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- EMDB-23720: ATPgS bound TnsC filament from ShCAST system -

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Basic information

Entry
Database: EMDB / ID: EMD-23720
TitleATPgS bound TnsC filament from ShCAST system
Map dataPostprocessed map
Sample
  • Complex: Helical filament of TnsC monomers bound to DNA
    • Protein or peptide: TnsC
    • DNA: DNA (5'-D(P*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*T)-3')
    • DNA: DNA (5'-D(P*AP*AP*AP*AP*AP*AP*AP*AP*AP*AP*AP*AP*AP*AP*AP*AP*AP*AP*A)-3')
  • Ligand: PHOSPHOTHIOPHOSPHORIC ACID-ADENYLATE ESTER
  • Ligand: MAGNESIUM ION
Biological speciesScytonema hofmannii (bacteria) / synthetic construct (others)
Methodhelical reconstruction / cryo EM / Resolution: 3.2 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)M 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-
UpdateJan 25, 2023-
Current statusJan 25, 2023Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.0241
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.0241
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-7m99
  • Surface level: 0.0241
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-7m99
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_23720.png

Downloads & links

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Map

FileDownload / File: emd_23720.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationPostprocessed map
Voxel sizeX=Y=Z: 1.33 Å
Density
Contour LevelBy AUTHOR: 0.0241 / Movie #1: 0.0241
Minimum - Maximum-0.110216305 - 0.23707424
Average (Standard dev.)0.0002818808 (±0.00465038)
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/NZ000
NX/NY/NZ660660660
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS256256256
D min/max/mean-0.1100.2370.000

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Supplemental data

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Mask #1

Fileemd_23720_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Full map

Fileemd_23720_additional_1.map
AnnotationFull map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Even half-map

Fileemd_23720_half_map_1.map
AnnotationEven half-map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Odd half-map

Fileemd_23720_half_map_2.map
AnnotationOdd half-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 : Helical filament of TnsC monomers bound to DNA

EntireName: Helical filament of TnsC monomers bound to DNA
Components
  • Complex: Helical filament of TnsC monomers bound to DNA
    • Protein or peptide: TnsC
    • DNA: DNA (5'-D(P*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*T)-3')
    • DNA: DNA (5'-D(P*AP*AP*AP*AP*AP*AP*AP*AP*AP*AP*AP*AP*AP*AP*AP*AP*AP*AP*A)-3')
  • Ligand: PHOSPHOTHIOPHOSPHORIC ACID-ADENYLATE ESTER
  • Ligand: MAGNESIUM ION

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Supramolecule #1: Helical filament of TnsC monomers bound to DNA

SupramoleculeName: Helical filament of TnsC monomers bound to DNA / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#3 / Details: Reconstituted with ATPgS
Source (natural)Organism: Scytonema hofmannii (bacteria)
Molecular weightTheoretical: 45.4 kDa/nm

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Macromolecule #1: TnsC

MacromoleculeName: TnsC / type: protein_or_peptide / ID: 1 / Number of copies: 8 / 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 (5'-D(P*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP...

MacromoleculeName: DNA (5'-D(P*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*T)-3')
type: dna / ID: 2 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 6.343091 KDa
SequenceString:
(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 #3: DNA (5'-D(P*AP*AP*AP*AP*AP*AP*AP*AP*AP*AP*AP*AP*AP*AP*AP*AP*AP*AP...

MacromoleculeName: DNA (5'-D(P*AP*AP*AP*AP*AP*AP*AP*AP*AP*AP*AP*AP*AP*AP*AP*AP*AP*AP*A)-3')
type: dna / ID: 3 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 6.532386 KDa
SequenceString:
(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 #4: PHOSPHOTHIOPHOSPHORIC ACID-ADENYLATE ESTER

MacromoleculeName: PHOSPHOTHIOPHOSPHORIC ACID-ADENYLATE ESTER / type: ligand / ID: 4 / Number of copies: 7 / Formula: AGS
Molecular weightTheoretical: 523.247 Da
Chemical component information

ChemComp-AGS:
PHOSPHOTHIOPHOSPHORIC ACID-ADENYLATE ESTER / ATP-gamma-S, energy-carrying molecule analogue*YM

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Macromolecule #5: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 5 / 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

Concentration1 mg/mL
BufferpH: 7.4
Component:
ConcentrationFormulaName
200.0 mMNaClSodium chloridesodium chloride
25.0 mMC8H18N2O4SHEPES
1.0 mMC4H10O2S2DTT
2.0 %C3H8O3glycerol
2.0 mMMgCl2Magnesium chloride
0.2 mMC10H12N5O12P3SATPgS

Details: Sample was diluted into low salt using dialysis.
GridModel: Quantifoil R1.2/1.3 / Material: GOLD / Mesh: 300 / Support film - Material: GOLD / Support film - topology: HOLEY / Support film - Film thickness: 50.0 nm / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 30 sec. / Pretreatment - Atmosphere: AIR / Pretreatment - Pressure: 0.038 kPa
Details: Grid cleaned with solvent and examined for defects under light microscope.
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277.15 K / Instrument: FEI VITROBOT MARK IV / Details: Blot for 6 seconds before plunging.
DetailsThis sample comprised monodisperse filaments reconstituted using dialysis.

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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
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
TemperatureMin: 93.15 K / Max: 103.2 K
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Digitization - Dimensions - Width: 5760 pixel / Digitization - Dimensions - Height: 4092 pixel / Number grids imaged: 1 / Number real images: 3474 / Average exposure time: 3.0 sec. / Average electron dose: 40.0 e/Å2
Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company

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Image processing

Segment selectionNumber selected: 1235415 / Software - Name: Warp (ver. 1.0.9) / Software - details: BoxNet2Mask_20180531 / Details: Neural net trained particle picking.
Startup modelType of model: INSILICO MODEL
Details: Used Relion generated helical filament of spheres with guesses for initial rise and twist.
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.1)
Final reconstructionNumber classes used: 1
Applied symmetry - Helical parameters - Δz: 6.83 Å
Applied symmetry - Helical parameters - Δ&Phi: 60 °
Applied symmetry - Helical parameters - Axial symmetry: C1 (asymmetric)
Algorithm: BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 3.2 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.1) / Number images used: 99247
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementSpace: REAL / Protocol: AB INITIO MODEL / Overall B value: 50 / Target criteria: Correlation coefficient
Output model

PDB-7m99:
ATPgS bound TnsC filament from ShCAST system

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