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- EMDB-19282: Conformational Landscape of the Type V-K CRISPR-associated Transp... -

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Basic information

Entry
Database: EMDB / ID: EMD-19282
TitleConformational Landscape of the Type V-K CRISPR-associated TransposonIntegration Assembly CAST V-K Cas12k domain local-refinement map
Map dataCAST V-K Cas12k domain local-refinement map
Sample
  • Complex: recognition complex
    • RNA: sgRNASubgenomic mRNA
    • DNA: Non-target strand - LE
    • DNA: Target strand - LE
    • Protein or peptide: ShCas12k
    • Protein or peptide: Small ribosomal subunit protein uS15
    • Protein or peptide: TniQ
  • Ligand: MAGNESIUM ION
  • Ligand: ZINC ION
  • Ligand: water
KeywordsCRISPR-associated Transposon genome editing transposition / DNA BINDING PROTEIN
Function / homology
Function and homology information


cytosolic small ribosomal subunit / rRNA binding / structural constituent of ribosome / translation
Similarity search - Function
TniQ / TniQ / Ribosomal protein S15, bacterial-type / Ribosomal protein S15 signature. / Ribosomal protein S15 / Ribosomal_S15 / Ribosomal protein S15 / S15/NS1, RNA-binding
Similarity search - Domain/homology
Small ribosomal subunit protein uS15 / TniQ (Homology model) / ShCas12k
Similarity search - Component
Biological speciesScytonema hofmannii (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.35 Å
AuthorsTenjo-Castano F / Mesa P / Montoya G
Funding support Denmark, 1 items
OrganizationGrant numberCountry
Novo Nordisk Foundation Denmark
CitationJournal: Mol Cell / Year: 2024
Title: Conformational landscape of the type V-K CRISPR-associated transposon integration assembly.
Authors: Francisco Tenjo-Castaño / Nicholas Sofos / Luisa S Stutzke / Piero Temperini / Anders Fuglsang / Tillmann Pape / Pablo Mesa / Guillermo Montoya /
Abstract: CRISPR-associated transposons (CASTs) are mobile genetic elements that co-opt CRISPR-Cas systems for RNA-guided DNA transposition. CASTs integrate large DNA cargos into the attachment (att) site ...CRISPR-associated transposons (CASTs) are mobile genetic elements that co-opt CRISPR-Cas systems for RNA-guided DNA transposition. CASTs integrate large DNA cargos into the attachment (att) site independently of homology-directed repair and thus hold promise for eukaryotic genome engineering. However, the functional diversity and complexity of CASTs hinder an understanding of their mechanisms. Here, we present the high-resolution cryoelectron microscopy (cryo-EM) structure of the reconstituted ∼1 MDa post-transposition complex of the type V-K CAST, together with different assembly intermediates and diverse TnsC filament lengths, thus enabling the recapitulation of the integration complex formation. The results of mutagenesis experiments probing the roles of specific residues and TnsB-binding sites show that transposition activity can be enhanced and suggest that the distance between the PAM and att sites is determined by the lengths of the TnsB C terminus and the TnsC filament. This singular model of RNA-guided transposition provides a foundation for repurposing the system for genome-editing applications.
History
DepositionDec 30, 2023-
Header (metadata) releaseJun 19, 2024-
Map releaseJun 19, 2024-
UpdateJun 19, 2024-
Current statusJun 19, 2024Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_19282.png

Downloads & links

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Map

FileDownload / File: emd_19282.map.gz / Format: CCP4 / Size: 106.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationCAST V-K Cas12k domain local-refinement map
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.73 Å/pix.
x 483 pix.
= 351.624 Å		0.73 Å/pix.
x 241 pix.
= 175.448 Å		0.73 Å/pix.
x 239 pix.
= 173.992 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

generated in cubic-lattice coordinate

Voxel sizeX=Y=Z: 0.728 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.24
Minimum - Maximum-1.0265458 - 1.9208239
Average (Standard dev.)0.0021386563 (±0.07536417)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin314315152
Dimensions241239483
Spacing239241483
CellA: 173.99199 Å / B: 175.448 Å / C: 351.624 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_19282_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: CAST V-K Cas12k domain local refinement, half-A map

Fileemd_19282_half_map_1.map
AnnotationCAST V-K Cas12k domain local refinement, half-A map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: CAST V-K Cas12k domain local refinement, half-B map

Fileemd_19282_half_map_2.map
AnnotationCAST V-K Cas12k domain local refinement, half-B map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : recognition complex

EntireName: recognition complex
Components
  • Complex: recognition complex
    • RNA: sgRNASubgenomic mRNA
    • DNA: Non-target strand - LE
    • DNA: Target strand - LE
    • Protein or peptide: ShCas12k
    • Protein or peptide: Small ribosomal subunit protein uS15
    • Protein or peptide: TniQ
  • Ligand: MAGNESIUM ION
  • Ligand: ZINC ION
  • Ligand: water

