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- EMDB-15975: Cas12k-sgRNA-dsDNA-S15-TniQ-TnsC transposon recruitment complex -

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Basic information

Entry
Database: EMDB / ID: EMD-15975
TitleCas12k-sgRNA-dsDNA-S15-TniQ-TnsC transposon recruitment complex
Map dataCas12k transposon recruitment complex
Sample
  • Complex: Target DNA bound Cas12k-sgRNA-S15-TniQ-TnsC transposon recruitment complex
    • Protein or peptide: ShCas12k
    • RNA: sgRNASubgenomic mRNA
    • DNA: DNA target strand
    • DNA: DNA non-target strand
    • Protein or peptide: TnsC
    • Protein or peptide: TniQ (Homology model)
    • Protein or peptide: 30S ribosomal protein S15
  • Ligand: ADENOSINE-5'-TRIPHOSPHATE
  • Ligand: MAGNESIUM ION
  • Ligand: ZINC ION
Function / homology
Function and homology information


mRNA base-pairing translational repressor activity / small ribosomal subunit rRNA binding / ribosomal small subunit assembly / cytosolic small ribosomal subunit / cytoplasmic translation / rRNA binding / structural constituent of ribosome / translation / cytosol / cytoplasm
Similarity search - Function
Bacterial TniB / Bacterial TniB protein / TniQ / TniQ / Ribosomal protein S15, bacterial-type / Ribosomal_S15 / Ribosomal protein S15 signature. / Ribosomal protein S15 / Ribosomal protein S15 / S15/NS1, RNA-binding / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
TnsC / TniQ (Homology model) / ShCas12k / Small ribosomal subunit protein uS15
Similarity search - Component
Biological speciesScytonema hofmannii (bacteria) / Escherichia coli K-12 (bacteria) / synthetic construct (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.3 Å
AuthorsSchmitz M / Querques I / Oberli S / Chanez C / Jinek M
Funding support Switzerland, European Union, United States, 4 items
OrganizationGrant numberCountry
Swiss National Science Foundation31003A_182567 Switzerland
European Research Council (ERC)ERC-CoG-820152European Union
Howard Hughes Medical Institute (HHMI)55008735 United States
European Molecular Biology Organization (EMBO)ALTF 296-2020European Union
CitationJournal: Cell / Year: 2022
Title: Structural basis for the assembly of the type V CRISPR-associated transposon complex.
Authors: Michael Schmitz / Irma Querques / Seraina Oberli / Christelle Chanez / Martin Jinek /
Abstract: CRISPR-Cas systems have been co-opted by Tn7-like transposable elements to direct RNA-guided transposition. Type V-K CRISPR-associated transposons rely on the concerted activities of the ...CRISPR-Cas systems have been co-opted by Tn7-like transposable elements to direct RNA-guided transposition. Type V-K CRISPR-associated transposons rely on the concerted activities of the pseudonuclease Cas12k, the AAA+ ATPase TnsC, the Zn-finger protein TniQ, and the transposase TnsB. Here we present a cryo-electron microscopic structure of a target DNA-bound Cas12k-transposon recruitment complex comprised of RNA-guided Cas12k, TniQ, a polymeric TnsC filament and, unexpectedly, the ribosomal protein S15. Complex assembly, mediated by a network of interactions involving the guide RNA, TniQ, and S15, results in R-loop completion. TniQ contacts two TnsC protomers at the Cas12k-proximal filament end, likely nucleating its polymerization. Transposition activity assays corroborate our structural findings, implying that S15 is a bona fide component of the type V crRNA-guided transposon machinery. Altogether, our work uncovers key mechanistic aspects underpinning RNA-mediated assembly of CRISPR-associated transposons to guide their development as programmable tools for site-specific insertion of large DNA payloads.
History
DepositionOct 18, 2022-
Header (metadata) releaseDec 28, 2022-
Map releaseDec 28, 2022-
UpdateFeb 1, 2023-
Current statusFeb 1, 2023Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_15975.png

Downloads & links

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Map

FileDownload / File: emd_15975.map.gz / Format: CCP4 / Size: 421.9 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationCas12k transposon recruitment complex
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.65 Å/pix.
x 480 pix.
= 312. Å		0.65 Å/pix.
x 480 pix.
= 312. Å		0.65 Å/pix.
x 480 pix.
= 312. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.65 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.005
Minimum - Maximum-0.008791034 - 0.031291883
Average (Standard dev.)8.494967e-05 (±0.0010291192)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions480480480
Spacing480480480
CellA=B=C: 312.0 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: Half2

Fileemd_15975_half_map_1.map
AnnotationHalf2
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half1

Fileemd_15975_half_map_2.map
AnnotationHalf1
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Target DNA bound Cas12k-sgRNA-S15-TniQ-TnsC transposon recruitmen...

EntireName: Target DNA bound Cas12k-sgRNA-S15-TniQ-TnsC transposon recruitment complex
Components
  • Complex: Target DNA bound Cas12k-sgRNA-S15-TniQ-TnsC transposon recruitment complex
    • Protein or peptide: ShCas12k
    • RNA: sgRNASubgenomic mRNA
    • DNA: DNA target strand
    • DNA: DNA non-target strand
    • Protein or peptide: TnsC
    • Protein or peptide: TniQ (Homology model)
    • Protein or peptide: 30S ribosomal protein S15
  • Ligand: ADENOSINE-5'-TRIPHOSPHATE
  • Ligand: MAGNESIUM ION
  • Ligand: ZINC ION

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Supramolecule #1: Target DNA bound Cas12k-sgRNA-S15-TniQ-TnsC transposon recruitmen...

