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- PDB-5vh9: Cryo-EM structure of yeast cytoplasmic dynein-1 with Lis1 and ATP -

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Basic information

Entry
Database: PDB / ID: 5vh9
TitleCryo-EM structure of yeast cytoplasmic dynein-1 with Lis1 and ATP
Components
  • Dynein heavy chain, cytoplasmic
  • Nuclear distribution protein PAC1
KeywordsMOTOR PROTEIN / cytoplasmic dynein / lis1
Function / homologyP-loop containing dynein motor region D3 / LIS1, N-terminal / WD40-repeat-containing domain / WD40 repeat, conserved site / G-protein beta WD-40 repeat / Dynein heavy chain, AAA module D4 / Dynein heavy chain, coiled coil stalk / Dynein heavy chain / P-loop containing nucleoside triphosphate hydrolase / Dynein heavy chain, hydrolytic ATP-binding dynein motor region D1 ...P-loop containing dynein motor region D3 / LIS1, N-terminal / WD40-repeat-containing domain / WD40 repeat, conserved site / G-protein beta WD-40 repeat / Dynein heavy chain, AAA module D4 / Dynein heavy chain, coiled coil stalk / Dynein heavy chain / P-loop containing nucleoside triphosphate hydrolase / Dynein heavy chain, hydrolytic ATP-binding dynein motor region D1 / Dynein heavy chain, ATP-binding dynein motor region D5 / WD40-repeat-containing domain superfamily / WD domain, G-beta repeat / Dynein heavy chain, domain-2 / Dynein heavy chain and region D6 of dynein motor / AAA domain (dynein-related subfamily) / Dynein heavy chain, N-terminal region 1 / Dynein heavy chain, N-terminal region 2 / Hydrolytic ATP binding site of dynein motor region D1 / Microtubule-binding stalk of dynein motor / P-loop containing dynein motor region D4 / ATP-binding dynein motor region D5 / Trp-Asp (WD) repeats signature. / Trp-Asp (WD) repeats profile. / Trp-Asp (WD) repeats circular profile. / Dynein regulator LIS1 / WD40/YVTN repeat-like-containing domain superfamily / Dynein heavy chain, domain-1 / ATPase, dynein-related, AAA domain / Dynein heavy chain domain / AAA+ ATPase domain / WD40 repeat / karyogamy / astral microtubule / nuclear migration along microtubule / establishment of mitotic spindle localization / minus-end-directed vesicle transport along microtubule / ATP-dependent microtubule motor activity, minus-end-directed / dynein light chain binding / dynein light intermediate chain binding / dynein intermediate chain binding / spindle pole body / cytoplasmic dynein complex / cytoplasmic microtubule organization / establishment of mitotic spindle orientation / mitotic sister chromatid segregation / cytoplasmic microtubule / spindle pole / cell cortex / microtubule / cell cycle / cell division / ATP binding / cytoplasm / Nuclear distribution protein PAC1 / Dynein heavy chain, cytoplasmic
Function and homology information
Specimen sourceSaccharomyces cerevisiae (baker's yeast)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / 7.7 Å resolution
AuthorsCianfrocco, M.A. / DeSantis, M.E. / Htet, Z.M. / Tran, P.T. / Reck-Peterson, S.L. / Leschziner, A.E.
CitationJournal: Cell / Year: 2017
Title: Lis1 Has Two Opposing Modes of Regulating Cytoplasmic Dynein.
Authors: Morgan E DeSantis / Michael A Cianfrocco / Zaw Min Htet / Phuoc Tien Tran / Samara L Reck-Peterson / Andres E Leschziner
Abstract: Regulation is central to the functional versatility of cytoplasmic dynein, a motor involved in intracellular transport, cell division, and neurodevelopment. Previous work established that Lis1, a ...Regulation is central to the functional versatility of cytoplasmic dynein, a motor involved in intracellular transport, cell division, and neurodevelopment. Previous work established that Lis1, a conserved regulator of dynein, binds to its motor domain and induces a tight microtubule-binding state in dynein. The work we present here-a combination of biochemistry, single-molecule assays, and cryoelectron microscopy-led to the surprising discovery that Lis1 has two opposing modes of regulating dynein, being capable of inducing both low and high affinity for the microtubule. We show that these opposing modes depend on the stoichiometry of Lis1 binding to dynein and that this stoichiometry is regulated by the nucleotide state of dynein's AAA3 domain. The low-affinity state requires Lis1 to also bind to dynein at a novel conserved site, mutation of which disrupts Lis1's function in vivo. We propose a new model for the regulation of dynein by Lis1.
Validation Report
SummaryFull reportAbout validation report
DateDeposition: Apr 12, 2017 / Release: Sep 6, 2017
RevisionDateData content typeGroupCategoryItemProviderType
1.0Sep 6, 2017Structure modelrepositoryInitial release
1.1Sep 20, 2017Structure modelDatabase referencescitation / citation_author_citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _citation_author.name
1.2Sep 27, 2017Structure modelAuthor supporting evidencepdbx_audit_support_pdbx_audit_support.funding_organization