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Supramolecule #1: recognition complex

SupramoleculeName: recognition complex / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#6
Source (natural)Organism: Scytonema hofmannii (bacteria)

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Macromolecule #1: sgRNA

MacromoleculeName: sgRNA / type: rna / ID: 1 / Number of copies: 1
Source (natural)Organism: Scytonema hofmannii (bacteria)
Molecular weightTheoretical: 84.006516 KDa
SequenceString: GGAUAUUAAU AGCGCCGCAA UUCAUGCUGC UUGCAGCCUC UGAAUUUUGU UAAAUGAGGG UUAGUUUGAC UGUAUAAAUA CAGUCUUGC UUUCUGACCC UGGUAGCUGC UCACCCUGAU GCUGCUGUCA AUAGACAGGA UAGGUGCGCU CCCAGCAAUA A GGGCGCGG ...String:
GGAUAUUAAU AGCGCCGCAA UUCAUGCUGC UUGCAGCCUC UGAAUUUUGU UAAAUGAGGG UUAGUUUGAC UGUAUAAAUA CAGUCUUGC UUUCUGACCC UGGUAGCUGC UCACCCUGAU GCUGCUGUCA AUAGACAGGA UAGGUGCGCU CCCAGCAAUA A GGGCGCGG AUGUACUGCU GUAGUGGCUA CUGAAUCACC CCCGAUCAAG GGGGAACCCU CCAAAAGGUG GGUUGAAAGG AG AAGUCAU UUAAUAAGGC CACU

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Macromolecule #2: Non-target strand - LE

MacromoleculeName: Non-target strand - LE / type: dna / ID: 2 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: Scytonema hofmannii (bacteria)
Molecular weightTheoretical: 20.961504 KDa
SequenceString: (DG)(DT)(DG)(DA)(DA)(DG)(DG)(DT)(DT)(DC) (DT)(DC)(DT)(DT)(DC)(DA)(DG)(DT)(DA)(DT) (DT)(DA)(DA)(DT)(DA)(DA)(DG)(DG)(DC) (DC)(DA)(DC)(DT)(DG)(DT)(DT)(DA)(DA)(DA) (DA) (DC)(DG)(DT)(DA)(DC)(DT) ...String:
(DG)(DT)(DG)(DA)(DA)(DG)(DG)(DT)(DT)(DC) (DT)(DC)(DT)(DT)(DC)(DA)(DG)(DT)(DA)(DT) (DT)(DA)(DA)(DT)(DA)(DA)(DG)(DG)(DC) (DC)(DA)(DC)(DT)(DG)(DT)(DT)(DA)(DA)(DA) (DA) (DC)(DG)(DT)(DA)(DC)(DT)(DA)(DT) (DA)(DT)(DA)(DG)(DA)(DC)(DA)(DT)(DC)(DT) (DC)(DC) (DA)(DC)(DA)(DA)(DA)(DA)(DG) (DG)

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Macromolecule #3: Target strand - LE

MacromoleculeName: Target strand - LE / type: dna / ID: 3 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: Scytonema hofmannii (bacteria)
Molecular weightTheoretical: 40.82216 KDa
SequenceString: (DA)(DA)(DT)(DT)(DA)(DA)(DA)(DT)(DA)(DG) (DT)(DC)(DA)(DC)(DA)(DA)(DT)(DG)(DA)(DC) (DA)(DT)(DT)(DA)(DA)(DT)(DC)(DT)(DG) (DT)(DC)(DA)(DC)(DC)(DG)(DA)(DC)(DG)(DA) (DC) (DA)(DG)(DA)(DT)(DA)(DA) ...String:
(DA)(DA)(DT)(DT)(DA)(DA)(DA)(DT)(DA)(DG) (DT)(DC)(DA)(DC)(DA)(DA)(DT)(DG)(DA)(DC) (DA)(DT)(DT)(DA)(DA)(DT)(DC)(DT)(DG) (DT)(DC)(DA)(DC)(DC)(DG)(DA)(DC)(DG)(DA) (DC) (DA)(DG)(DA)(DT)(DA)(DA)(DT)(DT) (DT)(DG)(DT)(DC)(DA)(DC)(DT)(DG)(DT)(DA) (DC)(DA) (DC)(DT)(DA)(DC)(DG)(DC)(DC) (DT)(DT)(DT)(DT)(DG)(DT)(DG)(DG)(DA)(DG) (DA)(DT)(DG) (DT)(DC)(DT)(DA)(DA)(DT) (DA)(DT)(DC)(DT)(DA)(DC)(DG)(DT)(DT)(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)(DT)(DC)(DA)(DC)

