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- EMDB-5439: Structural basis for microtubule binding and release by dynein -

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Basic information

Entry
Database: EMDB / ID: EMD-5439
TitleStructural basis for microtubule binding and release by dynein
Map dataConstruct of the high affinity dynein microtubule-binding domain fused to seryl-tRNA synthase-monomer
Sample
  • Sample: high affinity construct of dynein microtubule-binding domain fused to seryl-tRNA synthase monomer
  • Protein or peptide: High affinity dynein microtubule binding domain
  • Protein or peptide: tubulin
Keywordsdynein / microtubule bound / high affinity microtubule binding domain
Function / homology
Function and homology information


COPI-independent Golgi-to-ER retrograde traffic / HSP90 chaperone cycle for steroid hormone receptors (SHR) in the presence of ligand / Amplification of signal from unattached kinetochores via a MAD2 inhibitory signal / COPI-mediated anterograde transport / Aggrephagy / Mitotic Prometaphase / EML4 and NUDC in mitotic spindle formation / Resolution of Sister Chromatid Cohesion / cilium movement / RHO GTPases Activate Formins ...COPI-independent Golgi-to-ER retrograde traffic / HSP90 chaperone cycle for steroid hormone receptors (SHR) in the presence of ligand / Amplification of signal from unattached kinetochores via a MAD2 inhibitory signal / COPI-mediated anterograde transport / Aggrephagy / Mitotic Prometaphase / EML4 and NUDC in mitotic spindle formation / Resolution of Sister Chromatid Cohesion / cilium movement / RHO GTPases Activate Formins / Separation of Sister Chromatids / Loss of Nlp from mitotic centrosomes / Recruitment of mitotic centrosome proteins and complexes / Loss of proteins required for interphase microtubule organization from the centrosome / Recruitment of NuMA to mitotic centrosomes / Anchoring of the basal body to the plasma membrane / AURKA Activation by TPX2 / Regulation of PLK1 Activity at G2/M Transition / positive regulation of intracellular transport / regulation of metaphase plate congression / positive regulation of spindle assembly / MHC class II antigen presentation / establishment of spindle localization / dynein complex / positive regulation of axon guidance / P-body assembly / minus-end-directed microtubule motor activity / cytoplasmic dynein complex / dynein light intermediate chain binding / dynein intermediate chain binding / microtubule-based process / cytoplasmic microtubule / Neutrophil degranulation / stress granule assembly / cellular response to interleukin-4 / regulation of mitotic spindle organization / filopodium / structural constituent of cytoskeleton / microtubule cytoskeleton organization / neuron migration / mitotic cell cycle / double-stranded RNA binding / positive regulation of cold-induced thermogenesis / microtubule cytoskeleton / Hydrolases; Acting on acid anhydrides; Acting on GTP to facilitate cellular and subcellular movement / microtubule / cilium / protein heterodimerization activity / cell division / GTPase activity / ubiquitin protein ligase binding / centrosome / GTP binding / ATP hydrolysis activity / ATP binding / metal ion binding / identical protein binding / cytoplasm / cytosol
Similarity search - Function
Dynein heavy chain, AAA 5 extension domain / Dynein heavy chain AAA lid domain / Dynein heavy chain, C-terminal domain / Dynein heavy chain, C-terminal domain, barrel region / Dynein heavy chain C-terminal domain / : / Dynein heavy chain, ATPase lid domain / Dynein heavy chain, tail / Dynein heavy chain, N-terminal region 1 / P-loop containing dynein motor region ...Dynein heavy chain, AAA 5 extension domain / Dynein heavy chain AAA lid domain / Dynein heavy chain, C-terminal domain / Dynein heavy chain, C-terminal domain, barrel region / Dynein heavy chain C-terminal domain / : / Dynein heavy chain, ATPase lid domain / Dynein heavy chain, tail / Dynein heavy chain, N-terminal region 1 / P-loop containing dynein motor region / 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 / 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 / Alpha tubulin / Tubulin-beta mRNA autoregulation signal. / Beta tubulin, autoregulation binding site / Beta tubulin / Tubulin / Tubulin, C-terminal / Tubulin C-terminal domain / Tubulin, conserved site / Tubulin subunits alpha, beta, and gamma signature. / Tubulin/FtsZ family, C-terminal domain / Tubulin/FtsZ-like, C-terminal domain / Tubulin/FtsZ, C-terminal / Tubulin/FtsZ, 2-layer sandwich domain / Tubulin/FtsZ family, GTPase domain / Tubulin/FtsZ family, GTPase domain / Tubulin/FtsZ, GTPase domain / Tubulin/FtsZ, GTPase domain superfamily / ATPases associated with a variety of cellular activities / AAA+ ATPase domain / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
Tubulin alpha-1B chain / Tubulin beta-2B chain / Cytoplasmic dynein 1 heavy chain 1
Similarity search - Component
Biological speciesMus musculus (house mouse) / Bos taurus (domestic cattle)
Methodhelical reconstruction / cryo EM / Resolution: 9.7 Å
AuthorsRedwine WB / Hernandez-Lopez R / Zou S / Huang J / Reck-Peterson SL / Leschziner AE
CitationJournal: Science / Year: 2012
Title: Structural basis for microtubule binding and release by dynein.
Authors: W B Redwine / R Hernandez-Lopez / S Zou / J Huang / S L Reck-Peterson / A E Leschziner /
Abstract: Cytoplasmic dynein is a microtubule-based motor required for intracellular transport and cell division. Its movement involves coupling cycles of track binding and release with cycles of force- ...Cytoplasmic dynein is a microtubule-based motor required for intracellular transport and cell division. Its movement involves coupling cycles of track binding and release with cycles of force-generating nucleotide hydrolysis. How this is accomplished given the ~25 nanometers separating dynein's track- and nucleotide-binding sites is not understood. Here, we present a subnanometer-resolution structure of dynein's microtubule-binding domain bound to microtubules by cryo-electron microscopy that was used to generate a pseudo-atomic model of the complex with molecular dynamics. We identified large rearrangements triggered by track binding and specific interactions, confirmed by mutagenesis and single-molecule motility assays, which tune dynein's affinity for microtubules. Our results provide a molecular model for how dynein's binding to microtubules is communicated to the rest of the motor.
History
DepositionJun 24, 2012-
Header (metadata) releaseSep 26, 2012-
Map releaseSep 26, 2012-
UpdateOct 3, 2012-
Current statusOct 3, 2012Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.0036
  • Imaged by UCSF Chimera
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  • Surface view colored by height
  • Surface level: 0.0036
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-3j1t
  • Surface level: 0.0036
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-3j1u
  • Surface level: 0.0036
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_5439.png

