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- PDB-8s9u: CRISPR-Cas type III-D effector complex bound to a target RNA -

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Basic information

Entry
Database: PDB / ID: 8s9u
TitleCRISPR-Cas type III-D effector complex bound to a target RNA
Components
  • CRISPR RNA
  • Cas10
  • Cas7-2x
  • Cas7-Cas5-Cas11
  • TIGR03984 family CRISPR-associated protein
  • TIGR03986 family CRISPR-associated RAMP protein
  • Target RNA
KeywordsRNA BINDING PROTEIN/RNA / CRISPR / CRISPR-Cas / type III / complex / RNA / crRNA / RNA BINDING PROTEIN / RNA BINDING PROTEIN-RNA complex
Function / homology
Function and homology information


defense response to virus
Similarity search - Function
CRISPR-associated protein A791736 / CRISPR-associated RAMP BGP1436 / : / Cas10/Cmr2, second palm domain / : / CRISPR type III-associated protein / RAMP superfamily / Reverse transcriptase/Diguanylate cyclase domain
Similarity search - Domain/homology
RNA / RNA (> 10) / Cas10/Cmr2 second palm domain-containing protein / CRISPR type III-associated protein domain-containing protein / CRISPR type III-associated protein domain-containing protein / TIGR03984 family CRISPR-associated protein / CRISPR type III-associated protein domain-containing protein
Similarity search - Component
Biological speciesSynechocystis sp. PCC 6803 (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.77 Å
AuthorsSchwartz, E.A. / Taylor, D.W.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R35GM138348 United States
CitationJournal: Nat Commun / Year: 2024
Title: RNA targeting and cleavage by the type III-Dv CRISPR effector complex.
Authors: Evan A Schwartz / Jack P K Bravo / Mohd Ahsan / Luis A Macias / Caitlyn L McCafferty / Tyler L Dangerfield / Jada N Walker / Jennifer S Brodbelt / Giulia Palermo / Peter C Fineran / Robert D ...Authors: Evan A Schwartz / Jack P K Bravo / Mohd Ahsan / Luis A Macias / Caitlyn L McCafferty / Tyler L Dangerfield / Jada N Walker / Jennifer S Brodbelt / Giulia Palermo / Peter C Fineran / Robert D Fagerlund / David W Taylor /
Abstract: CRISPR-Cas are adaptive immune systems in bacteria and archaea that utilize CRISPR RNA-guided surveillance complexes to target complementary RNA or DNA for destruction. Target RNA cleavage at regular ...CRISPR-Cas are adaptive immune systems in bacteria and archaea that utilize CRISPR RNA-guided surveillance complexes to target complementary RNA or DNA for destruction. Target RNA cleavage at regular intervals is characteristic of type III effector complexes. Here, we determine the structures of the Synechocystis type III-Dv complex, an apparent evolutionary intermediate from multi-protein to single-protein type III effectors, in pre- and post-cleavage states. The structures show how multi-subunit fusion proteins in the effector are tethered together in an unusual arrangement to assemble into an active and programmable RNA endonuclease and how the effector utilizes a distinct mechanism for target RNA seeding from other type III effectors. Using structural, biochemical, and quantum/classical molecular dynamics simulation, we study the structure and dynamics of the three catalytic sites, where a 2'-OH of the ribose on the target RNA acts as a nucleophile for in line self-cleavage of the upstream scissile phosphate. Strikingly, the arrangement at the catalytic residues of most type III complexes resembles the active site of ribozymes, including the hammerhead, pistol, and Varkud satellite ribozymes. Our work provides detailed molecular insight into the mechanisms of RNA targeting and cleavage by an important intermediate in the evolution of type III effector complexes.
History
DepositionMar 30, 2023Deposition site: RCSB / Processing site: RCSB
Revision 1.0Apr 24, 2024Provider: repository / Type: Initial release
Revision 1.1May 14, 2025Group: Data collection / Database references / Structure summary
Category: citation / citation_author ...citation / citation_author / em_admin / pdbx_entry_details
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 / _em_admin.last_update

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Cas7-Cas5-Cas11
B: TIGR03984 family CRISPR-associated protein
C: Cas10
D: Cas7-2x
E: TIGR03986 family CRISPR-associated RAMP protein
F: CRISPR RNA
G: Target RNA


Theoretical massNumber of molelcules
Total (without water)352,1437
Polymers352,1437
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: mass spectrometry, electron microscopy
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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Protein , 5 types, 5 molecules ABCDE

#1: Protein Cas7-Cas5-Cas11


Mass: 87558.938 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Synechocystis sp. PCC 6803 (bacteria) / Gene: sll7066 / Production host: Escherichia coli (E. coli) / References: UniProt: Q6ZED2
#2: Protein TIGR03984 family CRISPR-associated protein


Mass: 21899.844 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Synechocystis sp. PCC 6803 (bacteria) / Gene: sll7064 / Production host: Escherichia coli (E. coli) / References: UniProt: Q6ZED4
#3: Protein Cas10


Mass: 64335.309 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Synechocystis sp. PCC 6803 (bacteria) / Gene: sll7067 / Production host: Escherichia coli (E. coli) / References: UniProt: Q6ZED1
#4: Protein Cas7-2x


Mass: 56820.832 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Synechocystis sp. PCC 6803 (bacteria) / Gene: sll7065 / Production host: Escherichia coli (E. coli) / References: UniProt: Q6ZED3
#5: Protein TIGR03986 family CRISPR-associated RAMP protein


Mass: 90334.984 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Synechocystis sp. PCC 6803 (bacteria) / Gene: sll7063 / Production host: Escherichia coli (E. coli) / References: UniProt: Q6ZED5

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RNA chain , 2 types, 2 molecules FG

#6: RNA chain CRISPR RNA


Mass: 11927.167 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Synechocystis sp. PCC 6803 (bacteria) / Production host: Escherichia coli (E. coli)
#7: RNA chain Target RNA


Mass: 19265.455 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Synechocystis sp. PCC 6803 (bacteria)

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Details

Has protein modificationN

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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: CRISPR-Cas type III-D effector complex bound to target RNA
Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Molecular weightValue: .351 MDa / Experimental value: YES
Source (natural)Organism: Synechocystis sp. PCC 6803 (bacteria)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7.5
Buffer component
IDConc.NameFormulaBuffer-ID
110 mMHEPES-NaOH1
2100 mMPotassium ChlorideKCl1
35 %Glycerol1
41 mMDTT1
SpecimenConc.: 0.3 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: Particles were monodisperse and homogeneous.
Specimen supportGrid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K

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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 / Nominal magnification: 105000 X / Nominal defocus max: 2200 nm / Nominal defocus min: 1200 nm / Cs: 2.7 mm / C2 aperture diameter: 70 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 80 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

Software
NameVersionClassification
phenix.real_space_refine1.18rc7_3834refinement
PHENIX1.18rc7_3834refinement
EM softwareName: cryoSPARC / Version: 3 / Category: 3D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 2.77 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 609722 / Symmetry type: POINT
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 54.97 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.010123209
ELECTRON MICROSCOPYf_angle_d0.922531759
ELECTRON MICROSCOPYf_chiral_restr0.063544
ELECTRON MICROSCOPYf_plane_restr0.0063899
ELECTRON MICROSCOPYf_dihedral_angle_d21.39958892

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