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Structure visualization

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Assembly

Deposited unit
A: Dynein heavy chain, cytoplasmic
B: Nuclear distribution protein PAC1


Theoretical massNumber of molelcules
Total (without water)313,3122
Polyers313,3122
Non-polymers00
Water0
1


TypeNameSymmetry operationNumber
identity operation1_5551
Buried area (Å2)520
ΔGint (kcal/M)-3
Surface area (Å2)124590

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Components

#1: Protein/peptide Dynein heavy chain, cytoplasmic / Dynein heavy chain / cytosolic / DYHC / Cytoplasmic dynein-1


Mass: 272666.250 Da / Num. of mol.: 1 / Fragment: UNP residues 1448-3028,3298-4092
Source: (gene. exp.) Saccharomyces cerevisiae (baker's yeast)
Gene: DYN1, DHC1, YKR054C / Production host: Saccharomyces cerevisiae (baker's yeast) / References: UniProt: P36022
#2: Protein/peptide Nuclear distribution protein PAC1 / Lissencephaly-1 homolog / LIS-1 / nudF homolog / Lis1


Mass: 40645.449 Da / Num. of mol.: 1 / Fragment: UNP residues 140-493
Source: (gene. exp.) Saccharomyces cerevisiae (baker's yeast)
Gene: PAC1, LIS1, SCY_5321 / Production host: Saccharomyces cerevisiae (baker's yeast) / References: UniProt: A6ZPA6

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Experimental details

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Experiment

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

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Sample preparation

ComponentName: Dynein-Lis1 co-complex with ATP / Type: COMPLEX / Entity ID: 1,2 / Source: RECOMBINANT
Molecular weightValue: 0.444 MDa / Experimental value: YES
Source (natural)Organism: Saccharomyces cerevisiae (baker's yeast)
Source (recombinant)Organism: Saccharomyces cerevisiae (baker's yeast) / Plasmid: RPY1302
Buffer solutionpH: 8
SpecimenConc.: 0.23 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: No pretreatment of grids / Grid material: GOLD / Grid mesh size: 300 / Grid type: Quantifoil UltrAuFoil 1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 276 kelvins

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Electron microscopy imaging

Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company
MicroscopyMicroscope model: FEI TALOS ARCTICA
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 36000 / Nominal defocus max: 5000 nm / Nominal defocus min: 2000 nm / Calibrated defocus min: 2000 nm / Calibrated defocus max: 5000 nm / Cs: 2.7 mm / C2 aperture diameter: 100 mm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 0.15 sec. / Electron dose: 82 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Number of grids imaged: 2 / Number of real images: 5614
Image scansMovie frames/image: 53 / Used frames/image: 3-53

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Processing

EM software
IDNameVersionCategoryDetails
2Leginon3.2image acquisitionAutomated hole and exposure targeting
7UCSF Chimera1.11.2model fittingInitial docking of atomic coordinates was performed in Chimera.
13Rosetta3.7model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 7.7 Å / Resolution method: FSC 0.143 CUT-OFF / Number of particles: 25520 / Symmetry type: POINT
Atomic model buildingOverall b value: 50 / Ref protocol: FLEXIBLE FIT / Ref space: REAL

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