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- EMDB-12827: Structure of the mini-RNA-guided endonuclease CRISPR-Cas_phi3 -

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Basic information

Entry
Database: EMDB / ID: EMD-12827
TitleStructure of the mini-RNA-guided endonuclease CRISPR-Cas_phi3
Map datamap 3 different conformation
Sample
  • Complex: cas_phi/R-loop complex
    • Complex: Cas_phi3
      • Protein or peptide: Cas_phi3
    • Complex: DNA and RNA
      • DNA: 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')
      • DNA: DNA (5'-D(P*GP*TP*AP*AP*TP*TP*CP*AP*G)-3')
      • DNA: DNA (5'-D(P*GP*G)-3')
      • RNA: RNA (43-MER)
  • Ligand: NICKEL (II) ION
  • Ligand: ZINC ION
  • Ligand: water
Biological speciesPhage (virus) / synthetic construct (others) / Phage #D (virus)
Methodsingle 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, 4 items
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, 2021-
Header (metadata) releaseJul 21, 2021-
Map releaseJul 21, 2021-
UpdateAug 11, 2021-
Current statusAug 11, 2021Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 4
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 4
  • Imaged by UCSF Chimera
  • Download
  • Surface view with fitted model
  • Atomic models: PDB-7odf
  • Surface level: 4
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_12827.png

Downloads & links

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Map

FileDownload / File: emd_12827.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationmap 3 different conformation
Voxel sizeX=Y=Z: 0.832 Å
Density
Contour LevelBy AUTHOR: 4.0 / Movie #1: 4
Minimum - Maximum-27.304577 - 44.54783
Average (Standard dev.)-6.3258287e-12 (±1.0)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderZYX
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 212.992 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z0.8320.8320.832
M x/y/z256256256
origin x/y/z0.0000.0000.000
length x/y/z212.992212.992212.992
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ256256256
MAP C/R/S321
start NC/NR/NS000
NC/NR/NS256256256
D min/max/mean-27.30544.548-0.000

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Supplemental data

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Additional map: #1

Fileemd_12827_additional_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: map 2 different conformation

Fileemd_12827_additional_2.map
Annotationmap 2 different conformation
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : cas_phi/R-loop complex

EntireName: cas_phi/R-loop complex
Components
  • Complex: cas_phi/R-loop complex
    • Complex: Cas_phi3
      • Protein or peptide: Cas_phi3
    • Complex: DNA and RNA
      • DNA: 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')
      • DNA: DNA (5'-D(P*GP*TP*AP*AP*TP*TP*CP*AP*G)-3')
      • DNA: DNA (5'-D(P*GP*G)-3')
      • RNA: RNA (43-MER)
  • Ligand: NICKEL (II) ION
  • Ligand: ZINC ION
  • Ligand: water

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Supramolecule #1: cas_phi/R-loop complex

SupramoleculeName: cas_phi/R-loop complex / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#5 / Details: crispr-cas rna-guided endonuclease
Molecular weightExperimental: 140 KDa

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Supramolecule #2: Cas_phi3

SupramoleculeName: Cas_phi3 / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1
Source (natural)Organism: Phage (virus)
Recombinant expressionOrganism: Escherichia coli (E. coli)

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Supramolecule #3: DNA and RNA

SupramoleculeName: DNA and RNA / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #2-#5
Source (natural)Organism: synthetic construct (others)
Recombinant expressionOrganism: synthetic construct (others)

