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- EMDB-24143: Cryo-EM structure of the Cas12k-sgRNA-dsDNA complex -

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Basic information

Entry
Database: EMDB / ID: EMD-24143
TitleCryo-EM structure of the Cas12k-sgRNA-dsDNA complex
Map dataCas12k-sgRNA-dsDNA complex
Sample
  • Complex: Cryo-EM structure of the Cas12k-sgRNA-dsDNA complex
    • Complex: Cas12k
      • Protein or peptide: Cas12k
    • Complex: single-guide RNA/Target DNA
      • RNA: Single guide RNA
      • DNA: DNA (5'-D(*CP*AP*TP*GP*AP*CP*TP*TP*CP*TP*CP*AP*AP*CP*CP*GP*AP*GP*TP*TP*T)-3')
      • DNA: DNA (5'-D(P*AP*AP*AP*CP*TP*CP*GP*GP*TP*T)-3')
Biological speciesScytonema hofmannii (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.65 Å
AuthorsChang L / Li Z / Xiao R / Wang S / Han R
CitationJournal: Mol Cell / Year: 2021
Title: Structural basis of target DNA recognition by CRISPR-Cas12k for RNA-guided DNA transposition.
Authors: Renjian Xiao / Shukun Wang / Ruijie Han / Zhuang Li / Clinton Gabel / Indranil Arun Mukherjee / Leifu Chang /
Abstract: The type V-K CRISPR-Cas system, featured by Cas12k effector with a naturally inactivated RuvC domain and associated with Tn7-like transposon for RNA-guided DNA transposition, is a promising tool for ...The type V-K CRISPR-Cas system, featured by Cas12k effector with a naturally inactivated RuvC domain and associated with Tn7-like transposon for RNA-guided DNA transposition, is a promising tool for precise DNA insertion. To reveal the mechanism underlying target DNA recognition, we determined a cryoelectron microscopy (cryo-EM) structure of Cas12k from cyanobacteria Scytonema hofmanni in complex with a single guide RNA (sgRNA) and a double-stranded target DNA. Coupled with mutagenesis and in vitro DNA transposition assay, our results revealed mechanisms for the recognition of the GGTT protospacer adjacent motif (PAM) sequence and the structural elements of Cas12k critical for RNA-guided DNA transposition. These structural and mechanistic insights should aid in the development of type V-K CRISPR-transposon systems as tools for genome editing.
History
DepositionJun 1, 2021-
Header (metadata) releaseSep 1, 2021-
Map releaseSep 1, 2021-
UpdateMar 16, 2022-
Current statusMar 16, 2022Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.467
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.467
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-7n3p
  • Surface level: 0.467
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_24143.png

Downloads & links

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Map

FileDownload / File: emd_24143.map.gz / Format: CCP4 / Size: 30.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationCas12k-sgRNA-dsDNA complex
Voxel sizeX=Y=Z: 1.05 Å
Density
Contour LevelBy AUTHOR: 0.467 / Movie #1: 0.467
Minimum - Maximum-1.6226313 - 2.8697736
Average (Standard dev.)0.003929524 (±0.108146586)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions200200200
Spacing200200200
CellA=B=C: 209.99998 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.051.051.05
M x/y/z200200200
origin x/y/z0.0000.0000.000
length x/y/z210.000210.000210.000
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS200200200
D min/max/mean-1.6232.8700.004

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Supplemental data

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Sample components

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Entire : Cryo-EM structure of the Cas12k-sgRNA-dsDNA complex

EntireName: Cryo-EM structure of the Cas12k-sgRNA-dsDNA complex
Components
  • Complex: Cryo-EM structure of the Cas12k-sgRNA-dsDNA complex
    • Complex: Cas12k
      • Protein or peptide: Cas12k
    • Complex: single-guide RNA/Target DNA
      • RNA: Single guide RNA
      • DNA: DNA (5'-D(*CP*AP*TP*GP*AP*CP*TP*TP*CP*TP*CP*AP*AP*CP*CP*GP*AP*GP*TP*TP*T)-3')
      • DNA: DNA (5'-D(P*AP*AP*AP*CP*TP*CP*GP*GP*TP*T)-3')

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Supramolecule #1: Cryo-EM structure of the Cas12k-sgRNA-dsDNA complex

SupramoleculeName: Cryo-EM structure of the Cas12k-sgRNA-dsDNA complex / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all

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Supramolecule #2: Cas12k

SupramoleculeName: Cas12k / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1
Source (natural)Organism: Scytonema hofmannii (bacteria)
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)

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Supramolecule #3: single-guide RNA/Target DNA

SupramoleculeName: single-guide RNA/Target DNA / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #2-#4
Source (natural)Organism: Scytonema hofmannii (bacteria)

