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- EMDB-6016: Dynein motor domain in complex with Lis1 in the presence of ATP and Vi -

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Basic information

Entry
Database: EMDB / ID: EMD-6016
TitleDynein motor domain in complex with Lis1 in the presence of ATP and ViMotor protein
Map dataReconstruction of dynein motor domain in complex with Lis1 in the presence of ATP and Vi
Sample
  • Sample: Dynein motor domain in complex with Lis1 in 500 uM ATP/NaVO4Motor protein
  • Protein or peptide: Dynein heavy chain
  • Protein or peptide: Lis1PAFAH1B1
Keywordsdynein / lis1 / regulation mechanism
Function / homology
Function and homology information


positive regulation of microtubule plus-end binding / microtubule sliding / microtubule organizing center organization / karyogamy / establishment of mitotic spindle localization / nuclear migration along microtubule / astral microtubule / vesicle transport along microtubule / Antigen processing: Ubiquitination & Proteasome degradation / microtubule plus-end binding ...positive regulation of microtubule plus-end binding / microtubule sliding / microtubule organizing center organization / karyogamy / establishment of mitotic spindle localization / nuclear migration along microtubule / astral microtubule / vesicle transport along microtubule / Antigen processing: Ubiquitination & Proteasome degradation / microtubule plus-end binding / minus-end-directed microtubule motor activity / cytoplasmic dynein complex / spindle pole body / dynein light intermediate chain binding / nuclear migration / microtubule associated complex / dynein intermediate chain binding / dynein complex binding / mitotic sister chromatid segregation / establishment of mitotic spindle orientation / cytoplasmic microtubule / cytoplasmic microtubule organization / Neutrophil degranulation / mitotic spindle organization / kinetochore / spindle pole / nuclear envelope / cell cortex / cell division / ATP hydrolysis activity / ATP binding / nucleus / identical protein binding / cytoplasm
Similarity search - Function
Dynein regulator LIS1 / LIS1, N-terminal / : / DYN1, AAA+ ATPase lid domain / Dynein heavy chain 3, AAA+ lid domain / AAA+ lid domain / P-loop containing dynein motor region / Dynein heavy chain, tail / Dynein heavy chain, N-terminal region 1 / Dynein heavy chain ...Dynein regulator LIS1 / LIS1, N-terminal / : / DYN1, AAA+ ATPase lid domain / Dynein heavy chain 3, AAA+ lid domain / AAA+ lid domain / P-loop containing dynein motor region / Dynein heavy chain, tail / Dynein heavy chain, N-terminal region 1 / Dynein heavy chain / Dynein heavy chain region D6 P-loop domain / Dynein heavy chain, linker / Dynein heavy chain, AAA module D4 / Dynein heavy chain, coiled coil stalk / Dynein heavy chain, hydrolytic ATP-binding dynein motor region / Dynein heavy chain, ATP-binding dynein motor region / Dynein heavy chain AAA lid domain / Dynein heavy chain AAA lid domain superfamily / Dynein heavy chain, domain 2, N-terminal / Dynein heavy chain, linker, subdomain 3 / Dynein heavy chain, AAA1 domain, small subdomain / Dynein heavy chain region D6 P-loop domain / Dynein heavy chain, N-terminal region 2 / Hydrolytic ATP binding site of dynein motor region / Microtubule-binding stalk of dynein motor / P-loop containing dynein motor region D4 / ATP-binding dynein motor region / Dynein heavy chain AAA lid domain / G-protein beta WD-40 repeat / WD40 repeat, conserved site / Trp-Asp (WD) repeats signature. / Trp-Asp (WD) repeats profile. / Trp-Asp (WD) repeats circular profile. / WD domain, G-beta repeat / WD40 repeats / WD40 repeat / WD40-repeat-containing domain superfamily / WD40/YVTN repeat-like-containing domain superfamily / ATPases associated with a variety of cellular activities / AAA+ ATPase domain / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
Dynein heavy chain, cytoplasmic / Nuclear distribution protein PAC1
Similarity search - Component
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
Methodsingle particle reconstruction / cryo EM / negative staining / Resolution: 23.1 Å
AuthorsToropova K / Zou S / Roberts AJ / Redwine WB / Goodman BS / Reck-Peterson SL / Leschziner AE
CitationJournal: Elife / Year: 2014
Title: Lis1 regulates dynein by sterically blocking its mechanochemical cycle.
Authors: Katerina Toropova / Sirui Zou / Anthony J Roberts / William B Redwine / Brian S Goodman / Samara L Reck-Peterson / Andres E Leschziner /
Abstract: Regulation of cytoplasmic dynein's motor activity is essential for diverse eukaryotic functions, including cell division, intracellular transport, and brain development. The dynein regulator Lis1 is ...Regulation of cytoplasmic dynein's motor activity is essential for diverse eukaryotic functions, including cell division, intracellular transport, and brain development. The dynein regulator Lis1 is known to keep dynein bound to microtubules; however, how this is accomplished mechanistically remains unknown. We have used three-dimensional electron microscopy, single-molecule imaging, biochemistry, and in vivo assays to help establish this mechanism. The three-dimensional structure of the dynein-Lis1 complex shows that binding of Lis1 to dynein's AAA+ ring sterically prevents dynein's main mechanical element, the 'linker', from completing its normal conformational cycle. Single-molecule experiments show that eliminating this block by shortening the linker to a point where it can physically bypass Lis1 renders single dynein motors insensitive to regulation by Lis1. Our data reveal that Lis1 keeps dynein in a persistent microtubule-bound state by directly blocking the progression of its mechanochemical cycle.
History
DepositionAug 3, 2014-
Header (metadata) releaseSep 24, 2014-
Map releaseNov 19, 2014-
UpdateNov 19, 2014-
Current statusNov 19, 2014Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 3
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 3
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

