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- EMDB-2098: Cryo-electron microscopy reconstruction of doublecortin-stabilise... -

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Basic information

Entry
Database: EMDB / ID: EMD-2098
TitleCryo-electron microscopy reconstruction of doublecortin-stabilised microtubules in presence of kinesin
Map dataReconstruction of doublecortin-stabilised microtubules decorated with kinesin motor domain (rigor)
Sample
  • Sample: Doublecortin-stabilised microtubules decorated with kinesin motor domains
  • Protein or peptide: Tubulin alpha-1D chain
  • Protein or peptide: Doublecortin
  • Protein or peptide: Tubulin beta-2B chain
  • Protein or peptide: Kinesin motor domain
Keywordsdoublecortin / kinesin / microtubule / Microtubule-Associated Protein
Function / homology
Function and homology information


RHO GTPases activate KTN1 / Kinesins / COPI-dependent Golgi-to-ER retrograde traffic / membrane-bounded organelle / cytoplasm organization / plus-end-directed vesicle transport along microtubule / anterograde dendritic transport of neurotransmitter receptor complex / positive regulation of voltage-gated sodium channel activity / retrograde neuronal dense core vesicle transport / positive regulation of vesicle fusion ...RHO GTPases activate KTN1 / Kinesins / COPI-dependent Golgi-to-ER retrograde traffic / membrane-bounded organelle / cytoplasm organization / plus-end-directed vesicle transport along microtubule / anterograde dendritic transport of neurotransmitter receptor complex / positive regulation of voltage-gated sodium channel activity / retrograde neuronal dense core vesicle transport / positive regulation of vesicle fusion / anterograde axonal protein transport / MHC class II antigen presentation / cytoskeleton-dependent intracellular transport / vesicle transport along microtubule / positive regulation of intracellular protein transport / positive regulation of potassium ion transport / JUN kinase binding / plus-end-directed microtubule motor activity / stress granule disassembly / positive regulation of axon guidance / microtubule lateral binding / ciliary rootlet / centrosome localization / kinesin complex / synaptic vesicle transport / microtubule motor activity / microtubule-based movement / positive regulation of insulin secretion involved in cellular response to glucose stimulus / endocytic vesicle / centriolar satellite / microtubule-based process / phagocytic vesicle / axonal growth cone / axon cytoplasm / regulation of membrane potential / dendrite cytoplasm / axon guidance / positive regulation of synaptic transmission, GABAergic / hippocampus development / positive regulation of protein localization to plasma membrane / Hydrolases; Acting on acid anhydrides; Acting on GTP to facilitate cellular and subcellular movement / brain development / structural constituent of cytoskeleton / microtubule cytoskeleton organization / cellular response to type II interferon / microtubule cytoskeleton / mitotic cell cycle / nervous system development / microtubule binding / vesicle / microtubule / hydrolase activity / neuron projection / protein heterodimerization activity / GTPase activity / GTP binding / perinuclear region of cytoplasm / ATP hydrolysis activity / ATP binding / identical protein binding / metal ion binding / cytosol / cytoplasm
Similarity search - Function
Doublecortin domain / Kinesin-like protein / Kinesin motor domain signature. / Kinesin motor domain, conserved site / Kinesin motor domain / Kinesin motor domain profile. / Kinesin motor, catalytic domain. ATPase. / Kinesin motor domain / Kinesin motor domain superfamily / Tubulin-beta mRNA autoregulation signal. ...Doublecortin domain / Kinesin-like protein / Kinesin motor domain signature. / Kinesin motor domain, conserved site / Kinesin motor domain / Kinesin motor domain profile. / Kinesin motor, catalytic domain. ATPase. / Kinesin motor domain / Kinesin motor domain superfamily / Tubulin-beta mRNA autoregulation signal. / Alpha tubulin / Alpha tubulin / Beta tubulin, autoregulation binding site / 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 / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
Tubulin alpha-1D chain / Kinesin-1 heavy chain / Tubulin beta-2B chain
Similarity search - Component
Biological speciesBos taurus (cattle) / Homo sapiens (human) / Rattus norvegicus (Norway rat)
Methodhelical reconstruction / cryo EM / negative staining / Resolution: 8.2 Å
AuthorsLiu JS / Schubert CR / Fu X / Fourniol FJ / Jaiswal JK / Houdusse A / Stultz CM / Moores CA / Walsh CA
CitationJournal: J Cell Biol / Year: 2010
Title: Template-free 13-protofilament microtubule-MAP assembly visualized at 8 A resolution.
Authors: Franck J Fourniol / Charles V Sindelar / Béatrice Amigues / Daniel K Clare / Geraint Thomas / Mylène Perderiset / Fiona Francis / Anne Houdusse / Carolyn A Moores /
Abstract: Microtubule-associated proteins (MAPs) are essential for regulating and organizing cellular microtubules (MTs). However, our mechanistic understanding of MAP function is limited by a lack of detailed ...Microtubule-associated proteins (MAPs) are essential for regulating and organizing cellular microtubules (MTs). However, our mechanistic understanding of MAP function is limited by a lack of detailed structural information. Using cryo-electron microscopy and single particle algorithms, we solved the 8 Å structure of doublecortin (DCX)-stabilized MTs. Because of DCX's unusual ability to specifically nucleate and stabilize 13-protofilament MTs, our reconstruction provides unprecedented insight into the structure of MTs with an in vivo architecture, and in the absence of a stabilizing drug. DCX specifically recognizes the corner of four tubulin dimers, a binding mode ideally suited to stabilizing both lateral and longitudinal lattice contacts. A striking consequence of this is that DCX does not bind the MT seam. DCX binding on the MT surface indirectly stabilizes conserved tubulin-tubulin lateral contacts in the MT lumen, operating independently of the nucleotide bound to tubulin. DCX's exquisite binding selectivity uncovers important insights into regulation of cellular MTs.
History
DepositionMay 10, 2012-
Header (metadata) releaseMay 17, 2012-
Map releaseAug 15, 2012-
UpdateSep 26, 2012-
Current statusSep 26, 2012Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 1.55
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 1.55
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-4atx
  • Surface level: 1.8
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-4atx
  • Imaged by Jmol
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Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

