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- PDB-9x25: Target analog-bound type III-B Cmr complex of Archaeoglobus fulgidus -

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Basic information

Entry
Database: PDB / ID: 9x25
TitleTarget analog-bound type III-B Cmr complex of Archaeoglobus fulgidus
Components
  • (CRISPR type III-associated protein domain-containing ...) x 2
  • CRISPR system Cmr subunit Cmr5
  • GGDEF domain-containing protein
  • Target analog DNA
  • Type III-B CRISPR module-associated protein Cmr3
  • crRNA
KeywordsRNA BINDING PROTEIN/RNA/DNA / Type III-B CRISPR-Cas effector / RNA BINDING PROTEIN / RNA BINDING PROTEIN-RNA-DNA complex
Function / homology
Function and homology information


defense response to virus / cytoplasm
Similarity search - Function
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-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 / : / : / Cas10/Cmr2, second palm domain / CRISPR type III-associated protein / RAMP superfamily / GGDEF domain profile. / GGDEF domain / Reverse transcriptase/Diguanylate cyclase domain
Similarity search - Domain/homology
DNA / DNA (> 10) / RNA / RNA (> 10) / GGDEF domain-containing protein / Type III-B CRISPR module-associated protein Cmr3 / CRISPR type III-associated protein domain-containing protein / CRISPR system Cmr subunit Cmr5 / CRISPR type III-associated protein domain-containing protein
Similarity search - Component
Biological speciesArchaeoglobus fulgidus DSM 4304 (archaea)
Pyrococcus furiosus DSM 3638 (archaea)
synthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.4 Å
AuthorsIshihara, K. / Numata, T.
Funding support Japan, 3items
OrganizationGrant numberCountry
Japan Society for the Promotion of Science (JSPS)20H02916 Japan
Japan Society for the Promotion of Science (JSPS)24H00505 Japan
Japan Society for the Promotion of Science (JSPS)23KJ1734 Japan
CitationJournal: Biochem Biophys Res Commun / Year: 2025
Title: Cryo-EM structure of Archaeoglobus fulgidus type III-B CRISPR-Cas effector and intermediate crRNA processing during effector assembly.
Authors: Kazuki Ishihara / Sumire Kitagawa / Naruhiko Adachi / Masato Akutsu / Toshiya Senda / Hideko Inanaga / Tomoyuki Numata /
Abstract: Type III CRISPR-Cas effectors recognize target RNAs complementary to the crRNA guide, activating diverse downstream antiviral responses. In contrast to type III-A systems, the architecture of the ...Type III CRISPR-Cas effectors recognize target RNAs complementary to the crRNA guide, activating diverse downstream antiviral responses. In contrast to type III-A systems, the architecture of the type III-B effector (Cmr), comprising six proteins (Cmr1-Cmr6) and a crRNA, remains incompletely defined. Moreover, although maturation of the 3' region of type III crRNA has been attributed to polynucleotide phosphorylase (PNPase), an alternative maturation pathway has been suggested but remains to be elucidated. Here we determined the cryo-EM structure of the Cmr1-lacking Archaeoglobus fulgidus Cmr (AfCmrΔ1) bound to a target analog at 3.4 Å resolution. The complex forms a continuous basic channel that accommodates a crRNA-target heteroduplex. Comparative interface analysis explains why the previously reported cross-species Cmr assembly retains activity, revealing interface flexibility that enables compatible Cmr3-Cmr4 and Cmr2-Cmr5 interactions. Furthermore, we show the cooperative, site-specific processing of an intermediate crRNA that requires both AfCmrΔ1 and AfCmr1 and proceeds without divalent cations. In addition to identifying the cleavage site within the intermediate crRNA, mutational analysis of AfCmr1 reveals residues critical for the reaction. These findings suggest an alternative pathway for crRNA maturation during type III effector assembly that complements PNPase-mediated trimming of the intermediate crRNA, thereby expanding the mechanistic landscape of type III CRISPR-Cas systems.
History
DepositionOct 3, 2025Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Dec 10, 2025Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: crRNA
B: GGDEF domain-containing protein
C: Type III-B CRISPR module-associated protein Cmr3
D: CRISPR type III-associated protein domain-containing protein
E: CRISPR type III-associated protein domain-containing protein
F: CRISPR type III-associated protein domain-containing protein
G: CRISPR system Cmr subunit Cmr5
H: CRISPR system Cmr subunit Cmr5
I: CRISPR type III-associated protein domain-containing protein
K: Target analog DNA


