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- PDB-9z7q: Cryo-EM Structure of the Type III-Bv CRISPR Complex from Dissulfu... -

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Basic information

Entry
Database: PDB / ID: 9z7q
TitleCryo-EM Structure of the Type III-Bv CRISPR Complex from Dissulfurispira thermophila bound to a crRNA
Components
  • (Type III-B CRISPR ...) x 2
  • CRISPR type III-B/RAMP module-associated protein Cmr5
  • CSD domain-containing protein Cmr6
  • Cmr1-MntA
  • RNA (38-MER)
  • Type III-B CRISPR-associated protein Cas10/Cmr2
KeywordsIMMUNE SYSTEM/RNA / CRISPR / complex / crRNA / IMMUNE SYSTEM / IMMUNE SYSTEM-RNA complex
Function / homology
Function and homology information


defense response to virus / nucleic acid binding / nucleotide binding / metal ion binding / cytosol / cytoplasm
Similarity search - Function
Polymerase beta, nucleotidyltransferase / Polymerase beta, Nucleotidyltransferase / CRISPR-associated protein TM1795 / CRISPR-associated protein, TM1793 / CRISPR-associated protein (Cas_Cmr5) / CRISPR-associated protein, TM1791 / CRISPR-associated protein, Cmr3 / CRISPR-associated protein (Cas_Cmr3) / CRISPR-associated protein, Cmr5 / CRISPR-associated RAMP Cmr4 ...Polymerase beta, nucleotidyltransferase / Polymerase beta, Nucleotidyltransferase / CRISPR-associated protein TM1795 / CRISPR-associated protein, TM1793 / CRISPR-associated protein (Cas_Cmr5) / CRISPR-associated protein, TM1791 / CRISPR-associated protein, Cmr3 / CRISPR-associated protein (Cas_Cmr3) / CRISPR-associated protein, Cmr5 / CRISPR-associated RAMP Cmr4 / AF1862-like domain superfamily / CRISPR-associated protein Cmr2 / CRISPR-associated protein Cmr2, N-terminal / CRISPR-Cas system, Cmr2 subunit, D1 domain, cysteine cluster / CRISPR-associated protein Cmr2, N-terminal / Cold-shock (CSD) domain profile. / Cold-shock protein, DNA-binding / 'Cold-shock' DNA-binding domain / : / Cas10/Cmr2, second palm domain / Cold shock domain / Cold shock protein domain / CRISPR type III-associated protein / RAMP superfamily / GGDEF domain profile. / GGDEF domain / Nucleotidyltransferase superfamily / Reverse transcriptase/Diguanylate cyclase domain / Nucleic acid-binding, OB-fold
Similarity search - Domain/homology
RNA / RNA (> 10) / Type III-B CRISPR module-associated protein Cmr3 / Uncharacterized protein / CSD domain-containing protein / CRISPR type III-B/RAMP module-associated protein Cmr5 / Type III-B CRISPR module RAMP protein Cmr4 / Type III-B CRISPR-associated protein Cas10/Cmr2
Similarity search - Component
Biological speciesDissulfurispira thermophila (bacteria)
Escherichia phage MS2 (virus)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3 Å
AuthorsBurman, N. / Pandey, S. / Wiedenheft, B.
Funding support United States, 2items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R35GM134867 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)F31GM153146 United States
CitationJournal: Structure / Year: 2026
Title: Identification and structure determination of a type III-Bv CRISPR complex that post-translationally modifies an associated toxin.
Authors: Shishir Pandey / Nathaniel Burman / William S Henriques / Tanner Wiegand / Trevor Zahl / Hannah Nyquist / Tanner Spreeuw / Murat Buyukyoruk / Blake Wiedenheft /
Abstract: Cas7-family proteins form the scaffolds of multi-subunit CRISPR RNA-guided surveillance complexes. To explore how Cas7 diversification expands CRISPR function, we identified Cas7 fusion proteins ...Cas7-family proteins form the scaffolds of multi-subunit CRISPR RNA-guided surveillance complexes. To explore how Cas7 diversification expands CRISPR function, we identified Cas7 fusion proteins linked to diverse accessory domains, including a type III-B variant (III-Bv) in which a Cas7 homolog (Cmr1) is fused to the MntA antitoxin and encoded adjacent to a HEPN-family toxin. Structures reveal that the core Cas proteins assemble into a stable surveillance complex in the absence of crRNA, whereas incorporation of the Cmr1-MntA fusion is crRNA-dependent. Target RNA recognition triggers conformational changes that expose the Cas10 cyclase active site and promote cyclic oligoadenylate synthesis. Biochemical analyses show that the CRISPR-associated MntA is enzymatically active and AMPylates the associated HEPN protein. Together, these findings establish the structural basis for assembly of a type III-Bv surveillance complex containing an enzymatically active toxin-antitoxin module.
History
DepositionNov 17, 2025Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jul 15, 2026Provider: repository / Type: Initial release
Revision 1.0Jul 15, 2026Data content type: EM metadata / Data content type: EM metadata / Provider: repository / Type: Initial release
Revision 1.0Jul 15, 2026Data content type: Additional map / Part number: 1 / Data content type: Additional map / Provider: repository / Type: Initial release
Revision 1.0Jul 15, 2026Data content type: FSC / Data content type: FSC / Provider: repository / Type: Initial release
Revision 1.0Jul 15, 2026Data content type: Half map / Part number: 1 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0Jul 15, 2026Data content type: Half map / Part number: 2 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0Jul 15, 2026Data content type: Image / Data content type: Image / Provider: repository / Type: Initial release
Revision 1.0Jul 15, 2026Data content type: Mask / Part number: 1 / Data content type: Mask / Provider: repository / Type: Initial release
Revision 1.0Jul 15, 2026Data content type: Primary map / Data content type: Primary map / Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Type III-B CRISPR-associated protein Cas10/Cmr2
B: Type III-B CRISPR module-associated protein Cmr3
C: Type III-B CRISPR module RAMP protein Cmr4
D: Type III-B CRISPR module RAMP protein Cmr4
E: Type III-B CRISPR module RAMP protein Cmr4
F: CRISPR type III-B/RAMP module-associated protein Cmr5
G: CRISPR type III-B/RAMP module-associated protein Cmr5
H: CSD domain-containing protein Cmr6
N: RNA (38-MER)
I: Cmr1-MntA
hetero molecules


