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- PDB-8e2l: Structure of Lates calcarifer Twinkle helicase with ATP and DNA -

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Basic information

Entry
Database: PDB / ID: 8e2l
TitleStructure of Lates calcarifer Twinkle helicase with ATP and DNA
Components
  • DNA (5'-D(P*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*T)-3')
  • Twinkle mtDNA helicase
KeywordsREPLICATION/DNA / Helicase Mitochondrion / REPLICATION-DNA complex
Function / homology
Function and homology information


single-stranded DNA binding / 5'-3' DNA helicase activity / DNA replication / ATP binding
Similarity search - Function
Twinkle-like protein / Archaeal primase DnaG/twinkle-like, TOPRIM domain / DNA helicase, DnaB-like, C-terminal / Superfamily 4 helicase domain profile. / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
ADENOSINE-5'-TRIPHOSPHATE / DNA / DNA (> 10) / Twinkle mtDNA helicase
Similarity search - Component
Biological speciesLates calcarifer (barramundi perch)
synthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.51 Å
AuthorsGao, Y. / Li, Z.
Funding support United States, 2items
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, 2022Deposition site: RCSB / Processing site: RCSB
Revision 1.0Nov 2, 2022Provider: repository / Type: Initial release
Revision 1.1Nov 30, 2022Group: Database references / Category: citation / citation_author
Item: _citation.pdbx_database_id_PubMed / _citation.title ..._citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID / _citation_author.name
Revision 1.2Dec 21, 2022Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation_author.identifier_ORCID
Revision 1.3Jun 12, 2024Group: Data collection / Category: chem_comp_atom / chem_comp_bond

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
C: Twinkle mtDNA helicase
M: DNA (5'-D(P*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*T)-3')
A: Twinkle mtDNA helicase
B: Twinkle mtDNA helicase
D: Twinkle mtDNA helicase
E: Twinkle mtDNA helicase
F: Twinkle mtDNA helicase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)374,96417
Polymers372,3077
Non-polymers2,65710
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein
Twinkle mtDNA helicase


Mass: 61298.160 Da / Num. of mol.: 6 / Mutation: E325Q
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Lates calcarifer (barramundi perch)
Production host: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
References: UniProt: A0A4W6C5C5
#2: DNA chain DNA (5'-D(P*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*T)-3')


Mass: 4517.935 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#3: Chemical
ChemComp-ATP / ADENOSINE-5'-TRIPHOSPHATE


Mass: 507.181 Da / Num. of mol.: 5 / Source method: obtained synthetically / Formula: C10H16N5O13P3 / Feature type: SUBJECT OF INVESTIGATION / Comment: ATP, energy-carrying molecule*YM
#4: Chemical
ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 5 / Source method: obtained synthetically / Formula: Mg / Feature type: SUBJECT OF INVESTIGATION
Has ligand of interestY

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: Twinkle protein, mitochondrial + DNA / Type: COMPLEX / Entity ID: #1-#2 / Source: MULTIPLE SOURCES
Source (natural)Organism: Lates calcarifer (barramundi perch)
Source (recombinant)Organism: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
Buffer solutionpH: 8
SpecimenConc.: 0.5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: 50 mM Tris (pH 8.0), 150 mM KCl, 3 mM DTT, 1 mM ATP, and 2 mM MgCl2
Specimen supportGrid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK I / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 298 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 3000 nm / Nominal defocus min: 600 nm
Image recordingElectron dose: 49 e/Å2 / Film or detector model: GATAN K2 SUMMIT (4k x 4k)

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Processing

SoftwareName: PHENIX / Version: 1.19.1_4122: / Classification: refinement
EM software
IDNameCategory
4GctfCTF correction
9cryoSPARCinitial Euler assignment
10cryoSPARCfinal Euler assignment
12cryoSPARC3D reconstruction
CTF correctionType: NONE
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.51 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 44814 / Symmetry type: POINT
Atomic model buildingProtocol: AB INITIO MODEL / Space: REAL
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00417389
ELECTRON MICROSCOPYf_angle_d0.72923723
ELECTRON MICROSCOPYf_dihedral_angle_d15.2945749
ELECTRON MICROSCOPYf_chiral_restr0.0452776
ELECTRON MICROSCOPYf_plane_restr0.0033058

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