Theoretical massNumber of molelcules
Total (without water)372,62910
Polymers372,62910
Non-polymers00
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 , 3 types, 4 molecules BCGH

#2: Protein GGDEF domain-containing protein


Mass: 117383.156 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Archaeoglobus fulgidus DSM 4304 (archaea)
Gene: AF_1867 / Plasmid: pET28b / Production host: Escherichia coli BL21(DE3) (bacteria) / Variant (production host): Gold CodonPlus RIL / References: UniProt: O28412
#3: Protein Type III-B CRISPR module-associated protein Cmr3


Mass: 39043.328 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Archaeoglobus fulgidus DSM 4304 (archaea)
Gene: AF_1866 / Plasmid: pET28b / Production host: Escherichia coli BL21(DE3) (bacteria) / Variant (production host): Gold CodonPlus RIL / References: UniProt: O28413
#5: Protein CRISPR system Cmr subunit Cmr5 / CRISPR type III-B/RAMP module-associated protein Cmr5


Mass: 17407.707 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Archaeoglobus fulgidus DSM 4304 (archaea)
Gene: cmr5, AF_1862 / Plasmid: pETDuet-1 / Production host: Escherichia coli BL21(DE3) (bacteria) / Variant (production host): Gold CodonPlus RIL / References: UniProt: O28417

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CRISPR type III-associated protein domain-containing ... , 2 types, 4 molecules DEFI

#4: Protein CRISPR type III-associated protein domain-containing protein


Mass: 39677.582 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Archaeoglobus fulgidus DSM 4304 (archaea)
Gene: AF_1863 / Plasmid: pETDuet-1 / Production host: Escherichia coli BL21(DE3) (bacteria) / Variant (production host): Gold CodonPlus RIL / References: UniProt: O28416
#6: Protein CRISPR type III-associated protein domain-containing protein


Mass: 40298.773 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Archaeoglobus fulgidus DSM 4304 (archaea)
Gene: AF_1861 / Plasmid: pETDuet-1 / Production host: Escherichia coli BL21(DE3) (bacteria) / Variant (production host): Gold CodonPlus RIL / References: UniProt: O28418

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RNA chain / DNA chain , 2 types, 2 molecules AK

#1: RNA chain crRNA


Mass: 12604.483 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Pyrococcus furiosus DSM 3638 (archaea)
#7: DNA chain Target analog DNA


Mass: 9451.103 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)

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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: Archaeoglobus fulgidus type III-B Cmr CRISPR-Cas effector
Type: COMPLEX / Entity ID: all / Source: MULTIPLE SOURCES
Molecular weightValue: 0.37 MDa / Experimental value: NO
Source (natural)Organism: Archaeoglobus fulgidus DSM 4304 (archaea)
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria) / Strain: Gold CodonPlus RIL / Plasmid: pET28b, pETDuet-1
Buffer solutionpH: 8
Buffer component
IDConc.NameBuffer-ID
120 mMTris-HCl1
2100 mMNaCl1
35 mMMgCl21
41 mMDTT1
SpecimenConc.: 0.77 mg/ml / Embedding 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: 291 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: 120000 X / Nominal defocus max: 2500 nm / Nominal defocus min: 1000 nm
Specimen holderCryogen: NITROGEN
Image recordingElectron dose: 50 e/Å2 / Film or detector model: FEI FALCON III (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 2559

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Processing

EM software
IDNameVersionCategory
1RELION4.0.1particle selection
2cryoSPARC4.7.0particle selection
3EPUimage acquisition
5cryoSPARC4.7.0CTF correction
8UCSF ChimeraX1.8model fitting
10PHENIX1.21.2_5419model refinement
11cryoSPARC4.7.0initial Euler assignment
12cryoSPARC4.7.0final Euler assignment
13cryoSPARC4.7.0classification
14cryoSPARC4.7.03D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.4 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 81835 / Symmetry type: POINT
Atomic model buildingSource name: AlphaFold / Type: in silico model
RefinementHighest resolution: 3.4 Å
Stereochemistry target values: REAL-SPACE (WEIGHTED MAP SUM AT ATOM CENTERS)
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00422315
ELECTRON MICROSCOPYf_angle_d0.49630609
ELECTRON MICROSCOPYf_dihedral_angle_d12.7473782
ELECTRON MICROSCOPYf_chiral_restr0.043655
ELECTRON MICROSCOPYf_plane_restr0.0033768

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