Theoretical massNumber of molelcules
Total (without water)351,65411
Polymers351,58910
Non-polymers651
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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Protein , 4 types, 5 molecules AFGHI

#1: Protein Type III-B CRISPR-associated protein Cas10/Cmr2


Mass: 70288.859 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Dissulfurispira thermophila (bacteria) / Gene: JZK55_14980 / Production host: Escherichia coli (E. coli) / Strain (production host): BL-21 AI / References: UniProt: A0A7G1H3Q2
#4: Protein CRISPR type III-B/RAMP module-associated protein Cmr5


Mass: 15953.378 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Dissulfurispira thermophila (bacteria) / Gene: JZK55_14940 / Production host: Escherichia coli (E. coli) / Strain (production host): BL-21 AI / References: UniProt: A0A7G1H353
#5: Protein CSD domain-containing protein Cmr6


Mass: 42396.605 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Dissulfurispira thermophila (bacteria) / Gene: JZK55_14930 / Production host: Escherichia coli (E. coli) / Strain (production host): BL-21 AI / References: UniProt: A0A7G1H339
#7: Protein Cmr1-MntA


Mass: 49646.605 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Dissulfurispira thermophila (bacteria) / Gene: JZK55_15000 / Production host: Escherichia coli (E. coli) / Strain (production host): BL-21 AI / References: UniProt: A0A7G1H1S1

