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- EMDB-28994: Cryo-EM structure of TnsC-TniQ-DNA complex in type I-B CAST system -

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Basic information

Entry
Database: EMDB / ID: EMD-28994
TitleCryo-EM structure of TnsC-TniQ-DNA complex in type I-B CAST system
Map data
Sample
  • Complex: Ternary complex of TnsC, TniQ and DNA in type I-B CAST system
    • DNA: DNA (60-MER)
    • DNA: DNA (60-MER)
    • Protein or peptide: TniQ
    • Protein or peptide: TnsC
  • Ligand: ADENOSINE-5'-TRIPHOSPHATE
  • Ligand: MAGNESIUM ION
KeywordsTnsC / TniQ / CRISPR / DNA BINDING PROTEIN
Function / homologyTniQ / TniQ / : / AAA domain / ATP hydrolysis activity / P-loop containing nucleoside triphosphate hydrolase / ORC1/DEAH AAA+ ATPase domain-containing protein / TniQ family protein
Function and homology information
Biological speciesPeltigera membranacea (fungus) / synthetic construct (others) / Nostoc sp. 'Peltigera membranacea cyanobiont' 210A (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.95 Å
AuthorsChang L / Wang S
Funding support United States, 1 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS) United States
CitationJournal: Cell / Year: 2023
Title: Molecular mechanism for Tn7-like transposon recruitment by a type I-B CRISPR effector.
Authors: Shukun Wang / Clinton Gabel / Romana Siddique / Thomas Klose / Leifu Chang /
Abstract: Tn7-like transposons have co-opted CRISPR-Cas systems to facilitate the movement of their own DNA. These CRISPR-associated transposons (CASTs) are promising tools for programmable gene knockin. A key ...Tn7-like transposons have co-opted CRISPR-Cas systems to facilitate the movement of their own DNA. These CRISPR-associated transposons (CASTs) are promising tools for programmable gene knockin. A key feature of CASTs is their ability to recruit Tn7-like transposons to nuclease-deficient CRISPR effectors. However, how Tn7-like transposons are recruited by diverse CRISPR effectors remains poorly understood. Here, we present the cryo-EM structure of a recruitment complex comprising the Cascade complex, TniQ, TnsC, and the target DNA in the type I-B CAST from Peltigera membranacea cyanobiont 210A. Target DNA recognition by Cascade induces conformational changes in Cas6 and primes TniQ recruitment through its C-terminal domain. The N-terminal domain of TniQ is bound to the seam region of the TnsC spiral heptamer. Our findings provide insights into the diverse mechanisms for the recruitment of Tn7-like transposons to CRISPR effectors and will aid in the development of CASTs as gene knockin tools.
History
DepositionDec 1, 2022-
Header (metadata) releaseAug 9, 2023-
Map releaseAug 9, 2023-
UpdateJan 31, 2024-
Current statusJan 31, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_28994.png

Downloads & links

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Map

FileDownload / File: emd_28994.map.gz / Format: CCP4 / Size: 216 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
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Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.05 Å/pix.
x 256 pix.
= 269.824 Å		1.05 Å/pix.
x 256 pix.
= 269.824 Å		1.05 Å/pix.
x 256 pix.
= 269.824 Å

Surface

Projections

Slices (1/3)

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Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.054 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 2.23
Minimum - Maximum-10.213709 - 25.765896000000001
Average (Standard dev.)0.0037339474 (±0.5108992)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions384384384
Spacing384384384
CellA=B=C: 404.73602 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #2

Fileemd_28994_half_map_1.map
Projections & Slices
AxesZYX

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Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_28994_half_map_2.map
Projections & Slices
AxesZYX

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Sample components

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Entire : Ternary complex of TnsC, TniQ and DNA in type I-B CAST system

EntireName: Ternary complex of TnsC, TniQ and DNA in type I-B CAST system
Components
  • Complex: Ternary complex of TnsC, TniQ and DNA in type I-B CAST system
    • DNA: DNA (60-MER)
    • DNA: DNA (60-MER)
    • Protein or peptide: TniQ
    • Protein or peptide: TnsC
  • Ligand: ADENOSINE-5'-TRIPHOSPHATE
  • Ligand: MAGNESIUM ION

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Supramolecule #1: Ternary complex of TnsC, TniQ and DNA in type I-B CAST system

SupramoleculeName: Ternary complex of TnsC, TniQ and DNA in type I-B CAST system
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#4
Source (natural)Organism: Peltigera membranacea (fungus)

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Macromolecule #1: DNA (60-MER)

MacromoleculeName: DNA (60-MER) / type: dna / ID: 1 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 18.500861 KDa
SequenceString: (DA)(DA)(DG)(DA)(DC)(DG)(DT)(DA)(DG)(DC) (DC)(DC)(DA)(DG)(DC)(DG)(DC)(DG)(DT)(DC) (DG)(DG)(DC)(DC)(DG)(DC)(DT)(DA)(DC) (DG)(DT)(DA)(DT)(DC)(DG)(DT)(DA)(DG)(DA) (DT) (DA)(DT)(DA)(DT)(DC)(DT) ...String:
(DA)(DA)(DG)(DA)(DC)(DG)(DT)(DA)(DG)(DC) (DC)(DC)(DA)(DG)(DC)(DG)(DC)(DG)(DT)(DC) (DG)(DG)(DC)(DC)(DG)(DC)(DT)(DA)(DC) (DG)(DT)(DA)(DT)(DC)(DG)(DT)(DA)(DG)(DA) (DT) (DA)(DT)(DA)(DT)(DC)(DT)(DA)(DC) (DG)(DC)(DG)(DT)(DA)(DG)(DA)(DT)(DA)(DT) (DA)(DT)

