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- EMDB-13489: Scytonema hofmannii TnsC bound to AMPPNP and DNA -

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Basic information

Entry
Database: EMDB / ID: EMD-13489
TitleScytonema hofmannii TnsC bound to AMPPNP and DNA
Map data
Sample
  • Complex: Complex of ShTnsC with AMPPNP and dsDNA
    • Protein or peptide: ShTnsC
    • DNA: DNA (5'-D(P*AP*TP*AP*TP*AP*TP*AP*TP*AP*TP*AP*TP*AP*TP*AP*TP*AP*TP*AP*TP*AP*T)-3')
  • Ligand: PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER
  • Ligand: MAGNESIUM ION
Biological speciesScytonema hofmannii (bacteria) / synthetic construct (others)
Methodhelical reconstruction / cryo EM / Resolution: 3.57 Å
AuthorsQuerques I / Jinek M
Funding supportEuropean Union, 2 items
OrganizationGrant numberCountry
Swiss National Science Foundation31003A_182567European Union
European Research Council (ERC)ERC-CoG-820152European Union
CitationJournal: Nature / Year: 2021
Title: Target site selection and remodelling by type V CRISPR-transposon systems.
Authors: Irma Querques / Michael Schmitz / Seraina Oberli / Christelle Chanez / Martin Jinek /
Abstract: Canonical CRISPR-Cas systems provide adaptive immunity against mobile genetic elements. However, type I-F, I-B and V-K systems have been adopted by Tn7-like transposons to direct RNA-guided ...Canonical CRISPR-Cas systems provide adaptive immunity against mobile genetic elements. However, type I-F, I-B and V-K systems have been adopted by Tn7-like transposons to direct RNA-guided transposon insertion. Type V-K CRISPR-associated transposons rely on the pseudonuclease Cas12k, the transposase TnsB, the AAA+ ATPase TnsC and the zinc-finger protein TniQ, but the molecular mechanism of RNA-directed DNA transposition has remained elusive. Here we report cryo-electron microscopic structures of a Cas12k-guide RNA-target DNA complex and a DNA-bound, polymeric TnsC filament from the CRISPR-associated transposon system of the photosynthetic cyanobacterium Scytonema hofmanni. The Cas12k complex structure reveals an intricate guide RNA architecture and critical interactions mediating RNA-guided target DNA recognition. TnsC helical filament assembly is ATP-dependent and accompanied by structural remodelling of the bound DNA duplex. In vivo transposition assays corroborate key features of the structures, and biochemical experiments show that TniQ restricts TnsC polymerization, while TnsB interacts directly with TnsC filaments to trigger their disassembly upon ATP hydrolysis. Together, these results suggest that RNA-directed target selection by Cas12k primes TnsC polymerization and DNA remodelling, generating a recruitment platform for TnsB to catalyse site-specific transposon insertion. Insights from this work will inform the development of CRISPR-associated transposons as programmable site-specific gene insertion tools.
History
DepositionAug 31, 2021-
Header (metadata) releaseDec 1, 2021-
Map releaseDec 1, 2021-
UpdateDec 1, 2021-
Current statusDec 1, 2021Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 3
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 3
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-7plh
  • Surface level: 3
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-7plh
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_13489.png

Downloads & links

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Map

FileDownload / File: emd_13489.map.gz / Format: CCP4 / Size: 166.4 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesX (Sec.)Y (Row.)Z (Col.)
0.65 Å/pix.
x 352 pix.
= 228.8 Å		0.65 Å/pix.
x 352 pix.
= 228.8 Å		0.65 Å/pix.
x 352 pix.
= 228.8 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: 2.1 / Movie #1: 3
Minimum - Maximum-9.278131 - 16.84585
Average (Standard dev.)-2.4413713e-12 (±1.0)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderZYX
Origin000
Dimensions352352352
Spacing352352352
CellA=B=C: 228.79999 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z0.650.650.65
M x/y/z352352352
origin x/y/z0.0000.0000.000
length x/y/z228.800228.800228.800
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ352352352
MAP C/R/S321
start NC/NR/NS000
NC/NR/NS352352352
D min/max/mean-9.27816.846-0.000

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Supplemental data

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Sample components

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Entire : Complex of ShTnsC with AMPPNP and dsDNA

EntireName: Complex of ShTnsC with AMPPNP and dsDNA
Components
  • Complex: Complex of ShTnsC with AMPPNP and dsDNA
    • Protein or peptide: ShTnsC
    • DNA: DNA (5'-D(P*AP*TP*AP*TP*AP*TP*AP*TP*AP*TP*AP*TP*AP*TP*AP*TP*AP*TP*AP*TP*AP*T)-3')
  • Ligand: PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER
  • Ligand: MAGNESIUM ION

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Supramolecule #1: Complex of ShTnsC with AMPPNP and dsDNA

SupramoleculeName: Complex of ShTnsC with AMPPNP and dsDNA / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#2

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Macromolecule #1: ShTnsC

MacromoleculeName: ShTnsC / type: protein_or_peptide / ID: 1 / 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 #2: DNA (5'-D(P*AP*TP*AP*TP*AP*TP*AP*TP*AP*TP*AP*TP*AP*TP*AP*TP*AP*TP...

MacromoleculeName: DNA (5'-D(P*AP*TP*AP*TP*AP*TP*AP*TP*AP*TP*AP*TP*AP*TP*AP*TP*AP*TP*AP*TP*AP*T)-3')
type: dna / ID: 2 / Number of copies: 2 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 6.746438 KDa
SequenceString:
(DA)(DT)(DA)(DT)(DA)(DT)(DA)(DT)(DA)(DT) (DA)(DT)(DA)(DT)(DA)(DT)(DA)(DT)(DA)(DT) (DA)(DT)

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Macromolecule #3: PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER

MacromoleculeName: PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER / type: ligand / ID: 3 / Number of copies: 7 / Formula: ANP
Molecular weightTheoretical: 506.196 Da
Chemical component information

ChemComp-ANP:
PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER / AMP-PNP, energy-carrying molecule analogue*YM

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Macromolecule #4: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 4 / 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
Processinghelical reconstruction
Aggregation statefilament

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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: 66.39 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: OTHER / Imaging mode: BRIGHT FIELD
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Final reconstructionApplied symmetry - Helical parameters - Δz: 6.78 Å
Applied symmetry - Helical parameters - Δ&Phi: 59.72 °
Applied symmetry - Helical parameters - Axial symmetry: C1 (asymmetric)
Resolution.type: BY AUTHOR / Resolution: 3.57 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 77544
Final angle assignmentType: NOT APPLICABLE

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