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Macromolecule #1: Cas12k

MacromoleculeName: Cas12k / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Scytonema hofmannii (bacteria)
Molecular weightTheoretical: 73.280719 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MSQITIQARL ISFESNRQQL WKLMADLNTP LINELLCQLG QHPDFEKWQQ KGKLPSTVVS QLCQPLKTDP RFAGQPSRLY MSAIHIVDY IYKSWLAIQK RLQQQLDGKT RWLEMLNSDA ELVELSGDTL EAIRVKAAEI LAIAMPASES DSASPKGKKG K KEKKPSSS ...String:
MSQITIQARL ISFESNRQQL WKLMADLNTP LINELLCQLG QHPDFEKWQQ KGKLPSTVVS QLCQPLKTDP RFAGQPSRLY MSAIHIVDY IYKSWLAIQK RLQQQLDGKT RWLEMLNSDA ELVELSGDTL EAIRVKAAEI LAIAMPASES DSASPKGKKG K KEKKPSSS SPKRSLSKTL FDAYQETEDI KSRSAISYLL KNGCKLTDKE EDSEKFAKRR RQVEIQIQRL TEKLISRMPK GR DLTNAKW LETLLTATTT VAEDNAQAKR WQDILLTRSS SLPFPLVFET NEDMVWSKNQ KGRLCVHFNG LSDLIFEVYC GNR QLHWFQ RFLEDQQTKR KSKNQHSSGL FTLRNGHLVW LEGEGKGEPW NLHHLTLYCC VDNRLWTEEG TEIVRQEKAD EITK FITNM KKKSDLSDTQ QALIQRKQST LTRINNSFER PSQPLYQGQS HILVGVSLGL EKPATVAVVD AIANKVLAYR SIKQL LGDN YELLNRQRRQ QQYLSHERHK AQKNFSPNQF GASELGQHID RLLAKAIVAL ARTYKAGSIV LPKLGDMREV VQSEIQ AIA EQKFPGYIEG QQKYAKQYRV NVHRWSYGRL IQSIQSKAAQ TGIVIEEGKQ PIRGSPHDKA KELALSAYNL RLTRRS

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Macromolecule #2: Single guide RNA

MacromoleculeName: Single guide RNA / type: rna / ID: 2 / Number of copies: 1
Source (natural)Organism: Scytonema hofmannii (bacteria)
Molecular weightTheoretical: 85.26125 KDa
SequenceString: AUAUUAAUAG CGCCGCAAUU CAUGCUGCUU GCAGCCUCUG AAUUUUGUUA AAUGAGGGUU AGUUUGACUG UAUAAAUACA GUCUUGCUU UCUGACCCUG GUAGCUGCUC ACCCUGAUGC UGCUGUCAAU AGACAGGAUA GGUGCGCUCC CAGCAAUAAG G GCGCGGAU ...String:
AUAUUAAUAG CGCCGCAAUU CAUGCUGCUU GCAGCCUCUG AAUUUUGUUA AAUGAGGGUU AGUUUGACUG UAUAAAUACA GUCUUGCUU UCUGACCCUG GUAGCUGCUC ACCCUGAUGC UGCUGUCAAU AGACAGGAUA GGUGCGCUCC CAGCAAUAAG G GCGCGGAU GUACUGCUGU AGUGGCUACU GAAUCACCCC CGAUCAAGGG GGAACCCUCC AAAAGGUGGG UUGAAAGGAG AA GUCAUUU AAUAAGGCCA CUGUUAAA

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Macromolecule #3: DNA (5'-D(*CP*AP*TP*GP*AP*CP*TP*TP*CP*TP*CP*AP*AP*CP*CP*GP*AP*GP*...

MacromoleculeName: DNA (5'-D(*CP*AP*TP*GP*AP*CP*TP*TP*CP*TP*CP*AP*AP*CP*CP*GP*AP*GP*TP*TP*T)-3')
type: dna / ID: 3 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: Scytonema hofmannii (bacteria)
Molecular weightTheoretical: 12.181854 KDa
SequenceString:
(DT)(DT)(DA)(DA)(DC)(DA)(DG)(DT)(DG)(DG) (DC)(DC)(DT)(DT)(DA)(DT)(DT)(DA)(DA)(DA) (DT)(DG)(DA)(DC)(DT)(DT)(DC)(DT)(DC) (DA)(DA)(DC)(DC)(DT)(DC)(DC)(DT)(DA)(DC) (DG)

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Macromolecule #4: DNA (5'-D(P*AP*AP*AP*CP*TP*CP*GP*GP*TP*T)-3')

MacromoleculeName: DNA (5'-D(P*AP*AP*AP*CP*TP*CP*GP*GP*TP*T)-3') / type: dna / ID: 4 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: Scytonema hofmannii (bacteria)
Molecular weightTheoretical: 3.429245 KDa
SequenceString:
(DC)(DG)(DT)(DA)(DG)(DG)(DA)(DG)(DG)(DT) (DT)

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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: 7.6
VitrificationCryogen name: ETHANE

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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
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average electron dose: 54.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 3021295
Initial angle assignmentType: RANDOM ASSIGNMENT
Final angle assignmentType: MAXIMUM LIKELIHOOD
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.65 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 114383

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