Downloads & links

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Map

FileDownload / File: emd_5439.map.gz / Format: CCP4 / Size: 492.2 KB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationConstruct of the high affinity dynein microtubule-binding domain fused to seryl-tRNA synthase-monomer
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.99 Å/pix.
x 60 pix.
= 119.28 Å		1.99 Å/pix.
x 39 pix.
= 77.532 Å		1.99 Å/pix.
x 55 pix.
= 109.34 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

generated in cubic-lattice coordinate

Voxel sizeX=Y=Z: 1.988 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.0036 / Movie #1: 0.0036
Minimum - Maximum-0.01287546 - 0.0133277
Average (Standard dev.)-0.00072928 (±0.00299425)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-9-17-12
Dimensions395560
Spacing395560
CellA: 109.340004 Å / B: 77.532 Å / C: 119.28 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.9881.9881.988
M x/y/z553960
origin x/y/z0.0000.0000.000
length x/y/z109.34077.532119.280
α/β/γ90.00090.00090.000
start NX/NY/NZ-150-1500
NX/NY/NZ301301151
MAP C/R/S123
start NC/NR/NS-17-9-12
NC/NR/NS553960
D min/max/mean-0.0130.013-0.001

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Supplemental data

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Sample components

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Entire : high affinity construct of dynein microtubule-binding domain fuse...