SupramoleculeName: Target DNA bound Cas12k-sgRNA-S15-TniQ-TnsC transposon recruitment complex
type: complex / ID: 1 / Chimera: Yes / Parent: 0 / Macromolecule list: #1-#7 / Details: Cas12k, sgRNA, target DNA, S15, TniQ, TnsC
Source (natural)Organism: Scytonema hofmannii (bacteria)

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Macromolecule #1: ShCas12k

MacromoleculeName: ShCas12k / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Scytonema hofmannii (bacteria)
Molecular weightTheoretical: 79.156773 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MGSSHHHHHH SGGGSGGSAW SHPQFEKGGG SGGGSGGSAW SHPQFEKSGG GENLYFQSNA SQITIQARLI SFESNRQQLW KLMADLNTP LINELLCQLG QHPDFEKWQQ KGKLPSTVVS QLCQPLKTDP RFAGQPSRLY MSAIHIVDYI YKSWLAIQKR L QQQLDGKT ...String:
MGSSHHHHHH SGGGSGGSAW SHPQFEKGGG SGGGSGGSAW SHPQFEKSGG GENLYFQSNA 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

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Macromolecule #5: TnsC

MacromoleculeName: TnsC / type: protein_or_peptide / ID: 5 / Number of copies: 7 / Enantiomer: LEVO
Source (natural)Organism: Scytonema hofmannii (bacteria)
Molecular weightTheoretical: 31.444617 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MTEAQAIAKQ LGGVKPDDEW LQAEIARLKG KSIVPLQQVK TLHDWLDGKR KARKSCRVVG ESRTGKTVAC DAYRYRHKPQ QEAGRPPTV PVVYIRPHQK CGPKDLFKKI TEYLKYRVTK GTVSDFRDRT IEVLKGCGVE MLIIDEADRL KPETFADVRD I AEDLGIAV ...String:
MTEAQAIAKQ LGGVKPDDEW LQAEIARLKG KSIVPLQQVK TLHDWLDGKR KARKSCRVVG ESRTGKTVAC DAYRYRHKPQ QEAGRPPTV PVVYIRPHQK CGPKDLFKKI TEYLKYRVTK GTVSDFRDRT IEVLKGCGVE MLIIDEADRL KPETFADVRD I AEDLGIAV VLVGTDRLDA VIKRDEQVLE RFRAHLRFGK LSGEDFKNTV EMWEQMVLKL PVSSNLKSKE MLRILTSATE GY IGRLDEI LREAAIRSLS RGLKKIDKAV LQEVAKEYK

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Macromolecule #6: TniQ (Homology model)

MacromoleculeName: TniQ (Homology model) / type: protein_or_peptide / ID: 6 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Scytonema hofmannii (bacteria)
Molecular weightTheoretical: 19.01124 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 MAKLQKV

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Macromolecule #7: 30S ribosomal protein S15

MacromoleculeName: 30S ribosomal protein S15 / type: protein_or_peptide / ID: 7 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Escherichia coli K-12 (bacteria)
Molecular weightTheoretical: 10.290816 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
MSLSTEATAK IVSEFGRDAN DTGSTEVQVA LLTAQINHLQ GHFAEHKKDH HSRRGLLRMV SQRRKLLDYL KRKDVARYTQ LIERLGLRR

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Macromolecule #2: sgRNA

MacromoleculeName: sgRNA / type: rna / ID: 2 / Number of copies: 1
Source (natural)Organism: Scytonema hofmannii (bacteria)
Molecular weightTheoretical: 82.376547 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 AAAUGGGUUG AAAGGAGAAG UC AUUUAAU AAGGCCACU

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Macromolecule #3: DNA target strand

MacromoleculeName: DNA target strand / type: dna / ID: 3 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 14.916607 KDa
SequenceString:
(DA)(DT)(DA)(DT)(DC)(DT)(DA)(DC)(DG)(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)(DC)(DC)(DT)(DA) (DC)

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Macromolecule #4: DNA non-target strand

MacromoleculeName: DNA non-target strand / type: dna / ID: 4 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 15.125739 KDa
SequenceString:
(DG)(DT)(DA)(DG)(DG)(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) (DC)(DG)(DT)(DA)(DC)(DT)(DA)(DT) (DA)

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Macromolecule #8: ADENOSINE-5'-TRIPHOSPHATE

MacromoleculeName: ADENOSINE-5'-TRIPHOSPHATE / type: ligand / ID: 8 / Number of copies: 7 / Formula: ATP
Molecular weightTheoretical: 507.181 Da
Chemical component information

ChemComp-ATP:
ADENOSINE-5'-TRIPHOSPHATE / ATP, energy-carrying molecule*YM / Adenosine triphosphate

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Macromolecule #9: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 9 / Number of copies: 7 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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Macromolecule #10: ZINC ION

MacromoleculeName: ZINC ION / type: ligand / ID: 10 / Number of copies: 2 / Formula: ZN
Molecular weightTheoretical: 65.409 Da

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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: 7.5
VitrificationCryogen name: ETHANE / Chamber humidity: 75 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV

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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: 2.4 µm / Nominal defocus min: 1.0 µm
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 67.68 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: EMDB MAP
EMDB ID:

13486

Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.3 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.1.2) / Number images used: 75000

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