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- PDB-7odf: Structure of the mini-RNA-guided endonuclease CRISPR-Cas_phi3 -

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Basic information

Entry
Database: PDB / ID: 7odf
TitleStructure of the mini-RNA-guided endonuclease CRISPR-Cas_phi3
Components
  • Cas_phi3
  • DNA (5'-D(P*GP*G)-3')
  • DNA (5'-D(P*GP*TP*AP*AP*TP*TP*CP*AP*G)-3')
  • DNA (5'-D(P*GP*TP*AP*TP*CP*CP*CP*AP*TP*TP*AP*CP*CP*AP*GP*CP*TP*GP*AP*AP*TP*TP*AP*C)-3')
  • RNA (43-MER)
KeywordsRNA BINDING PROTEIN / CRISPR-Cas / RNA-guided endonuclease / R-loop
Function / homologyNICKEL (II) ION / DNA / DNA (> 10) / RNA / RNA (> 10)
Function and homology information
Biological speciesPhage #D (virus)
synthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.66 Å
AuthorsCarabias del Rey, A. / Fugilsang, A. / Temperini, P. / Pape, T. / Sofos, N. / Stella, S. / Erledsson, S. / Montoya, G.
Funding support Denmark, 4items
OrganizationGrant numberCountry
Novo Nordisk FoundationNNF14CC0001 Denmark
Novo Nordisk FoundationNNF0024386 Denmark
Novo Nordisk FoundationNNF17SA0030214 Denmark
Novo Nordisk FoundationNNF18OC0055061 Denmark
CitationJournal: Nat Commun / Year: 2021
Title: Structure of the mini-RNA-guided endonuclease CRISPR-Cas12j3.
Authors: Arturo Carabias / Anders Fuglsang / Piero Temperini / Tillmann Pape / Nicholas Sofos / Stefano Stella / Simon Erlendsson / Guillermo Montoya /
Abstract: CRISPR-Cas12j is a recently identified family of miniaturized RNA-guided endonucleases from phages. These ribonucleoproteins provide a compact scaffold gathering all key activities of a genome ...CRISPR-Cas12j is a recently identified family of miniaturized RNA-guided endonucleases from phages. These ribonucleoproteins provide a compact scaffold gathering all key activities of a genome editing tool. We provide the first structural insight into the Cas12j family by determining the cryoEM structure of Cas12j3/R-loop complex after DNA cleavage. The structure reveals the machinery for PAM recognition, hybrid assembly and DNA cleavage. The crRNA-DNA hybrid is directed to the stop domain that splits the hybrid, guiding the T-strand towards the catalytic site. The conserved RuvC insertion is anchored in the stop domain and interacts along the phosphate backbone of the crRNA in the hybrid. The assembly of a hybrid longer than 12-nt activates catalysis through key functional residues in the RuvC insertion. Our findings suggest why Cas12j unleashes unspecific ssDNA degradation after activation. A site-directed mutagenesis analysis supports the DNA cutting mechanism, providing new avenues to redesign CRISPR-Cas12j nucleases for genome editing.
History
DepositionApr 29, 2021Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jul 21, 2021Provider: repository / Type: Initial release
Revision 1.0Jul 21, 2021Data content type: EM metadata / Data content type: EM metadata / Provider: repository / Type: Initial release
Revision 1.0Jul 21, 2021Data content type: Additional map / Part number: 1 / Data content type: Additional map / Provider: repository / Type: Initial release
Revision 1.0Jul 21, 2021Data content type: Additional map / Part number: 2 / Data content type: Additional map / Provider: repository / Type: Initial release
Revision 1.0Jul 21, 2021Data content type: FSC / Data content type: FSC / Provider: repository / Type: Initial release
Revision 1.0Jul 21, 2021Data content type: Image / Data content type: Image / Provider: repository / Type: Initial release
Revision 1.0Jul 21, 2021Data content type: Primary map / Data content type: Primary map / Provider: repository / Type: Initial release
Revision 1.0Jul 21, 2021Data content type: Additional map / Part number: 1 / Data content type: Additional map / Provider: repository / Type: Initial release
Revision 1.0Jul 21, 2021Data content type: Additional map / Part number: 2 / Data content type: Additional map / Provider: repository / Type: Initial release
Revision 1.0Jul 21, 2021Data content type: FSC / Data content type: FSC / Provider: repository / Type: Initial release
Revision 1.0Jul 21, 2021Data content type: Image / Data content type: Image / Provider: repository / Type: Initial release
Revision 1.0Jul 21, 2021Data content type: Primary map / Data content type: Primary map / Provider: repository / Type: Initial release
Revision 1.0Jul 21, 2021Data content type: Additional map / Part number: 1 / Data content type: Additional map / Provider: repository / Type: Initial release
Revision 1.0Jul 21, 2021Data content type: Additional map / Part number: 2 / Data content type: Additional map / Provider: repository / Type: Initial release
Revision 1.0Jul 21, 2021Data content type: FSC / Data content type: FSC / Provider: repository / Type: Initial release
Revision 1.0Jul 21, 2021Data content type: Image / Data content type: Image / Provider: repository / Type: Initial release
Revision 1.0Jul 21, 2021Data content type: Primary map / Data content type: Primary map / Provider: repository / Type: Initial release
Revision 1.1Aug 11, 2021Group: Database references / Source and taxonomy
Category: citation / citation_author ...citation / citation_author / database_2 / entity_src_gen
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 / _database_2.pdbx_DOI / _database_2.pdbx_database_accession / _entity_src_gen.pdbx_gene_src_scientific_name
Revision 1.2Jul 10, 2024Group: Data collection / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / em_admin / refine
Item: _em_admin.last_update / _refine.ls_d_res_high / _refine.ls_d_res_low
Revision 1.3Jul 2, 2025Group: Data collection / Structure summary / Category: em_admin / em_software / pdbx_entry_details
Item: _em_admin.last_update / _em_software.name / _pdbx_entry_details.has_protein_modification
Revision 1.1Jul 2, 2025Data content type: EM metadata / Data content type: EM metadata / EM metadata / Group: Data processing / Experimental summary / Data content type: EM metadata / EM metadata / Category: em_admin / em_software / Data content type: EM metadata / EM metadata / Item: _em_admin.last_update / _em_software.name

