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- PDB-9l7d: Type III-B CRISPR-Cas effector from Archaeoglobus fulgidus (Form 2) -

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Basic information

Entry
Database: PDB / ID: 9l7d
TitleType III-B CRISPR-Cas effector from Archaeoglobus fulgidus (Form 2)
Components
  • (Type III-B CRISPR module RAMP protein ...) x 2
  • CRISPR system Cmr subunit Cmr5
  • Cmr1 C-terminal domain-containing protein
  • GGDEF domain-containing protein
  • RNA (5'-R(*UP*GP*UP*AP*GP*UP*AP*UP*GP*CP*GP*GP*UP*CP*CP*UP*UP*GP*CP*GP*GP*C)-3')
  • Type III-B CRISPR module-associated protein Cmr3
KeywordsANTIVIRAL PROTEIN / CRISPR-Cas effector
Function / homology
Function and homology information


defense response to virus / cytoplasm
Similarity search - Function
: / Cmr1, C-terminal domain / 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 ...: / Cmr1, C-terminal domain / 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 / : / : / 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
ADENOSINE-5'-TRIPHOSPHATE / RNA / RNA (> 10) / Type III-B CRISPR module RAMP protein Cmr1 / 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)
Archaeoglobus fulgidus (archaea)
Pyrococcus furiosus DSM 3638 (archaea)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.1 Å
AuthorsOuchi, R. / Numata, T.
Funding support Japan, 4items
OrganizationGrant numberCountry
Japan Society for the Promotion of Science (JSPS)16H04759 Japan
Japan Society for the Promotion of Science (JSPS)16KK0166 Japan
Japan Society for the Promotion of Science (JSPS)20H02916 Japan
Japan Society for the Promotion of Science (JSPS)24H00505 Japan
CitationJournal: To Be Published
Title: A new in vitro reconstitution system of type III-B CRISPR-Cas effector for functional and structural studies
Authors: Ouchi, R. / Numata, T.
History
DepositionDec 26, 2024Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Dec 31, 2025Provider: repository / Type: Initial release
Revision 1.0Dec 31, 2025Data content type: EM metadata / Data content type: EM metadata / Provider: repository / Type: Initial release
Revision 1.0Dec 31, 2025Data content type: Additional map / Part number: 1 / Data content type: Additional map / Provider: repository / Type: Initial release
Revision 1.0Dec 31, 2025Data content type: FSC / Data content type: FSC / Provider: repository / Type: Initial release
Revision 1.0Dec 31, 2025Data content type: Half map / Part number: 1 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0Dec 31, 2025Data content type: Half map / Part number: 2 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0Dec 31, 2025Data content type: Image / Data content type: Image / Provider: repository / Type: Initial release
Revision 1.0Dec 31, 2025Data content type: Mask / Part number: 1 / Data content type: Mask / Provider: repository / Type: Initial release
Revision 1.0Dec 31, 2025Data content type: Primary map / Data content type: Primary map / Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
F: Type III-B CRISPR module RAMP protein Cmr4
A: Cmr1 C-terminal domain-containing protein
B: GGDEF domain-containing protein
C: Type III-B CRISPR module-associated protein Cmr3
I: Type III-B CRISPR module RAMP protein Cmr6
G: CRISPR system Cmr subunit Cmr5
E: Type III-B CRISPR module RAMP protein Cmr4
V: RNA (5'-R(*UP*GP*UP*AP*GP*UP*AP*UP*GP*CP*GP*GP*UP*CP*CP*UP*UP*GP*CP*GP*GP*C)-3')
hetero molecules


Theoretical massNumber of molelcules
Total (without water)347,26012
Polymers346,1568
Non-polymers1,1044
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

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Type III-B CRISPR module RAMP protein ... , 2 types, 3 molecules FEI

#1: Protein Type III-B CRISPR module RAMP protein Cmr4


Mass: 41252.254 Da / Num. of mol.: 2 / Mutation: D31A
Source method: isolated from a genetically manipulated source
Details: His-tagged protein
Source: (gene. exp.) Archaeoglobus fulgidus DSM 4304 (archaea)
Gene: AF_1863 / Plasmid: pACYCDuet-1 / Production host: Escherichia coli BL21(DE3) (bacteria) / Variant (production host): Gold CodonPlus RIL / References: UniProt: O28416
#5: Protein Type III-B CRISPR module RAMP protein Cmr6