FileDownload / File: emd_6016.map.gz / Format: CCP4 / Size: 21.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationReconstruction of dynein motor domain in complex with Lis1 in the presence of ATP and Vi
Voxel sizeX=Y=Z: 1.73 Å
Density
Contour LevelBy AUTHOR: 3.0 / Movie #1: 3
Minimum - Maximum-6.25472641 - 10.28431892
Average (Standard dev.)0.0 (±1.0)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-90-90-90
Dimensions180180180
Spacing180180180
CellA=B=C: 311.4 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.731.731.73
M x/y/z180180180
origin x/y/z0.0000.0000.000
length x/y/z311.400311.400311.400
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ969680
MAP C/R/S123
start NC/NR/NS-90-90-90
NC/NR/NS180180180
D min/max/mean-6.25510.2840.000

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Supplemental data

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Supplemental map: run4 half1 class001 unfil.map

Filerun4_half1_class001_unfil.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

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Supplemental map: run4 half2 class001 unfil.map

Filerun4_half2_class001_unfil.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

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Sample components

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Entire : Dynein motor domain in complex with Lis1 in 500 uM ATP/NaVO4

EntireName: Dynein motor domain in complex with Lis1 in 500 uM ATP/NaVO4Motor protein
Components
  • Sample: Dynein motor domain in complex with Lis1 in 500 uM ATP/NaVO4Motor protein
  • Protein or peptide: Dynein heavy chain
  • Protein or peptide: Lis1PAFAH1B1

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Supramolecule #1000: Dynein motor domain in complex with Lis1 in 500 uM ATP/NaVO4

SupramoleculeName: Dynein motor domain in complex with Lis1 in 500 uM ATP/NaVO4
type: sample / ID: 1000
Oligomeric state: One motor domain to one Lis1 propeller domain
Number unique components: 2
Molecular weightExperimental: 445 KDa / Theoretical: 445 KDa

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Macromolecule #1: Dynein heavy chain

MacromoleculeName: Dynein heavy chain / type: protein_or_peptide / ID: 1 / Name.synonym: DYN1 / Number of copies: 1 / Oligomeric state: Monomer / Recombinant expression: Yes
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast) / Strain: RPY1302 / synonym: Yeast / Location in cell: Cytoplasmic
Molecular weightExperimental: 331 KDa / Theoretical: 331 KDa
Recombinant expressionOrganism: Saccharomyces cerevisiae (brewer's yeast) / Recombinant strain: RPY1302
SequenceUniProtKB: Dynein heavy chain, cytoplasmic

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Macromolecule #2: Lis1

MacromoleculeName: Lis1 / type: protein_or_peptide / ID: 2 / Name.synonym: Pac1 / Details: Only a single copy of Lis1 is resolved in the map. / Number of copies: 2 / Oligomeric state: Dimer / Recombinant expression: Yes
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast) / Strain: RPY816 / synonym: Yeast / Location in cell: Cytoplasmic
Molecular weightExperimental: 57 KDa / Theoretical: 57 KDa
Recombinant expressionOrganism: Saccharomyces cerevisiae (brewer's yeast) / Recombinant strain: RPY816
SequenceUniProtKB: Nuclear distribution protein PAC1

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Experimental details

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Structure determination

Methodnegative staining, cryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration0.03 mg/mL
BufferpH: 8
Details: 50 mM Tris-HCl, 150 mM potassium acetate, 2 mM magnesium acetate, 1 mM EGTA, 1 mM DTT, 500 uM Mg-ATP/NaVO4
StainingType: NEGATIVE
Details: Grids with adsorbed protein were floated on 2% w/v uranyl formate, then sandwiched with a thin layer of carbon, blotted, and frozen in liquid nitrogen.
GridDetails: 200 mesh C-flat grid with thin carbon support
VitrificationCryogen name: NITROGEN / Instrument: OTHER
Method: Manually blot, wait 10-20 seconds, then manually plunge into liquid nitrogen.

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Electron microscopy

MicroscopeFEI TECNAI 20
Electron beamAcceleration voltage: 120 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 86800 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2 mm / Nominal defocus max: 1.2 µm / Nominal defocus min: 0.51 µm / Nominal magnification: 62000
Sample stageSpecimen holder model: GATAN LIQUID NITROGEN
DateMar 19, 2013
Image recordingCategory: CCD / Film or detector model: GATAN ULTRASCAN 4000 (4k x 4k) / Digitization - Sampling interval: 15 µm / Number real images: 103 / Average electron dose: 25 e/Å2

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Image processing

CTF correctionDetails: phase flip
Final reconstructionResolution.type: BY AUTHOR / Resolution: 23.1 Å / Resolution method: OTHER / Software - Name: RELION
Details: The dataset was first 3D-classified in RELION. Particles in this map displayed a resolved linker domain at lower contour levels.
Number images used: 1072

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Atomic model buiding 1

Initial modelPDB ID:

Chain - Chain ID: A
SoftwareName: Chimera
RefinementSpace: REAL / Protocol: RIGID BODY FIT

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