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Macromolecule #2: DNA (60-MER)

MacromoleculeName: DNA (60-MER) / type: dna / ID: 2 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 18.482832 KDa
SequenceString: (DA)(DT)(DA)(DT)(DA)(DT)(DC)(DT)(DA)(DC) (DG)(DC)(DG)(DT)(DA)(DG)(DA)(DT)(DA)(DT) (DA)(DT)(DC)(DT)(DA)(DC)(DG)(DA)(DT) (DA)(DC)(DG)(DT)(DA)(DG)(DC)(DG)(DG)(DC) (DC) (DG)(DA)(DC)(DG)(DC)(DG) ...String:
(DA)(DT)(DA)(DT)(DA)(DT)(DC)(DT)(DA)(DC) (DG)(DC)(DG)(DT)(DA)(DG)(DA)(DT)(DA)(DT) (DA)(DT)(DC)(DT)(DA)(DC)(DG)(DA)(DT) (DA)(DC)(DG)(DT)(DA)(DG)(DC)(DG)(DG)(DC) (DC) (DG)(DA)(DC)(DG)(DC)(DG)(DC)(DT) (DG)(DG)(DG)(DC)(DT)(DA)(DC)(DG)(DT)(DC) (DT)(DT)

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Macromolecule #3: TniQ

MacromoleculeName: TniQ / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Nostoc sp. 'Peltigera membranacea cyanobiont' 210A (bacteria)
Molecular weightTheoretical: 37.591961 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MTIYSEHWSL NPLEIPQRSR LFSLEPVAVG TPYAESLSSY LHRLAQAHCL TSEKLVMGEI APLILKDEDK SELLSKNLSH LLGNSDAKP AINGMREMTE KLVTVLEELT MRQDLRFLTL LSWKGMIYDK GLFRNYRAWC PCCCEEWMQK NKTIYEPLSW S FKDVEFCL ...String:
MTIYSEHWSL NPLEIPQRSR LFSLEPVAVG TPYAESLSSY LHRLAQAHCL TSEKLVMGEI APLILKDEDK SELLSKNLSH LLGNSDAKP AINGMREMTE KLVTVLEELT MRQDLRFLTL LSWKGMIYDK GLFRNYRAWC PCCCEEWMQK NKTIYEPLSW S FKDVEFCL IHKQRLIEEC SHCGARLPVM ARLSPAGFCS RCYGWLGQEI KGEEEIEKYR VNIQGISELI ALTPQLGYKP IP IELTRKL QLILLVFEQA IGKDVKLLGD LGGIMESLRI ASTTNQSQPY HLVKLIIPVC EKAKISVFQL FGSDFKELGK ILF GNFSLE LKL

UniProtKB: TniQ family protein

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Macromolecule #4: TnsC

MacromoleculeName: TnsC / type: protein_or_peptide / ID: 4 / Number of copies: 7 / Enantiomer: LEVO
Source (natural)Organism: Nostoc sp. 'Peltigera membranacea cyanobiont' 210A (bacteria)
Molecular weightTheoretical: 43.521934 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MTKSTGFPLE LLTRPATERL AYFENYTVAH PRLKEVYEIL MRTIAEPAGA SFIFVYGASG VGKTTLRLRV EQKLTELALP KLESDRARV PVVGIEAIAP ESRYFNWKEY YTRALITLEE PLIDHKFDYG VRGISRDNFG KINVESKVVA PALRRALENA L IHRHPDVF ...String:
MTKSTGFPLE LLTRPATERL AYFENYTVAH PRLKEVYEIL MRTIAEPAGA SFIFVYGASG VGKTTLRLRV EQKLTELALP KLESDRARV PVVGIEAIAP ESRYFNWKEY YTRALITLEE PLIDHKFDYG VRGISRDNFG KINVESKVVA PALRRALENA L IHRHPDVF FVDEAQHFGK VASGYKLQDQ LDCLKSLANM TGILHCLLGT YELLTFRNLS GQLSRRSVDI HFRRYCADSP ED VQAFKSV LLTFQQHLPL AETPNLVDHW EYFYERTLGC IGTLKDWLKR VLSDALDREA TTITLKDLQK RALSVAQCQK MFK EIQEGE RQLSETEADV QNLRSALGLG AKPIVLPEET PKTTRPPGKV GKRKPKRDPI GVQQDVS

UniProtKB: ORC1/DEAH AAA+ ATPase domain-containing protein

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Macromolecule #5: ADENOSINE-5'-TRIPHOSPHATE

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

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

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Macromolecule #6: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 6 / Number of copies: 7 / Formula: MG
Molecular weightTheoretical: 24.305 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

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average electron dose: 54.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.0 µm / Nominal defocus min: 0.8 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: INSILICO MODEL
Final reconstructionResolution.type: BY AUTHOR / Resolution: 2.95 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 185087
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD

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