num. of mol.: 2 / Fragment: Cas8c C-terminal domain (UNP residues 489-612)
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Desulfovibrio vulgaris str. Hildenborough (bacteria)
Strain: Hildenborough / ATCC 29579 / DSM 644 / NCIMB 8303 / Gene: DVUA0131 / Production host: Escherichia coli (E. coli) / References: UniProt: Q72WF8

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RNA chain , 1 types, 1 molecules L

#5: RNA chain RNA (48-MER)


Mass: 15476.264 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Desulfovibrio vulgaris (bacteria) / Strain: Hildenborough / 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

Component
IDNameTypeEntity IDParent-IDSource
1type I-C Cascade bound to AcrIF2COMPLEXall0MULTIPLE SOURCES
2type I-C CascadeCOMPLEX#1-#51RECOMBINANT
3AcrIF2COMPLEX#61RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-IDStrain
22Desulfovibrio vulgaris (bacteria)882Hildenborough
33Casadabanvirus D311210708
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
22Escherichia coli (E. coli)562
33Escherichia coli (E. coli)562
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

MicroscopyModel: TFS GLACIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 2200 nm / Nominal defocus min: 1200 nm
Image recordingElectron dose: 40.5 e/Å2 / Film or detector model: FEI FALCON IV (4k x 4k)

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Processing

SoftwareName: PHENIX / Version: 1.20.1_4487: / Classification: refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 21625 / Symmetry type: POINT
Atomic model buildingPDB-ID: 5UZ9

5uz9


Pdb chain-ID: K / Accession code: 5UZ9 / Source name: PDB / Type: experimental model
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00325871
ELECTRON MICROSCOPYf_angle_d0.55635173
ELECTRON MICROSCOPYf_dihedral_angle_d14.0393949
ELECTRON MICROSCOPYf_chiral_restr0.0463869
ELECTRON MICROSCOPYf_plane_restr0.0044459

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