Mass: 39469.902 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: pACYCDuet-1 / Production host: Escherichia coli BL21(DE3) (bacteria) / Variant (production host): Gold CodonPlus RIL / References: UniProt: O28418

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Protein , 4 types, 4 molecules ABCG

#2: Protein Cmr1 C-terminal domain-containing protein


Mass: 38016.969 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Archaeoglobus fulgidus (archaea) / Gene: AF_1868 / Plasmid: pETDuet-1 / Production host: Escherichia coli BL21(DE3) (bacteria) / Variant (production host): Gold CodonPlus RIL / References: UniProt: O28411
#3: Protein GGDEF domain-containing protein


Mass: 115211.852 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: pETDuet-1 / Production host: Escherichia coli BL21(DE3) (bacteria) / Variant (production host): Gold CodonPlus RIL / References: UniProt: O28412
#4: 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: pETDuet-1 / Production host: Escherichia coli BL21(DE3) (bacteria) / Variant (production host): Gold CodonPlus RIL / References: UniProt: O28413
#6: Protein CRISPR system Cmr subunit Cmr5 / CRISPR type III-B/RAMP module-associated protein Cmr5


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

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RNA chain , 1 types, 1 molecules V

#7: RNA chain RNA (5'-R(*UP*GP*UP*AP*GP*UP*AP*UP*GP*CP*GP*GP*UP*CP*CP*UP*UP*GP*CP*GP*GP*C)-3')


Mass: 14501.589 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Pyrococcus furiosus DSM 3638 (archaea)

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Non-polymers , 3 types, 4 molecules

#8: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Mg / Feature type: SUBJECT OF INVESTIGATION
#9: Chemical ChemComp-ATP / ADENOSINE-5'-TRIPHOSPHATE


Mass: 507.181 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C10H16N5O13P3 / Feature type: SUBJECT OF INVESTIGATION / Comment: ATP, energy-carrying molecule*YM
#10: Chemical ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Zn / Feature type: SUBJECT OF INVESTIGATION

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Details

Has ligand of interestY
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 CRISPR-Cas effector / Type: COMPLEX / Entity ID: #1-#7 / Source: RECOMBINANT
Molecular weightValue: 0.346 MDa / Experimental value: NO
Source (natural)Organism: Archaeoglobus fulgidus DSM 4304 (archaea)
Source (recombinant)Organism: Escherichia coli (E. coli) / Strain: BL21(DE3) Gold CodonPlus RIL / Plasmid: pACYCDuet-1 and pETDuet-1
Buffer solutionpH: 7
Buffer component
IDConc.NameBuffer-ID
120 mMTris-HCl1
2200 mMNaCl1
31 mMDTT1
42 mMMgCl21
SpecimenConc.: 0.8 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 200 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 281 K

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Electron microscopy imaging

MicroscopyModel: JEOL CRYO ARM 300
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 60000 X / Nominal defocus max: 1600 nm / Nominal defocus min: 1400 nm / Cs: 2.7 mm / C2 aperture diameter: 50 µm
Specimen holderCryogen: NITROGEN
Image recordingAverage exposure time: 2.597 sec. / Electron dose: 50 e/Å2 / Film or detector model: GATAN K3 BIOCONTINUUM (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 5500
EM imaging opticsEnergyfilter name: In-column Omega Filter

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Processing

EM software
IDNameVersionCategory
1cryoSPARC4.3.1particle selection
2SerialEMimage acquisition
7UCSF ChimeraX1.7.1model fitting
9PHENIX1.20.1model refinement
10cryoSPARC4.3.1initial Euler assignment
11cryoSPARC4.3.1final Euler assignment
13cryoSPARC4.3.13D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.1 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 66164 / Symmetry type: POINT
Atomic model buildingDetails: The initial model containing all protein subunits was constructed by AlphaFold2
Source name: AlphaFold / Type: in silico model
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 142.01 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00221865
ELECTRON MICROSCOPYf_angle_d0.475129802
ELECTRON MICROSCOPYf_chiral_restr0.03843478
ELECTRON MICROSCOPYf_plane_restr0.00313676
ELECTRON MICROSCOPYf_dihedral_angle_d6.06973237

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