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- EMDB-27843: Structure of Lates calcarifer Twinkle helicase with ATP and DNA, ... -

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Basic information

Entry
Database: EMDB / ID: EMD-27843
TitleStructure of Lates calcarifer Twinkle helicase with ATP and DNA, conformer 2
Map data
Sample
  • Complex: Twinkle protein, mitochondrial
    • Protein or peptide: Twinkle protein, mitochondrial
Biological speciesLates calcarifer (barramundi perch)
Methodsingle particle reconstruction / cryo EM / Resolution: 7.5 Å
AuthorsGao Y / Li Z
Funding support United States, 2 items
OrganizationGrant numberCountry
Cancer Prevention and Research Institute of Texas (CPRIT)RR190046 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM142722 United States
CitationJournal: Nucleic Acids Res / Year: 2022
Title: Structural and dynamic basis of DNA capture and translocation by mitochondrial Twinkle helicase.
Authors: Zhuo Li / Parminder Kaur / Chen-Yu Lo / Neil Chopra / Jamie Smith / Hong Wang / Yang Gao /
Abstract: Twinkle is a mitochondrial replicative helicase which can self-load onto and unwind mitochondrial DNA. Nearly 60 mutations on Twinkle have been linked to human mitochondrial diseases. Using cryo- ...Twinkle is a mitochondrial replicative helicase which can self-load onto and unwind mitochondrial DNA. Nearly 60 mutations on Twinkle have been linked to human mitochondrial diseases. Using cryo-electron microscopy (cryo-EM) and high-speed atomic force microscopy (HS-AFM), we obtained the atomic-resolution structure of a vertebrate Twinkle homolog with DNA and captured in real-time how Twinkle is self-loaded onto DNA. Our data highlight the important role of the non-catalytic N-terminal domain of Twinkle. The N-terminal domain directly contacts the C-terminal helicase domain, and the contact interface is a hotspot for disease-related mutations. Mutations at the interface destabilize Twinkle hexamer and reduce helicase activity. With HS-AFM, we observed that a highly dynamic Twinkle domain, which is likely to be the N-terminal domain, can protrude ∼5 nm to transiently capture nearby DNA and initialize Twinkle loading onto DNA. Moreover, structural analysis and subunit doping experiments suggest that Twinkle hydrolyzes ATP stochastically, which is distinct from related helicases from bacteriophages.
History
DepositionAug 15, 2022-
Header (metadata) releaseNov 2, 2022-
Map releaseNov 2, 2022-
UpdateDec 21, 2022-
Current statusDec 21, 2022Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_27843.png

Downloads & links

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Map

FileDownload / File: emd_27843.map.gz / Format: CCP4 / Size: 125 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
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AxesZ (Sec.)Y (Row.)X (Col.)
1.07 Å/pix.
x 320 pix.
= 342.4 Å		1.07 Å/pix.
x 320 pix.
= 342.4 Å		1.07 Å/pix.
x 320 pix.
= 342.4 Å

Surface

Projections

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Images are generated by Spider.

Voxel sizeX=Y=Z: 1.07 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.13
Minimum - Maximum-0.5495947 - 1.039519
Average (Standard dev.)0.0002683517 (±0.04075132)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions320320320
Spacing320320320
CellA=B=C: 342.40002 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #1

Fileemd_27843_half_map_1.map
Projections & Slices
AxesZYX

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Density Histograms

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Histogram (log scale)

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Half map: #2

Fileemd_27843_half_map_2.map
Projections & Slices
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Histogram

Histogram (log scale)

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Sample components

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Entire : Twinkle protein, mitochondrial

EntireName: Twinkle protein, mitochondrial
Components
  • Complex: Twinkle protein, mitochondrial
    • Protein or peptide: Twinkle protein, mitochondrial

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Supramolecule #1: Twinkle protein, mitochondrial

SupramoleculeName: Twinkle protein, mitochondrial / type: complex / ID: 1 / Chimera: Yes / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Lates calcarifer (barramundi perch)

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Macromolecule #1: Twinkle protein, mitochondrial

MacromoleculeName: Twinkle protein, mitochondrial / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO
Source (natural)Organism: Lates calcarifer (barramundi perch)
SequenceString: MQKEEQDFLS PHVMLGYPES LDEQEEGERE LREVQRIWSS AVPFNDLPED EAQLIKTMFQ ITKVSNATLK KFGVRLFKP TKSLVFPWFA GPDSSLKGLK LLSAQNTDTE KVTYNEATVP KISSYYNLFG LTLVGRMDSE V VLTGHELD TLAVSQATGL PSVALPRGVS ...String:
MQKEEQDFLS PHVMLGYPES LDEQEEGERE LREVQRIWSS AVPFNDLPED EAQLIKTMFQ ITKVSNATLK KFGVRLFKP TKSLVFPWFA GPDSSLKGLK LLSAQNTDTE KVTYNEATVP KISSYYNLFG LTLVGRMDSE V VLTGHELD TLAVSQATGL PSVALPRGVS CLPPMLLPYL EQFKRVTLWL GHDIRSWEAS KIFSRKLGLR RC SLVRPGE DRPCPLEALA RGKNLSRIIK TSIPAAHKSI VSFKQLREDV YGELLNTEQV AGVKWTRFPE LNR ILKGHR KGELTVFTGP TGSGKTTFIS EVALDLCIQG VNTLWGSFEI NNVRLAKIML TQFAMQRLEE NLEQ YDFWA DKFEELPLYF MTFHGQQNIK TVLDTMQHAV YLYDINHVII DNLQFMMGQE NLSIDKYAVQ DHIIG AFRK FATNTSCHVT LIIHPRKEED DRELQTASIF GSAKASQEAD NVLILQEKKL VTCPGRRSLQ VTKNRF DGD VGIFPLDFIK SSLTFSAPIK GKVKLRKVST KPENEEVGGE RGGSEEGRG

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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

Concentration0.5 mg/mL
BufferpH: 8
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 298 K / Instrument: FEI VITROBOT MARK I
Details50 mM Tris (pH 8.0), 150 mM KCl, 3 mM DTT, 1 mM ATP, and 2 mM MgCl2

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Average electron dose: 49.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 3.0 µm / Nominal defocus min: 0.6 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Final reconstructionResolution.type: BY AUTHOR / Resolution: 7.5 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC / Number images used: 18243
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC

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Atomic model buiding 1

RefinementSpace: REAL / Protocol: AB INITIO MODEL

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