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Macromolecule #1: Cas_phi3

MacromoleculeName: Cas_phi3 / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Phage #D (virus)
Molecular weightTheoretical: 87.411664 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MEKEITELTK IRREFPNKKF SSTDMKKAGK LLKAEGPDAV RDFLNSCQEI IGDFKPPVKT NIVSISRPFE EWPVSMVGRA IQEYYFSLT KEELESVHPG TSSEDHKSFF NITGLSNYNY TSVQGLNLIF KNAKAIYDGT LVKANNKNKK LEKKFNEINH K RSLEGLPI ...String:
MEKEITELTK IRREFPNKKF SSTDMKKAGK LLKAEGPDAV RDFLNSCQEI IGDFKPPVKT NIVSISRPFE EWPVSMVGRA IQEYYFSLT KEELESVHPG TSSEDHKSFF NITGLSNYNY TSVQGLNLIF KNAKAIYDGT LVKANNKNKK LEKKFNEINH K RSLEGLPI ITPDFEEPFD ENGHLNNPPG INRNIYGYQG CAAKVFVPSK HKMVSLPKEY EGYNRDPNLS LAGFRNRLEI PE GEPGHVP WFQRMDIPEG QIGHVNKIQR FNFVHGKNSG KVKFSDKTGR VKRYHHSKYK DATKPYKFLE ESKKVSALDS ILA IITIGD DWVVFDIRGL YRNVFYRELA QKGLTAVQLL DLFTGDPVID PKKGVVTFSY KEGVVPVFSQ KIVPRFKSRD TLEK LTSQG PVALLSVDLG QNEPVAARVC SLKNINDKIT LDNSCRISFL DDYKKQIKDY RDSLDELEIK IRLEAINSLE TNQQV EIRD LDVFSADRAK ANTVDMFDID PNLISWDSMS DARVSTQISD LYLKNGGDES RVYFEINNKR IKRSDYNISQ LVRPKL SDS TRKNLNDSIW KLKRTSEEYL KLSKRKLELS RAVVNYTIRQ SKLLSGINDI VIILEDLDVK KKFNGRGIRD IGWDNFF SS RKENRWFIPA FHKAFSELSS NRGLCVIEVN PAWTSATCPD CGFCSKENRD GINFTCRKCG VSYHADIDVA TLNIARVA V LGKPMSGPAD RERLGDTKKP RVARSRKTMK RKDISNSTVE AMVTA

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Macromolecule #2: DNA (5'-D(P*GP*TP*AP*TP*CP*CP*CP*AP*TP*TP*AP*CP*CP*AP*GP*CP*TP*GP...

MacromoleculeName: 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')
type: dna / ID: 2 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 7.28873 KDa
SequenceString:
(DG)(DT)(DA)(DT)(DC)(DC)(DC)(DA)(DT)(DT) (DA)(DC)(DC)(DA)(DG)(DC)(DT)(DG)(DA)(DA) (DT)(DT)(DA)(DC)

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Macromolecule #3: DNA (5'-D(P*GP*TP*AP*AP*TP*TP*CP*AP*G)-3')

MacromoleculeName: DNA (5'-D(P*GP*TP*AP*AP*TP*TP*CP*AP*G)-3') / type: dna / ID: 3 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 2.754835 KDa
SequenceString:
(DG)(DT)(DA)(DA)(DT)(DT)(DC)(DA)(DG)

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Macromolecule #4: DNA (5'-D(P*GP*G)-3')

MacromoleculeName: DNA (5'-D(P*GP*G)-3') / type: dna / ID: 4 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 613.454 Da
SequenceString:
(DG)(DG)

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Macromolecule #5: RNA (43-MER)

MacromoleculeName: RNA (43-MER) / type: rna / ID: 5 / Number of copies: 1
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 13.849232 KDa
SequenceString:
AUUGAUUGCC CAGUACGCUG GGACAGCUGG UAAUGGGAUA CCU

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Macromolecule #6: NICKEL (II) ION

MacromoleculeName: NICKEL (II) ION / type: ligand / ID: 6 / Number of copies: 1 / Formula: NI
Molecular weightTheoretical: 58.693 Da
Chemical component information

ChemComp-NI:
NICKEL (II) ION / Nickel

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Macromolecule #7: ZINC ION

MacromoleculeName: ZINC ION / type: ligand / ID: 7 / Number of copies: 1 / Formula: ZN
Molecular weightTheoretical: 65.409 Da

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Macromolecule #8: water

MacromoleculeName: water / type: ligand / ID: 8 / Number of copies: 5 / Formula: HOH
Molecular weightTheoretical: 18.015 Da
Chemical component information

ChemComp-HOH:
WATER / Water

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

BufferpH: 7.4
GridMaterial: GOLD
VitrificationCryogen name: ETHANE-PROPANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: OTHER / Imaging mode: OTHER / Cs: 2.7 mm
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: FEI FALCON III (4k x 4k) / Detector mode: COUNTING / Average exposure time: 1.05 sec. / Average electron dose: 42.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 3504102
CTF correctionSoftware - Name: Warp
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC
Final 3D classificationSoftware - Name: cryoSPARC
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 2.66 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC / Number images used: 178412
FSC plot (resolution estimation)

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