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- EMDB-1788: Doublecortin-stabilised microtubules at secondary structure resolution -

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Basic information

Entry
Database: EMDB / ID: EMD-1788
TitleDoublecortin-stabilised microtubules at secondary structure resolution
Map dataThis is the corner between 4 tubulin dimers bound by doublecortin, in 13-protofilament microtubules
Sample
  • Sample: Microtubules co-polymerised with doublecortin
  • Protein or peptide: Tubulin alpha-1D chain
  • Protein or peptide: Tubulin beta-2B chain
  • Protein or peptide: Doublecortin
KeywordsTubulin / MAP / microtubule / stabilisation / doublecortin
Function / homology
Function and homology information


axoneme assembly / Neurofascin interactions / positive regulation of axon guidance / microtubule associated complex / microtubule-based process / central nervous system development / Hydrolases; Acting on acid anhydrides; Acting on GTP to facilitate cellular and subcellular movement / neuron migration / structural constituent of cytoskeleton / microtubule cytoskeleton organization ...axoneme assembly / Neurofascin interactions / positive regulation of axon guidance / microtubule associated complex / microtubule-based process / central nervous system development / Hydrolases; Acting on acid anhydrides; Acting on GTP to facilitate cellular and subcellular movement / neuron migration / structural constituent of cytoskeleton / microtubule cytoskeleton organization / microtubule cytoskeleton / retina development in camera-type eye / mitotic cell cycle / nervous system development / microtubule binding / microtubule / cytoskeleton / hydrolase activity / neuron projection / intracellular signal transduction / protein heterodimerization activity / GTPase activity / GTP binding / protein kinase binding / metal ion binding / cytosol / cytoplasm
Similarity search - Function
Neuronal migration protein doublecortin, chordata / : / Doublecortin / Doublecortin domain profile. / Domain in the Doublecortin (DCX) gene product / Doublecortin domain / Doublecortin domain / Doublecortin domain superfamily / Tubulin-beta mRNA autoregulation signal. / Alpha tubulin ...Neuronal migration protein doublecortin, chordata / : / Doublecortin / Doublecortin domain profile. / Domain in the Doublecortin (DCX) gene product / Doublecortin domain / Doublecortin domain / Doublecortin 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
Similarity search - Domain/homology
Neuronal migration protein doublecortin / Tubulin alpha-1D chain / Tubulin beta-2B chain
Similarity search - Component
Biological speciesBos taurus (cattle) / Homo sapiens (human)
Methodhelical reconstruction / cryo EM / negative staining / Resolution: 8.2 Å
AuthorsFourniol FJ / Sindelar CV / Amigues B / Clare D / Thomas G / Perderiset M / Francis F / Houdusse A / Moores 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
DepositionSep 15, 2010-
Header (metadata) releaseSep 24, 2010-
Map releaseOct 29, 2010-
UpdateSep 9, 2011-
Current statusSep 9, 2011Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.92
  • Imaged by UCSF Chimera
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  • Surface view colored by height
  • Surface level: 0.92
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-2xrp
  • Surface level: 0.92
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

EMD-1788.png

Downloads & links

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Map

FileDownload / File: emd_1788.map.gz / Format: CCP4 / Size: 319.3 KB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationThis is the corner between 4 tubulin dimers bound by doublecortin, in 13-protofilament microtubules
Voxel sizeX=Y=Z: 2.8 Å
Density
Contour LevelBy AUTHOR: 0.92 / Movie #1: 0.92
Minimum - Maximum-4.73387 - 5.69783
Average (Standard dev.)0.106293 (±0.755376)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin0150
Dimensions503449
Spacing503449
CellA: 95.2 Å / B: 140 Å / C: 137.2 Å
α=β=γ: 90 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z2.82.82.8
M x/y/z345049
origin x/y/z0.0000.0000.000
length x/y/z95.200140.000137.200
α/β/γ90.00090.00090.000
start NX/NY/NZ-184-184-183
NX/NY/NZ368368368
MAP C/R/S123
start NC/NR/NS1500
NC/NR/NS345049
D min/max/mean-4.7345.6980.106

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Supplemental data

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Sample components

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Entire : Microtubules co-polymerised with doublecortin

EntireName: Microtubules co-polymerised with doublecortin
Components
  • Sample: Microtubules co-polymerised with doublecortin
  • Protein or peptide: Tubulin alpha-1D chain
  • Protein or peptide: Tubulin beta-2B chain
  • Protein or peptide: Doublecortin

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Supramolecule #1000: Microtubules co-polymerised with doublecortin

SupramoleculeName: Microtubules co-polymerised with doublecortin / type: sample / ID: 1000 / Oligomeric state: 13-protofilament microtubule / Number unique components: 2

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Macromolecule #1: Tubulin alpha-1D chain

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

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Macromolecule #2: Tubulin beta-2B chain

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

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Macromolecule #3: Doublecortin

MacromoleculeName: Doublecortin / type: protein_or_peptide / ID: 3 / Name.synonym: DCX / Oligomeric state: Monomer / Recombinant expression: Yes
Source (natural)Organism: Homo sapiens (human) / synonym: Human / Location in cell: Cytoplasm
Recombinant expressionOrganism: Spodoptera frugiperda (Sf9) / Recombinant plasmid: pFastBac
SequenceInterPro: Doublecortin domain

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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: 80mM 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: OTHER / Details: Vitrification instrument: Vitrobot (FEI) / Method: Chamber at 37 degrees C, blot 2s

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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: Eucentric / Specimen holder model: OTHER
TemperatureAverage: 93 K
Alignment procedureLegacy - Astigmatism: Objective lens astigmatism was corrected at 150,000 times magnification
Image recordingCategory: FILM / Film or detector model: KODAK SO-163 FILM / Digitization - Scanner: ZEISS SCAI / Digitization - Sampling interval: 7 µm / Number real images: 63 / Average electron dose: 15 e/Å2 / Details: Images were binned with a factor of 2 / Bits/pixel: 8
Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company

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Image processing

CTF correctionDetails: done with FREALIGN
Final reconstructionAlgorithm: 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

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Atomic model buiding 1

Initial modelPDB ID:

1jff

SoftwareName: UCSF Chimera, Flex-EM
DetailsProtocol: Rigid body. 4 tubulin monomers were fitted separately in 3D map, which was zoned around the subunits, and multiple subunit fitting was refined in Flex-EM
RefinementSpace: REAL / Protocol: RIGID BODY FIT / Target criteria: Cross-correlation
Output model

PDB-2xrp:
Human Doublecortin N-DC Repeat (1MJD) and Mammalian Tubulin (1JFF and 3HKE) Docked into the 8-Angstrom Cryo-EM Map of Doublecortin- Stabilised Microtubules

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Atomic model buiding 2

Initial modelPDB ID:

1mjd


Chain - Chain ID: A
SoftwareName: UCSF Chimera, Flex-EM
DetailsPDBEntryID_givenInChain. Protocol: Rigid body. 4 tubulin monomers were fitted separately in 3D map, which was zoned around the subunits, and multiple subunit fitting was refined in Flex-EM
RefinementSpace: REAL / Protocol: RIGID BODY FIT / Target criteria: Cross-correlation
Output model

PDB-2xrp:
Human Doublecortin N-DC Repeat (1MJD) and Mammalian Tubulin (1JFF and 3HKE) Docked into the 8-Angstrom Cryo-EM Map of Doublecortin- Stabilised Microtubules

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