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Type III-B CRISPR ... , 2 types, 4 molecules BCDE

#2: Protein Type III-B CRISPR module-associated protein Cmr3


Mass: 40201.371 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Dissulfurispira thermophila (bacteria) / Gene: JZK55_14970 / Production host: Escherichia coli (E. coli) / Strain (production host): BL-21 AI / References: UniProt: A0A7G1H1A4
#3: Protein Type III-B CRISPR module RAMP protein Cmr4


Mass: 34966.660 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Dissulfurispira thermophila (bacteria) / Gene: JZK55_14950 / Production host: Escherichia coli (E. coli) / Strain (production host): BL-21 AI / References: UniProt: A0A7G1H376

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RNA chain / Non-polymers , 2 types, 2 molecules N

#6: RNA chain RNA (38-MER)


Mass: 12248.350 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia phage MS2 (virus) / Production host: Escherichia coli (E. coli) / Strain (production host): BL-21 AI
#8: Chemical ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Zn

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Details

Has ligand of interestN
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: Type III-Bv CRISPR Complex from Dissulfurispira thermophila bound to a crRNA
Type: COMPLEX / Entity ID: #1-#7 / Source: MULTIPLE SOURCES
Molecular weightExperimental value: NO
Source (natural)Organism: Dissulfurispira thermophila (bacteria)
Source (recombinant)Organism: Escherichia coli (E. coli) / Strain: BL-21 AI
Buffer solutionpH: 8
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
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: 277.15 K

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Electron microscopy imaging

Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company
MicroscopyModel: FEI TALOS ARCTICA
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 45000 X / Nominal defocus max: 2000 nm / Nominal defocus min: 500 nm / Cs: 2.7 mm / C2 aperture diameter: 50 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 65 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 4871
Details: Micrographs are the average of 50 subframes with dose of 1.3 electrons per subframe.

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Processing

EM software
IDNameVersionCategoryDetails (eV)
1cryoSPARC4.7.1particle selectionTemplate picker was used to identify particle images
2SerialEM4.2.0image acquisition
4cryoSPARC4.7.1CTF correctioncryoSPARC's patch CTF estimation was used to determine CTF correction
7UCSF ChimeraX1.10.1model fittingChimeraX's Fit in map command was used to dock protein subunits into the experimental density
9cryoSPARC4.7.1initial Euler assignment
10cryoSPARC4.7.1final Euler assignment
11cryoSPARC4.7.1classification3-D classification was used without a focus mask to sort particles in the final stage of classification
12cryoSPARC4.7.13D reconstructionnon-uniform refinement was used to generate the final reconstruction
13PHENIX1.21.2_5419model refinementRealspace refine was used to refine the structure using the starting model as a reference following manual editing in COOT.
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 1621549
Details: Particles identified using cryoSPARC's template picker. Templates generated from a de novo volume that contained 105809 particles.
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 194795 / Algorithm: FOURIER SPACE
Details: cryoSPARC's non-uniform refinement with per particle CTF correction was used to generate the final reconstruction.
Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL / Target criteria: Cross-correlation coefficient
Details: Initial fitting was done in ChimeraX, followed by refinement in PHENIX. COOT was used to manually edit the structure to better fit the density before a final refinement in PHENIX
Atomic model buildingDetails: Alphafold predicted strucutres of protein subunits were fit into the experimental density using ChimeraX. The crRNA was build de novo using COOT.
Source name: AlphaFold / Type: in silico model
RefinementHighest resolution: 3 Å
Stereochemistry target values: REAL-SPACE (WEIGHTED MAP SUM AT ATOM CENTERS)
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00322769
ELECTRON MICROSCOPYf_angle_d0.62531011
ELECTRON MICROSCOPYf_dihedral_angle_d11.8533583
ELECTRON MICROSCOPYf_chiral_restr0.0443569
ELECTRON MICROSCOPYf_plane_restr0.0043817

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