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- EMDB-1787: Asymmetric reconstruction of doublecortin-stabilised microtubules... -

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Basic information

Entry
Database: EMDB / ID: EMD-1787
TitleAsymmetric reconstruction of doublecortin-stabilised microtubules decorated with kinesin motor domain
Map dataThis is a C1 reconstruction of doublecortin-stabilised microtubule decorated with kinesin motor domain
Sample
  • Sample: Microtubules co-polymerised with doublecortin and bound with kinesin motor domain
  • Protein or peptide: Alpha-beta tubulin dimer
  • Protein or peptide: Doublecortin
  • Protein or peptide: Kinesin motor domain
KeywordsTubulin / MAP / microtubule / stabilisation / doublecortin / kinesin
Biological speciesBos taurus (domestic cattle) / Homo sapiens (human) / Rattus norvegicus (Norway rat)
Methodsingle particle reconstruction / cryo EM / negative staining / Resolution: 13.5 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 19, 2012-
Current statusSep 19, 2012Processing site: PDBe / Status: Released

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Structure visualization

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

1787_image.png

Downloads & links

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Map

FileDownload / File: emd_1787.map.gz / Format: CCP4 / Size: 37.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationThis is a C1 reconstruction of doublecortin-stabilised microtubule decorated with kinesin motor domain
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
2.8 Å/pix.
x 216 pix.
= 604.8 Å		2.8 Å/pix.
x 216 pix.
= 604.8 Å		2.8 Å/pix.
x 216 pix.
= 604.8 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 2.8 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 3.0 / Movie #1: 3
Minimum - Maximum-3.84556389 - 7.45892239
Average (Standard dev.)0.0 (±1.0)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions216216216
Spacing216216216
CellA=B=C: 604.8 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z2.82.82.8
M x/y/z216216216
origin x/y/z0.0000.0000.000
length x/y/z604.800604.800604.800
α/β/γ90.00090.00090.000
start NX/NY/NZ-184-184-183
NX/NY/NZ368368368
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS216216216
D min/max/mean-3.8467.459-0.000

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Supplemental data

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Sample components

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Entire : Microtubules co-polymerised with doublecortin and bound with kine...

EntireName: Microtubules co-polymerised with doublecortin and bound with kinesin motor domain
Components
  • Sample: Microtubules co-polymerised with doublecortin and bound with kinesin motor domain
  • Protein or peptide: Alpha-beta tubulin dimer
  • Protein or peptide: Doublecortin
  • Protein or peptide: Kinesin motor domain

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Supramolecule #1000: Microtubules co-polymerised with doublecortin and bound with kine...

SupramoleculeName: Microtubules co-polymerised with doublecortin and bound with kinesin motor domain
type: sample / ID: 1000 / Oligomeric state: 13-protofilament microtubule / Number unique components: 3

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Macromolecule #1: Alpha-beta tubulin dimer

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

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Macromolecule #2: Doublecortin

MacromoleculeName: Doublecortin / type: protein_or_peptide / ID: 2 / 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 (fall armyworm) / Recombinant plasmid: pFastBac

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Macromolecule #3: Kinesin motor domain

MacromoleculeName: Kinesin motor domain / type: protein_or_peptide / ID: 3 / Name.synonym: Kinesin head / Oligomeric state: Monomer / Recombinant expression: Yes
Source (natural)Organism: Rattus norvegicus (Norway rat) / synonym: Rat / Location in cell: Cytoplasm
Recombinant expressionOrganism: Escherichia coli (E. coli)

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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

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
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
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / 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
Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company

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Image processing

CTF correctionDetails: FREALIGN
Final reconstructionAlgorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 13.5 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: SPIDER, FREALIGN
Details: C1 map calculated from approximately 13,000 one-dimer-long microtubule segments

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