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Structure visualization

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  • Deposited structure unit
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  • Superimposition on EM map
  • EMDB-12827
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Structure viewerMolecule:
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Assembly

Deposited unit
A: Cas_phi3
E: DNA (5'-D(P*GP*TP*AP*TP*CP*CP*CP*AP*TP*TP*AP*CP*CP*AP*GP*CP*TP*GP*AP*AP*TP*TP*AP*C)-3')
G: DNA (5'-D(P*GP*TP*AP*AP*TP*TP*CP*AP*G)-3')
C: DNA (5'-D(P*GP*G)-3')
F: RNA (43-MER)
hetero molecules


Theoretical massNumber of molelcules
Total (without water)112,0427
Polymers111,9185
Non-polymers1242
Water905
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration, assay for oligomerization, mass photometry
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area15320 Å2
ΔGint-95 kcal/mol
Surface area41180 Å2

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Components

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DNA chain , 3 types, 3 molecules EGC

#2: DNA chain DNA (5'-D(P*GP*TP*AP*TP*CP*CP*CP*AP*TP*TP*AP*CP*CP*AP*GP*CP*TP*GP*AP*AP*TP*TP*AP*C)-3')


Mass: 7288.730 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#3: DNA chain DNA (5'-D(P*GP*TP*AP*AP*TP*TP*CP*AP*G)-3')


Mass: 2754.835 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#4: DNA chain DNA (5'-D(P*GP*G)-3')


Mass: 613.454 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)

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Protein / RNA chain , 2 types, 2 molecules AF

#1: Protein Cas_phi3


Mass: 87411.664 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Phage #D (virus) / Production host: Escherichia coli (E. coli)
#5: RNA chain RNA (43-MER)


Mass: 13849.232 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)

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Non-polymers , 3 types, 7 molecules

#6: Chemical ChemComp-NI / NICKEL (II) ION


Mass: 58.693 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Ni / Feature type: SUBJECT OF INVESTIGATION
#7: Chemical ChemComp-ZN / ZINC ION


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


Mass: 18.015 Da / Num. of mol.: 5 / 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
IDNameTypeDetailsEntity IDParent-IDSource
1cas_phi/R-loop complexCOMPLEXcrispr-cas rna-guided endonuclease#1-#50RECOMBINANT
2Cas_phi3COMPLEX#11RECOMBINANT
3DNA and RNACOMPLEX#2-#51RECOMBINANT
Molecular weightValue: 0.14 MDa / Experimental value: YES
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
22Phage (virus)77920
33synthetic construct (others)32630
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
22Escherichia coli (E. coli)562
33synthetic construct (others)32630
Buffer solutionpH: 7.4
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD
VitrificationCryogen name: ETHANE-PROPANE

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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: OTHER
Electron lensMode: OTHER / Cs: 2.7 mm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 1.05 sec. / Electron dose: 42 e/Å2 / Detector mode: COUNTING / Film or detector model: FEI FALCON III (4k x 4k)

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Processing

SoftwareName: PHENIX / Version: 1.19.1_4122: / Classification: refinement
EM software
IDNameCategory
1Warpparticle selection
2EPUimage acquisition
4WarpCTF correction
8PHENIXmodel refinement
10cryoSPARCinitial Euler assignment
11cryoSPARCfinal Euler assignment
12cryoSPARCclassification
13cryoSPARC3D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 3504102
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 2.66 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 178412 / Algorithm: FOURIER SPACE / Symmetry type: POINT
RefinementResolution: 2.66→2.66 Å / Cor.coef. Fo:Fc: 0.811 / SU B: 12.457 / SU ML: 0.229 / ESU R: 0.342
Stereochemistry target values: MAXIMUM LIKELIHOOD WITH PHASES
RfactorNum. reflection% reflection
Rwork0.40225 --
obs0.40225 96543 100 %
Solvent computationSolvent model: PARAMETERS FOR MASK CACLULATION
Displacement parametersBiso mean: 95.528 Å2
Baniso -1Baniso -2Baniso -3
1--2.59 Å20.44 Å2-0.51 Å2
2--4.1 Å20.51 Å2
3----1.51 Å2
Refinement stepCycle: 1 / Total: 6846
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0027146
ELECTRON MICROSCOPYf_angle_d0.44910057
ELECTRON MICROSCOPYf_dihedral_angle_d16.9451613
ELECTRON MICROSCOPYf_chiral_restr0.0351185
ELECTRON MICROSCOPYf_plane_restr0.0031014
LS refinement shellResolution: 2.7→2.77 Å / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rfree0 0 -
Rwork3.055 7140 -
obs--100 %

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