EMD-2098.jpg

Downloads & links

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Map

FileDownload / File: emd_2098.map.gz / Format: CCP4 / Size: 1.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationReconstruction of doublecortin-stabilised microtubules decorated with kinesin motor domain (rigor)
Voxel sizeX=Y=Z: 2.8 Å
Density
Contour LevelBy AUTHOR: 1.55 / Movie #1: 1.55
Minimum - Maximum-8.071179389999999 - 11.104262350000001
Average (Standard dev.)0.54360223 (±1.79660368)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin416675
Dimensions5085101
Spacing5085101
CellA: 238.0 Å / B: 140.0 Å / C: 282.8 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z2.82.82.8
M x/y/z8550101
origin x/y/z0.0000.0000.000
length x/y/z238.000140.000282.800
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ128128168
MAP C/R/S123
start NC/NR/NS664175
NC/NR/NS8550101
D min/max/mean-8.07111.1040.544

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Supplemental data

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Sample components

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Entire : Doublecortin-stabilised microtubules decorated with kinesin motor...

EntireName: Doublecortin-stabilised microtubules decorated with kinesin motor domains
Components
  • Sample: Doublecortin-stabilised microtubules decorated with kinesin motor domains
  • Protein or peptide: Tubulin alpha-1D chain
  • Protein or peptide: Doublecortin
  • Protein or peptide: Tubulin beta-2B chain
  • Protein or peptide: Kinesin motor domain

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Supramolecule #1000: Doublecortin-stabilised microtubules decorated with kinesin motor...

