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- EMDB-23726: ATP-bound TnsC-TniQ complex from ShCAST system -

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Basic information

Entry
Database: EMDB / ID: EMD-23726
TitleATP-bound TnsC-TniQ complex from ShCAST system
Map datalocal resolution filtered map
Sample
  • Complex: Helical filament of TnsC monomers bound to DNA and TniQ
    • Protein or peptide: TnsC
    • Protein or peptide: TniQ
Biological speciesScytonema hofmannii (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.9 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-
UpdateAug 25, 2021-
Current statusAug 25, 2021Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.012
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.012
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-7n6i
  • Surface level: 0.012
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-7n6i
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_23726.png

Downloads & links

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Map

FileDownload / File: emd_23726.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationlocal resolution filtered map
Voxel sizeX=Y=Z: 1.33 Å
Density
Contour LevelBy AUTHOR: 0.012 / Movie #1: 0.012
Minimum - Maximum-0.037898265 - 0.07712847
Average (Standard dev.)0.00020632187 (±0.0020992646)
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
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS256256256
D min/max/mean-0.0380.0770.000

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Supplemental data

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Mask #1

Fileemd_23726_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_23726_additional_1.map
AnnotationFull map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: sharpened map using uniform B-factor

Fileemd_23726_additional_2.map
Annotationsharpened map using uniform B-factor
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: half map 1

Fileemd_23726_half_map_1.map
Annotationhalf map 1
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: half map 2

Fileemd_23726_half_map_2.map
Annotationhalf map 2
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 and TniQ

EntireName: Helical filament of TnsC monomers bound to DNA and TniQ
Components
  • Complex: Helical filament of TnsC monomers bound to DNA and TniQ
    • Protein or peptide: TnsC
    • Protein or peptide: TniQ

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

SupramoleculeName: Helical filament of TnsC monomers bound to DNA and TniQ
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all / Details: Reconstituted with ATP
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 / Enantiomer: LEVO
Source (natural)Organism: Scytonema hofmannii (bacteria)
Recombinant expressionOrganism: Escherichia coli BL21 (bacteria)
SequenceString: MTEAQAIAKQ LGGVKPDDEW LQAEIARLKG KSIVPLQQVK TLHDWLDGKR KARKSCRVVG ESRTGKTVAC DAYRYRHKPQ QEAGRPPTVP VVYIRPHQKC GPKDLFKKIT EYLKYRVTKG TVSDFRDRTI EVLKGCGVEM LIIDEADRLK PETFADVRDI AEDLGIAVVL ...String:
MTEAQAIAKQ LGGVKPDDEW LQAEIARLKG KSIVPLQQVK TLHDWLDGKR KARKSCRVVG ESRTGKTVAC DAYRYRHKPQ QEAGRPPTVP VVYIRPHQKC GPKDLFKKIT EYLKYRVTKG TVSDFRDRTI EVLKGCGVEM LIIDEADRLK PETFADVRDI AEDLGIAVVL VGTDRLDAVI KRDEQVLERF RAHLRFGKLS GEDFKNTVEM WEQMVLKLPV SSNLKSKEML RILTSATEGY IGRLDEILRE AAIRSLSRGL KKIDKAVLQE VAKEYK

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Macromolecule #2: TniQ

MacromoleculeName: TniQ / type: protein_or_peptide / ID: 2 / Enantiomer: LEVO
Source (natural)Organism: Scytonema hofmannii (bacteria)
Recombinant expressionOrganism: Escherichia coli BL21 (bacteria)
SequenceString:
MIEAPDVKPW LFLIKPYEGE SLSHFLGRFR RANHLSASGL GTLAGIGAIV ARWERFHFNP RPSQQELEAI ASVVEVAAQR LAQMLPPAGV GMQHEPIRLC GACYAESPCH RIEWQYKSVW KCDRHQLKIL AKCPNCQAPF KMPALWEDGC CHRCRMPFAE MAKLQKV

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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

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
2.0 mMC10H16N5O13P3ATPAdenosine triphosphate
GridModel: UltrAuFoil R2/2 / 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 / Pretreatment - Pressure: 0.038 kPa
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277.15 K / Instrument: FEI VITROBOT MARK IV / Details: Blot for 7 seconds before plunging.
DetailsExcess of TniQ was added to ATP-bound TnsC filaments

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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
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: 4172 / Average exposure time: 3.0 sec. / Average electron dose: 50.0 e/Å2
Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 648550 / Details: Neural net based particle picking
CTF correctionSoftware: (Name: Warp (ver. 1.0.9), RELION (ver. 3.1))
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software: (Name: cryoSPARC (ver. 3.0), RELION (ver. 3.1))
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.1)
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Algorithm: BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 3.9 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.1) / Number images used: 34546
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementProtocol: RIGID BODY FIT
Output model

PDB-7n6i:
ATP-bound TnsC-TniQ complex from ShCAST system

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