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Macromolecule #4: ShCas12k

MacromoleculeName: ShCas12k / type: protein_or_peptide / ID: 4 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Scytonema hofmannii (bacteria)
Molecular weightTheoretical: 79.479312 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MGSSHHHHHH SSGLVPRGSH MASWSHPQFE KGGGSGGGSG GSAWSHPQFE KENLYFQGGS SQITIQARLI SFESNRQQLW KLMADLNTP LINELLCQLG QHPDFEKWQQ KGKLPSTVVS QLCQPLKTDP RFAGQPSRLY MSAIHIVDYI YKSWLAIQKR L QQQLDGKT ...String:
MGSSHHHHHH SSGLVPRGSH MASWSHPQFE KGGGSGGGSG GSAWSHPQFE KENLYFQGGS SQITIQARLI SFESNRQQLW KLMADLNTP LINELLCQLG QHPDFEKWQQ KGKLPSTVVS QLCQPLKTDP RFAGQPSRLY MSAIHIVDYI YKSWLAIQKR L QQQLDGKT RWLEMLNSDA ELVELSGDTL EAIRVKAAEI LAIAMPASES DSASPKGKKG KKEKKPSSSS PKRSLSKTLF DA YQETEDI KSRSAISYLL KNGCKLTDKE EDSEKFAKRR RQVEIQIQRL TEKLISRMPK GRDLTNAKWL ETLLTATTTV AED NAQAKR WQDILLTRSS SLPFPLVFET NEDMVWSKNQ KGRLCVHFNG LSDLIFEVYC GNRQLHWFQR FLEDQQTKRK SKNQ HSSGL FTLRNGHLVW LEGEGKGEPW NLHHLTLYCC VDNRLWTEEG TEIVRQEKAD EITKFITNMK KKSDLSDTQQ ALIQR KQST LTRINNSFER PSQPLYQGQS HILVGVSLGL EKPATVAVVD AIANKVLAYR SIKQLLGDNY ELLNRQRRQQ QYLSHE RHK AQKNFSPNQF GASELGQHID RLLAKAIVAL ARTYKAGSIV LPKLGDMREV VQSEIQAIAE QKFPGYIEGQ QKYAKQY RV NVHRWSYGRL IQSIQSKAAQ TGIVIEEGKQ PIRGSPHDKA KELALSAYNL RLTRRS

UniProtKB: ShCas12k

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Macromolecule #5: Small ribosomal subunit protein uS15

MacromoleculeName: Small ribosomal subunit protein uS15 / type: protein_or_peptide / ID: 5 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Scytonema hofmannii (bacteria)
Molecular weightTheoretical: 10.376003 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
SMALTQERKQ EIIVNYQVHE TDTGSADVQV AMLTERINRL SLHLQANKKD HSSRRGLLKL IGQRKRLLAY IQKDSREKYQ ALIGRLGIR G

UniProtKB: Small ribosomal subunit protein uS15

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Macromolecule #6: TniQ

MacromoleculeName: TniQ / type: protein_or_peptide / ID: 6 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Scytonema hofmannii (bacteria)
Molecular weightTheoretical: 20.468783 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
MIEAPDVKPW LFLIKPYEGE SLSHFLGRFR RANHLSASGL GTLAGIGAIV ARWERFHFNP RPSQQELEAI ASVVEVDAQR LAQMLPPAG VGMQHEPIRL CGACYAESPC HRIEWQYKSV WKCDRHQLKI LAKCPNCQAP FKMPALWEDG CCHRCRMPFA E MAKLQKVG SEFELENLYF Q

UniProtKB: TniQ (Homology model)

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Macromolecule #7: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 7 / Number of copies: 1 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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Macromolecule #8: ZINC ION

MacromoleculeName: ZINC ION / type: ligand / ID: 8 / Number of copies: 2 / Formula: ZN
Molecular weightTheoretical: 65.409 Da

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Macromolecule #9: water

MacromoleculeName: water / type: ligand / ID: 9 / Number of copies: 323 / 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
GridModel: UltrAuFoil R0./1 / Material: GOLD / Mesh: 300
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: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 2.0 µm / Nominal defocus min: 0.8 µm
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: FEI FALCON IV (4k x 4k) / Number real images: 19936 / Average electron dose: 40.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: INSILICO MODEL
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
Final reconstructionResolution.type: BY AUTHOR / Resolution: 2.35 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 4.4) / Number images used: 144000
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementProtocol: AB INITIO MODEL
Output model

PDB-8rkt:
Conformational Landscape of the Type V-K CRISPR-associated TransposonIntegration Assembly CAST V-K Cas12k domain local-refinement map

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