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- EMDB-6015: Dynein motor domain in the presence of ADP, with linker at position 1 -

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Basic information

Entry
Database: EMDB / ID: EMD-6015
TitleDynein motor domain in the presence of ADP, with linker at position 1Motor protein
Map dataReconstruction of dynein motor domain in 100 uM ADP
Sample
  • Sample: Dynein motor domain in 100 uM ADPMotor protein
  • Protein or peptide: Dynein heavy chain
Keywordsdynein / lis1 / regulation mechanism
Function / homology
Function and homology information


karyogamy / establishment of mitotic spindle localization / nuclear migration along microtubule / astral microtubule / minus-end-directed microtubule motor activity / cytoplasmic dynein complex / dynein light intermediate chain binding / spindle pole body / nuclear migration / dynein intermediate chain binding ...karyogamy / establishment of mitotic spindle localization / nuclear migration along microtubule / astral microtubule / minus-end-directed microtubule motor activity / cytoplasmic dynein complex / dynein light intermediate chain binding / spindle pole body / nuclear migration / dynein intermediate chain binding / mitotic sister chromatid segregation / establishment of mitotic spindle orientation / cytoplasmic microtubule / cytoplasmic microtubule organization / Neutrophil degranulation / mitotic spindle organization / cell cortex / ATP hydrolysis activity / ATP binding / cytoplasm
Similarity search - Function
: / 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 ...: / 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 / 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
Similarity search - Component
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
Methodsingle particle reconstruction / cryo EM / negative staining / Resolution: 18.3 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: 3.5
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 3.5
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

400_6015.gif

Downloads & links

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Map

FileDownload / File: emd_6015.map.gz / Format: CCP4 / Size: 21.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationReconstruction of dynein motor domain in 100 uM ADP
Voxel sizeX=Y=Z: 1.73 Å
Density
Contour LevelBy AUTHOR: 3.5 / Movie #1: 3.5
Minimum - Maximum-7.06270647 - 12.442327499999999
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-7.06312.442-0.000

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Supplemental data

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Supplemental map: relion half1 class001 unfil.map

Filerelion_half1_class001_unfil.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Supplemental map: relion half2 class001 unfil.map

Filerelion_half2_class001_unfil.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Dynein motor domain in 100 uM ADP

EntireName: Dynein motor domain in 100 uM ADPMotor protein
Components
  • Sample: Dynein motor domain in 100 uM ADPMotor protein
  • Protein or peptide: Dynein heavy chain

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Supramolecule #1000: Dynein motor domain in 100 uM ADP

SupramoleculeName: Dynein motor domain in 100 uM ADP / type: sample / ID: 1000 / Oligomeric state: monomer / Number unique components: 1
Molecular weightExperimental: 361 KDa / Theoretical: 361 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: RPY844 / synonym: Yeast / Location in cell: Cytoplasmic
Molecular weightExperimental: 361 KDa / Theoretical: 361 KDa
Recombinant expressionOrganism: Saccharomyces cerevisiae (brewer's yeast) / Recombinant strain: RPY844
SequenceUniProtKB: Dynein heavy chain, cytoplasmic

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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, 100 uM ADP
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: 1.31 µm / Nominal defocus min: 0.39 µm / Nominal magnification: 62000
Sample stageSpecimen holder model: GATAN LIQUID NITROGEN
DateJan 14, 2013
Image recordingCategory: CCD / Film or detector model: GATAN ULTRASCAN 4000 (4k x 4k) / Digitization - Sampling interval: 15 µm / Number real images: 200 / Average electron dose: 25 e/Å2

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Image processing

CTF correctionDetails: phase flip
Final reconstructionResolution.type: BY AUTHOR / Resolution: 18.3 Å / Resolution method: OTHER / Software - Name: RELION
Details: The dataset was first 3D-classified in RELION to sort different linker positions. Particles in this map displayed a linker unshifted relative to its position in the absence of nucleotide.
Number images used: 7630

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Atomic model buiding 1

Initial modelPDB ID:

4akg


Chain - Chain ID: A
SoftwareName: Chimera
DetailsTo compare linker positions between PDB file and EM map, only coordinates for the dynein ring (AAA1-6) were fit into the EM density.
RefinementSpace: REAL / Protocol: RIGID BODY FIT

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