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- EMDB-23722: ADP-AlF3 bound TnsC structure in closed form -

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Basic information

Entry
Database: EMDB / ID: EMD-23722
TitleADP-AlF3 bound TnsC structure in closed form
Map datalocal filtered map
Sample
  • Complex: Head to head dimer of TnsC hexamers bound to DNA
    • Protein or peptide: TnsC
    • DNA: DNA (27-MER)
    • DNA: DNA (27-MER)
  • Ligand: ADENOSINE-5'-DIPHOSPHATE
Biological speciesScytonema hofmannii (bacteria) / synthetic construct (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.8 Å
AuthorsPark J / Tsai AWL / Mehrotra E / Kellogg EH
Funding support United States, 1 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R00-GM124463 United States
CitationJournal: Science / Year: 2021
Title: Structural basis for target site selection in RNA-guided DNA transposition systems.
Authors: Jung-Un Park / Amy Wei-Lun Tsai / Eshan Mehrotra / Michael T Petassi / Shan-Chi Hsieh / Ailong Ke / Joseph E Peters / Elizabeth H Kellogg /
Abstract: CRISPR-associated transposition systems allow guide RNA-directed integration of a single DNA cargo in one orientation at a fixed distance from a programmable target sequence. We used cryo-electron ...CRISPR-associated transposition systems allow guide RNA-directed integration of a single DNA cargo in one orientation at a fixed distance from a programmable target sequence. We used cryo-electron microscopy (cryo-EM) to define the mechanism that underlies this process by characterizing the transposition regulator, TnsC, from a type V-K CRISPR-transposase system. In this scenario, polymerization of adenosine triphosphate-bound TnsC helical filaments could explain how polarity information is passed to the transposase. TniQ caps the TnsC filament, representing a universal mechanism for target information transfer in Tn7/Tn7-like elements. Transposase-driven disassembly establishes delivery of the element only to unused protospacers. Finally, TnsC transitions to define the fixed point of insertion, as revealed by structures with the transition state mimic ADP•AlF These mechanistic findings provide the underpinnings for engineering CRISPR-associated transposition systems for research and therapeutic applications.
History
DepositionMar 30, 2021-
Header (metadata) releaseJul 28, 2021-
Map releaseJul 28, 2021-
UpdateNov 16, 2022-
Current statusNov 16, 2022Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.0078
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.0078
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-7m9b
  • Surface level: 0.0078
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_23722.png

Downloads & links

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Map

FileDownload / File: emd_23722.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationlocal filtered map
Voxel sizeX=Y=Z: 1.33 Å
Density
Contour LevelBy AUTHOR: 0.0078 / Movie #1: 0.0078
Minimum - Maximum-0.03385488 - 0.06482591
Average (Standard dev.)0.00015108493 (±0.0019425051)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 340.48 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.331.331.33
M x/y/z256256256
origin x/y/z0.0000.0000.000
length x/y/z340.480340.480340.480
α/β/γ90.00090.00090.000
start NX/NY/NZ1331310
NX/NY/NZ223226424
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS256256256
D min/max/mean-0.0340.0650.000

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Supplemental data

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Mask #1

Fileemd_23722_msk_1.map
Projections & Slices
AxesZYX

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Slices (1/2)
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Additional map: full map

Fileemd_23722_additional_1.map
Annotationfull map
Projections & Slices
AxesZYX

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Additional map: sharpened map

Fileemd_23722_additional_2.map
Annotationsharpened map
Projections & Slices
AxesZYX

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Half map: half map 1

Fileemd_23722_half_map_1.map
Annotationhalf map 1
Projections & Slices
AxesZYX

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Histogram

Histogram (log scale)

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Half map: half map 2

Fileemd_23722_half_map_2.map
Annotationhalf map 2
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Sample components

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Entire : Head to head dimer of TnsC hexamers bound to DNA

EntireName: Head to head dimer of TnsC hexamers bound to DNA
Components
  • Complex: Head to head dimer of TnsC hexamers bound to DNA
    • Protein or peptide: TnsC
    • DNA: DNA (27-MER)
    • DNA: DNA (27-MER)
  • Ligand: ADENOSINE-5'-DIPHOSPHATE

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Supramolecule #1: Head to head dimer of TnsC hexamers bound to DNA

SupramoleculeName: Head to head dimer of TnsC hexamers bound to DNA / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#3 / Details: Reconstituted with ADP-AlF3, in closed state
Source (natural)Organism: Scytonema hofmannii (bacteria)
Recombinant expressionOrganism: Escherichia coli BL21 (bacteria)
Molecular weightTheoretical: 400 KDa

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Macromolecule #1: TnsC

MacromoleculeName: TnsC / type: protein_or_peptide / ID: 1 / Number of copies: 12 / Enantiomer: LEVO
Source (natural)Organism: Scytonema hofmannii (bacteria)
Molecular weightTheoretical: 31.444617 KDa
Recombinant expressionOrganism: Escherichia coli BL21 (bacteria)
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 (27-MER)

MacromoleculeName: DNA (27-MER) / type: dna / ID: 2 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 8.411627 KDa
SequenceString:
(DA)(DA)(DA)(DA)(DA)(DA)(DA)(DA)(DA)(DA) (DA)(DA)(DA)(DA)(DA)(DA)(DA)(DA)(DA)(DA) (DA)(DA)(DA)(DA)(DA)(DA)(DA)

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Macromolecule #3: DNA (27-MER)

MacromoleculeName: DNA (27-MER) / type: dna / ID: 3 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 8.168248 KDa
SequenceString:
(DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT) (DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT) (DT)(DT)(DT)(DT)(DT)(DT)(DT)

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Macromolecule #4: ADENOSINE-5'-DIPHOSPHATE

MacromoleculeName: ADENOSINE-5'-DIPHOSPHATE / type: ligand / ID: 4 / Number of copies: 10 / Formula: ADP
Molecular weightTheoretical: 427.201 Da
Chemical component information

ChemComp-ADP:
ADENOSINE-5'-DIPHOSPHATE / ADP, energy-carrying molecule*YM / Adenosine diphosphate

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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

Concentration10 mg/mL
BufferpH: 7.4
Component:
ConcentrationFormulaName
25.0 mMC8H18N2O4SHEPES
1.0 mMC4H10O2S2DTT
2.0 %C3H8O3glycerol
6.0 mMMgCl2Magnesium Chloride
2.0 mMC10H15N5O10P2ADPAdenosine diphosphate
200.0 mMNaClSodium chlorideSodium Chloride
6.0 mMAlCl3Aluminum Chloride
60.0 mMNaFSodium Fluoride
GridModel: UltrAuFoil R1.2/1.3 / Material: GOLD / Mesh: 400 / Support film - Material: GOLD / Support film - topology: HOLEY / Support film - Film thickness: 50.0 nm / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: AIR
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277.15 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TALOS ARCTICA
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 2.5 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 63000
Specialist opticsEnergy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV
Sample stageCooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average electron dose: 45.0 e/Å2
Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 754099
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC / Details: cryosparc stochastic gradient descent
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.1)
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 3.8 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 121512
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

6az0

RefinementSpace: REAL / Protocol: FLEXIBLE FIT
Output model

PDB-7m9b:
ADP-AlF3 bound TnsC structure in closed form

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