SupramoleculeName: Doublecortin-stabilised microtubules decorated with kinesin motor domains
type: sample / ID: 1000 / Oligomeric state: 13-protofilament microtubule / Number unique components: 4

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Macromolecule #1: Tubulin alpha-1D chain

MacromoleculeName: Tubulin alpha-1D chain / type: protein_or_peptide / ID: 1 / Oligomeric state: Heterodimer / Recombinant expression: No / Database: NCBI
Source (natural)Organism: Bos taurus (cattle) / synonym: Cattle / Tissue: Brain / Location in cell: Cytoplasm
Molecular weightTheoretical: 50 KDa
SequenceInterPro: Alpha tubulin

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Macromolecule #2: Doublecortin

MacromoleculeName: Doublecortin / type: protein_or_peptide / ID: 2 / Name.synonym: DCX / Recombinant expression: Yes
Source (natural)Organism: Homo sapiens (human) / synonym: Human
Molecular weightTheoretical: 40 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm) / Recombinant plasmid: pFastBac
SequenceInterPro: Doublecortin domain

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Macromolecule #3: Tubulin beta-2B chain

MacromoleculeName: Tubulin beta-2B chain / type: protein_or_peptide / ID: 3 / Oligomeric state: Heterodimer / Recombinant expression: No / Database: NCBI
Source (natural)Organism: Bos taurus (cattle) / synonym: Cattle / Tissue: Brain / Location in cell: Cytoplasm
Molecular weightTheoretical: 50 KDa
SequenceInterPro: Beta tubulin, autoregulation binding site

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Macromolecule #4: Kinesin motor domain

MacromoleculeName: Kinesin motor domain / type: protein_or_peptide / ID: 4 / Details: mutant T93N / Recombinant expression: Yes
Source (natural)Organism: Rattus norvegicus (Norway rat) / synonym: Norway rat
Molecular weightTheoretical: 40 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)

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Experimental details

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Structure determination

Methodnegative staining, cryo EM
Processinghelical reconstruction
Aggregation statefilament

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Sample preparation

BufferpH: 6.8
Details: 20mM Pipes, 1mM EGTA, 3mM MgCl2, 1mM TCEP, 0.5mM GTP
StainingType: NEGATIVE / Details: cryo-EM
GridDetails: 300 mesh lacey carbon grids
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Instrument: FEI VITROBOT MARK I

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Electron microscopy

MicroscopeFEI TECNAI F20
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.0 mm / Nominal defocus max: 2.9 µm / Nominal defocus min: 0.76 µm / Nominal magnification: 50000
Sample stageSpecimen holder model: SIDE ENTRY, EUCENTRIC
TemperatureAverage: 93 K
Alignment procedureLegacy - Astigmatism: Objective lens astigmatism was corrected at 150,000 times magnification
DateSep 1, 2009
Image recordingCategory: FILM / Film or detector model: KODAK SO-163 FILM / Digitization - Scanner: ZEISS SCAI / Number real images: 63 / Average electron dose: 15 e/Å2 / Bits/pixel: 8
Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company

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Image processing

CTF correctionDetails: done with FREALIGN
Final reconstructionApplied symmetry - Helical parameters - Δz: 9.2 Å
Applied symmetry - Helical parameters - Δ&Phi: 27.7 °
Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 8.2 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: SPIDER, FREALIGN
Details: Approximately 168,000 asymmetric units were averaged together in the final map
DetailsParticles were aligned using Spider and Frealign (Sindelar and Downing, 2010)

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Atomic model buiding 1

Initial modelPDB ID:

2xrp


Chain - #0 - Chain ID: E / Chain - #1 - Chain ID: F
SoftwareName: Chimera
DetailsProtocol: Rigid body
RefinementSpace: REAL / Protocol: RIGID BODY FIT / Target criteria: Cross correlation
Output model

PDB-4atx:
Rigor kinesin motor domain with an ordered neck-linker, docked on tubulin dimer, modelled into the 8A cryo-EM map of doublecortin- microtubules decorated with kinesin

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Atomic model buiding 2

Initial modelPDB ID:

1bg2


Chain - Chain ID: A
SoftwareName: Chimera, Flex-EM
DetailsProtocol: Flexible fitting. Kinesin neck-linker (aa 324-329) was modeled into the EM density, and its position refined using Flex-EM, along with loop 2, loop 9 and helix alpha6
RefinementSpace: REAL / Protocol: FLEXIBLE FIT / Target criteria: Cross correlation
Output model

PDB-4atx:
Rigor kinesin motor domain with an ordered neck-linker, docked on tubulin dimer, modelled into the 8A cryo-EM map of doublecortin- microtubules decorated with kinesin

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