EntireName: high affinity construct of dynein microtubule-binding domain fused to seryl-tRNA synthase monomer
Components
  • Sample: high affinity construct of dynein microtubule-binding domain fused to seryl-tRNA synthase monomer
  • Protein or peptide: High affinity dynein microtubule binding domain
  • Protein or peptide: tubulin

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Supramolecule #1000: high affinity construct of dynein microtubule-binding domain fuse...

SupramoleculeName: high affinity construct of dynein microtubule-binding domain fused to seryl-tRNA synthase monomer
type: sample / ID: 1000 / Number unique components: 2

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Macromolecule #1: High affinity dynein microtubule binding domain

MacromoleculeName: High affinity dynein microtubule binding domain / type: protein_or_peptide / ID: 1 / Recombinant expression: Yes
Source (natural)Organism: Mus musculus (house mouse) / synonym: mouse
Recombinant expressionOrganism: Escherichia coli (E. coli)

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Macromolecule #2: tubulin

MacromoleculeName: tubulin / type: protein_or_peptide / ID: 2 / Recombinant expression: No / Database: NCBI
Source (natural)Organism: Bos taurus (domestic cattle)

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Experimental details

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Structure determination

Methodcryo EM
Processinghelical reconstruction
Aggregation statefilament

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Sample preparation

Concentration15 mg/mL
BufferpH: 8 / Details: 50 mM Tris-HCl, 1 mM MgCl2, 1 mM EGTA, 1 mM DTT
GridDetails: C-flat 2/2-2C holey carbon grids (Protochips) were glow-discharged for 20 seconds at 30 mA in an Edwards carbon evaporator.
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 120 K / Instrument: FEI VITROBOT MARK IV
Method: The solution was blotted manually, and the process of addition and blotting of SRS-MTBD was repeated a total of three times. The final blotting was done inside the humidity chamber of a ...Method: The solution was blotted manually, and the process of addition and blotting of SRS-MTBD was repeated a total of three times. The final blotting was done inside the humidity chamber of a Vitrobot Mark IV (FEI) at 22 Celsius.

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Electron microscopy

MicroscopeFEI TECNAI F20
TemperatureAverage: 100 K
DateApr 10, 2011
Image recordingCategory: FILM / Film or detector model: KODAK SO-163 FILM / Digitization - Scanner: OTHER / Digitization - Sampling interval: 6.35 µm / Number real images: 225 / Average electron dose: 15 e/Å2 / Bits/pixel: 8
Electron beamAcceleration voltage: 120 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 63377 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.2 mm / Nominal defocus max: 2.5 µm / Nominal defocus min: 0.5 µm / Nominal magnification: 62000
Sample stageSpecimen holder model: GATAN LIQUID NITROGEN
Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company

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Image processing

DetailsParticles were aligned using customized scripts in SPIDER and 3D reconstruction was done using Frealign.
Final reconstructionApplied symmetry - Helical parameters - Δz: 9.26 Å
Applied symmetry - Helical parameters - Δ&Phi: 25.76 °
Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 9.7 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: Frealign
CTF correctionDetails: each particle

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Atomic model buiding 1

Initial modelPDB ID:

3err


Chain - Chain ID: A
SoftwareName: Chimera
DetailsProtocol: Rigid Body. The components of the complex were separately fitted by manual docking
RefinementSpace: REAL / Protocol: RIGID BODY FIT
Output model

PDB-3j1t:
High affinity dynein microtubule binding domain - tubulin complex

PDB-3j1u:
Low affinity dynein microtubule binding domain - tubulin complex

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Atomic model buiding 2

Initial modelPDB ID:

1jff


Chain - #0 - Chain ID: A / Chain - #1 - Chain ID: B
SoftwareName: Chimera
DetailsProtocol: Rigid Body. The components of the complex were separately fitted by manual docking
RefinementSpace: REAL / Protocol: RIGID BODY FIT
Output model

PDB-3j1t:
High affinity dynein microtubule binding domain - tubulin complex

PDB-3j1u:
Low affinity dynein microtubule binding domain - tubulin complex

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