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- PDB-7t8c: Heptameric Human Twinkle Helicase Clinical Variant W315L -

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Basic information

Entry
Database: PDB / ID: 7t8c
TitleHeptameric Human Twinkle Helicase Clinical Variant W315L
ComponentsTwinkle mtDNA helicase
KeywordsDNA BINDING PROTEIN / Helicase / walker A / walker B / DNA binding
Function / homology
Function and homology information


mitochondrial DNA replication / mitochondrial transcription / DNA 5'-3' helicase / protein hexamerization / DNA unwinding involved in DNA replication / mitochondrial nucleoid / Mitochondrial protein degradation / DNA helicase activity / isomerase activity / cellular response to glucose stimulus ...mitochondrial DNA replication / mitochondrial transcription / DNA 5'-3' helicase / protein hexamerization / DNA unwinding involved in DNA replication / mitochondrial nucleoid / Mitochondrial protein degradation / DNA helicase activity / isomerase activity / cellular response to glucose stimulus / Transcriptional activation of mitochondrial biogenesis / single-stranded DNA binding / 5'-3' DNA helicase activity / protease binding / mitochondrial inner membrane / mitochondrial matrix / lipid binding / ATP hydrolysis activity / mitochondrion / ATP binding / identical protein binding
Similarity search - Function
AAA domain / 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
Twinkle mtDNA helicase
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.5 Å
AuthorsRiccio, A.A. / Bouvette, J. / Krahn, J. / Borgnia, M.J. / Copeland, W.C.
Funding support United States, 2items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of Environmental Health Sciences (NIH/NIEHS)Z01 ES065078 United States
National Institutes of Health/National Institute of Environmental Health Sciences (NIH/NIEHS)Z01 ES065080 United States
CitationJournal: Proc Natl Acad Sci U S A / Year: 2022
Title: Structural insight and characterization of human Twinkle helicase in mitochondrial disease.
Authors: Amanda A Riccio / Jonathan Bouvette / Lalith Perera / Matthew J Longley / Juno M Krahn / Jason G Williams / Robert Dutcher / Mario J Borgnia / William C Copeland /
Abstract: Twinkle is the mammalian helicase vital for replication and integrity of mitochondrial DNA. Over 90 Twinkle helicase disease variants have been linked to progressive external ophthalmoplegia and ...Twinkle is the mammalian helicase vital for replication and integrity of mitochondrial DNA. Over 90 Twinkle helicase disease variants have been linked to progressive external ophthalmoplegia and ataxia neuropathies among other mitochondrial diseases. Despite the biological and clinical importance, Twinkle represents the only remaining component of the human minimal mitochondrial replisome that has yet to be structurally characterized. Here, we present 3-dimensional structures of human Twinkle W315L. Employing cryo-electron microscopy (cryo-EM), we characterize the oligomeric assemblies of human full-length Twinkle W315L, define its multimeric interface, and map clinical variants associated with Twinkle in inherited mitochondrial disease. Cryo-EM, crosslinking-mass spectrometry, and molecular dynamics simulations provide insight into the dynamic movement and molecular consequences of the W315L clinical variant. Collectively, this ensemble of structures outlines a framework for studying Twinkle function in mitochondrial DNA replication and associated disease states.
History
DepositionDec 16, 2021Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jul 27, 2022Provider: repository / Type: Initial release
Revision 1.1Aug 24, 2022Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID / _citation_author.name
Revision 1.2Feb 28, 2024Group: Data collection / Category: chem_comp_atom / chem_comp_bond

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Twinkle mtDNA helicase
B: Twinkle mtDNA helicase
C: Twinkle mtDNA helicase
D: Twinkle mtDNA helicase
E: Twinkle mtDNA helicase
F: Twinkle mtDNA helicase
G: Twinkle mtDNA helicase


Theoretical massNumber of molelcules
Total (without water)549,2867
Polymers549,2867
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, gel filtration, cross-linking
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein
Twinkle mtDNA helicase / Progressive external ophthalmoplegia 1 protein / T7 gp4-like protein with intramitochondrial ...Progressive external ophthalmoplegia 1 protein / T7 gp4-like protein with intramitochondrial nucleoid localization / T7-like mitochondrial DNA helicase / Twinkle protein / mitochondrial


Mass: 78469.422 Da / Num. of mol.: 7 / Mutation: W315L
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: TWNK, C10orf2, PEO1 / Production host: Escherichia coli (E. coli) / References: UniProt: Q96RR1, DNA helicase

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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: Heptameric Human Twinkle Clinical Variant W315L / Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Molecular weightValue: 0.504 MDa / Experimental value: YES
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Escherichia coli (E. coli) / Strain: Rosetta 2 (DE3) / Plasmid: pET21a
Buffer solutionpH: 8
SpecimenConc.: 1.2 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: TFS KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 2500 nm / Nominal defocus min: 1000 nm
Image recordingElectron dose: 60 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)

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Processing

SoftwareName: PHENIX / Version: 1.19.2_4158: / Classification: refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C7 (7 fold cyclic)
3D reconstructionResolution: 4.5 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 55655 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00129610
ELECTRON MICROSCOPYf_angle_d0.39240103
ELECTRON MICROSCOPYf_dihedral_angle_d7.59510857
ELECTRON MICROSCOPYf_chiral_restr0.0374438
ELECTRON MICROSCOPYf_plane_restr0.0035187

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