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- PDB-9wxj: Cryo-EM structure of the type III-D2 CRISPR-Cas effector complex ... -

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Basic information

Entry
Database: PDB / ID: 9wxj
TitleCryo-EM structure of the type III-D2 CRISPR-Cas effector complex bound to a non-cognate target RNA in the pre-cleavage state
Components
  • CRISPR RNA
  • CRISPR type III-associated protein domain-containing protein
  • Cas10
  • Non-cognate target RNA (NTR)
  • TIGR03986 family CRISPR-associated RAMP protein
KeywordsIMMUNE SYSTEM / CRISPR / Anti-phage defense / Second messenger / SAM-AMP
Function / homologyPHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER / S-ADENOSYLMETHIONINE / RNA / RNA (> 10) / : / : / :
Function and homology information
Biological speciesGammaproteobacteria bacterium (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.7 Å
AuthorsMitsuda, Y. / Ishikawa, J. / Nagahata, N. / Hiraizumi, M. / Yamashita, K. / Nishimasu, H.
Funding support Japan, 2items
OrganizationGrant numberCountry
Japan Science and TechnologyJPMJCR23B6 Japan
Japan Society for the Promotion of Science (JSPS)25H00436 Japan
CitationJournal: To Be Published
Title: Structural mechanism of SAM-AMP synthesis by the type III-D2 CRISPR effector complex
Authors: Mitsuda, Y. / Sugaya, M. / Ishikawa, J. / Nagahata, N. / Okazaki, S. / Hiraizumi, M. / Kato, K. / Gootenberg, J.S. / Abudayyeh, O.O. / Osawa, T. / Yamashita, K. / Nishimasu, H.
History
DepositionSep 25, 2025Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Jun 24, 2026Provider: repository / Type: Initial release
Revision 1.0Jun 24, 2026Data content type: EM metadata / Data content type: EM metadata / Provider: repository / Type: Initial release
Revision 1.0Jun 24, 2026Data content type: FSC / Data content type: FSC / Provider: repository / Type: Initial release
Revision 1.0Jun 24, 2026Data content type: Half map / Part number: 1 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0Jun 24, 2026Data content type: Half map / Part number: 2 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0Jun 24, 2026Data content type: Image / Data content type: Image / Provider: repository / Type: Initial release
Revision 1.0Jun 24, 2026Data content type: Mask / Part number: 1 / Data content type: Mask / Provider: repository / Type: Initial release
Revision 1.0Jun 24, 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: CRISPR RNA
B: Cas10
C: CRISPR type III-associated protein domain-containing protein
D: TIGR03986 family CRISPR-associated RAMP protein
E: Non-cognate target RNA (NTR)
hetero molecules


Theoretical massNumber of molelcules
Total (without water)306,84810
Polymers305,8295
Non-polymers1,0195
Water181
1


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

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Components

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RNA chain , 2 types, 2 molecules AE

#1: RNA chain CRISPR RNA


Mass: 13718.080 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Gammaproteobacteria bacterium (bacteria)
#5: RNA chain Non-cognate target RNA (NTR)


Mass: 15054.008 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Gammaproteobacteria bacterium (bacteria)

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Protein , 3 types, 3 molecules BCD

#2: Protein Cas10


Mass: 67985.797 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Gammaproteobacteria bacterium (bacteria)
Gene: ENJ84_12795 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: A0A9D0P2E0
#3: Protein CRISPR type III-associated protein domain-containing protein


Mass: 70690.969 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Gammaproteobacteria bacterium (bacteria)
Gene: ENJ84_12800 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: A0A9D0P213
#4: Protein TIGR03986 family CRISPR-associated RAMP protein


Mass: 138380.078 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Gammaproteobacteria bacterium (bacteria)
Gene: ENJ84_12805 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: A0A9D0P2B2

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Non-polymers , 5 types, 6 molecules

#6: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Mg / Feature type: SUBJECT OF INVESTIGATION
#7: Chemical ChemComp-ANP / PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER


Mass: 506.196 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C10H17N6O12P3 / Feature type: SUBJECT OF INVESTIGATION / Comment: AMP-PNP, energy-carrying molecule analogue*YM
#8: Chemical ChemComp-SAM / S-ADENOSYLMETHIONINE


Mass: 398.437 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C15H22N6O5S / Feature type: SUBJECT OF INVESTIGATION
#9: Chemical ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Zn / Feature type: SUBJECT OF INVESTIGATION
#10: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Formula: H2O

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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

Component
IDNameTypeEntity IDParent-IDSource
1Cryo-EM structure of the type III-D2 CRISPR-Cas effector complex bound to a non-cognate target RNA in the pre-cleavage stateCOMPLEX#1-#50MULTIPLE SOURCES
2RNACOMPLEX#1, #51SYNTHETIC
3Cas7x3-Cas7-5-Cas10COMPLEX#2-#41RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Gammaproteobacteria bacterium (bacteria)
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria)
Buffer solutionpH: 7
Buffer component
IDConc.NameFormulaBuffer-ID
1150 mMsodium chlorideNaCl1
220 mMHEPES-NaOHC8H17N2O4SNa1
35 mMmagnesium chlorideMgCl21
41 mMS-adenosylmethinonineC15H22N6O5S1
51 mMAdenylyl imidodiphosphateC10H17N6O12P31
SpecimenConc.: 0.15 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: TFS KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 2000 nm / Nominal defocus min: 800 nm
Image recordingElectron dose: 50.6 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

EM software
IDNameCategory
1cryoSPARCparticle selection
9Servalcatmodel refinement
11cryoSPARCfinal Euler assignment
13cryoSPARC3D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 2.7 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 135266 / Symmetry type: POINT

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