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- EMDB-8673: Cryo-EM structure of yeast cytoplasmic dynein-1 with Lis1 and ATP -

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Basic information

Entry
Database: EMDB / ID: 8673
TitleCryo-EM structure of yeast cytoplasmic dynein-1 with Lis1 and ATP
Map dataCryo-EM structure of yeast dynein motor domain bound to Lis1 in the presence of ATP
SampleDynein-Lis1 co-complex with ATP:
Dynein heavy chain, cytoplasmic / Nuclear distribution protein PAC1
Function / homologyDynein heavy chain, domain-2 / Dynein heavy chain, AAA module D4 / Dynein heavy chain domain / ATPase, dynein-related, AAA domain / Dynein heavy chain, domain-1 / WD40/YVTN repeat-like-containing domain superfamily / Dynein regulator LIS1 / WD40-repeat-containing domain / WD40 repeat, conserved site / G-protein beta WD-40 repeat ...Dynein heavy chain, domain-2 / Dynein heavy chain, AAA module D4 / Dynein heavy chain domain / ATPase, dynein-related, AAA domain / Dynein heavy chain, domain-1 / WD40/YVTN repeat-like-containing domain superfamily / Dynein regulator LIS1 / WD40-repeat-containing domain / WD40 repeat, conserved site / G-protein beta WD-40 repeat / Dynein heavy chain, coiled coil stalk / Dynein heavy chain, N-terminal region 1 / 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 / LIS1, N-terminal / WD domain, G-beta repeat / Dynein heavy chain and region D6 of dynein motor / AAA+ ATPase domain / WD40 repeat / AAA domain (dynein-related subfamily) / Hydrolytic ATP binding site of dynein motor region D1 / Trp-Asp (WD) repeats circular profile. / Trp-Asp (WD) repeats profile. / Trp-Asp (WD) repeats signature. / ATP-binding dynein motor region D5 / P-loop containing dynein motor region D4 / Microtubule-binding stalk of dynein motor / Dynein heavy chain, N-terminal region 2 / 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
SourceSaccharomyces cerevisiae (baker's yeast)
Methodsingle particle reconstruction / cryo EM / 7.7 Å resolution
AuthorsCianfrocco MA / DeSantis ME
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 ReportPDB-ID: 5vh9

SummaryFull reportAbout validation report
DateDeposition: Apr 12, 2017 / Header (metadata) release: Aug 23, 2017 / Map release: Sep 6, 2017 / Last update: Sep 27, 2017

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.0119
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.0119
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: : PDB-5vh9
  • Surface level: 0.0119
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

Fileemd_8673.map.gz (map file in CCP4 format, 136049 KB)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
324 pix
1.2 Å/pix.
= 388.8 Å
324 pix
1.2 Å/pix.
= 388.8 Å
324 pix
1.2 Å/pix.
= 388.8 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.2 Å
Density
Contour Level:0.0119 (by author), 0.0119 (movie #1):
Minimum - Maximum-0.018116549 - 0.06178146
Average (Standard dev.)0.00017972072 (0.0020816762)
Details

EMDB XML:

Space Group Number1
Map Geometry
Axis orderXYZ
Dimensions324324324
Origin000
Limit323323323
Spacing324324324
CellA=B=C: 388.80002 Å
α=β=γ: 90 deg.

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.21.21.2
M x/y/z324324324
origin x/y/z0.0000.0000.000
length x/y/z388.800388.800388.800
α/β/γ90.00090.00090.000
start NX/NY/NZ
NX/NY/NZ
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS324324324
D min/max/mean-0.0180.0620.000

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Supplemental data

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Sample components

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Entire Dynein-Lis1 co-complex with ATP

EntireName: Dynein-Lis1 co-complex with ATP / Number of components: 3
MassExperimental: 444 kDa

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Component #1: protein, Dynein-Lis1 co-complex with ATP

ProteinName: Dynein-Lis1 co-complex with ATP / Recombinant expression: No
MassExperimental: 444 kDa
SourceSpecies: Saccharomyces cerevisiae (baker's yeast)
Source (engineered)Expression System: Saccharomyces cerevisiae (baker's yeast) / Vector: RPY1302

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Component #2: protein, Dynein heavy chain, cytoplasmic

ProteinName: Dynein heavy chain, cytoplasmic / Recombinant expression: No
MassTheoretical: 272.66625 kDa
Source (engineered)Expression System: Saccharomyces cerevisiae (baker's yeast)

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Component #3: protein, Nuclear distribution protein PAC1

ProteinName: Nuclear distribution protein PAC1 / Recombinant expression: No
MassTheoretical: 40.645449 kDa
Source (engineered)Expression System: Saccharomyces cerevisiae (baker's yeast)

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Experimental details

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Sample preparation

SpecimenSpecimen state: particle / Method: cryo EM
Sample solutionSpecimen conc.: 0.23 mg/ml / pH: 8
Support filmNo pretreatment of grids
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Temperature: 276 K / Humidity: 100 %

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Electron microscopy imaging

Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company
ImagingMicroscope: FEI TALOS ARCTICA
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Electron dose: 82 e/Å2 / Illumination mode: FLOOD BEAM
LensMagnification: 36000 X (nominal) / Cs: 2.7 mm / Imaging mode: BRIGHT FIELD / Defocus: 2000 - 5000 nm
Specimen HolderModel: FEI TITAN KRIOS AUTOGRID HOLDER
CameraDetector: GATAN K2 (4k x 4k)

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Image acquisition

Image acquisitionNumber of digital images: 5614

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Image processing

ProcessingMethod: single particle reconstruction / Number of projections: 25520
3D reconstructionResolution: 7.7 Å / Resolution method: FSC 0.143 CUT-OFF

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Atomic model buiding

Output model

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