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- EMDB-2004: Asymmetric reconstruction of 13-protofilament GTPgammaS microtubu... -

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Entry
Database: EMDB / ID: EMD-2004
TitleAsymmetric reconstruction of 13-protofilament GTPgammaS microtubules decorated with Mal3 CH domain
Map dataAsymmetric reconstruction of 13-protofilament GTPgammaS microtubules decorated with Mal3 CH domain
Sample
  • Sample: 13-protofilament GTPgammaS microtubule decorated with monomeric Mal3
  • Protein or peptide: Alpha-beta tubulin dimer
  • Protein or peptide: Mal3
  • Ligand: guanosine 5'-O-(gamma-thio)triphosphate
Keywordscytoskeleton / GTPase / end binding / calponin homology
Biological speciesSus scrofa (pig) / Saccharomyces pombe / synthetic construct (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 15.0 Å
AuthorsMaurer SP / Fourniol FJ / Bohner G / Moores CA / Surrey T
CitationJournal: Cell / Year: 2012
Title: EBs recognize a nucleotide-dependent structural cap at growing microtubule ends.
Authors: Sebastian P Maurer / Franck J Fourniol / Gergő Bohner / Carolyn A Moores / Thomas Surrey /
Abstract: Growing microtubule ends serve as transient binding platforms for essential proteins that regulate microtubule dynamics and their interactions with cellular substructures. End-binding proteins (EBs) ...Growing microtubule ends serve as transient binding platforms for essential proteins that regulate microtubule dynamics and their interactions with cellular substructures. End-binding proteins (EBs) autonomously recognize an extended region at growing microtubule ends with unknown structural characteristics and then recruit other factors to the dynamic end structure. Using cryo-electron microscopy, subnanometer single-particle reconstruction, and fluorescence imaging, we present a pseudoatomic model of how the calponin homology (CH) domain of the fission yeast EB Mal3 binds to the end regions of growing microtubules. The Mal3 CH domain bridges protofilaments except at the microtubule seam. By binding close to the exchangeable GTP-binding site, the CH domain is ideally positioned to sense the microtubule's nucleotide state. The same microtubule-end region is also a stabilizing structural cap protecting the microtubule from depolymerization. This insight supports a common structural link between two important biological phenomena, microtubule dynamic instability and end tracking.
History
DepositionDec 2, 2011-
Header (metadata) releaseJan 6, 2012-
Map releaseMay 31, 2012-
UpdateOct 24, 2012-
Current statusOct 24, 2012Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.05
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.05
  • Imaged by UCSF Chimera
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Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

2004.jpg

Downloads & links

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Map

FileDownload / File: emd_2004.map.gz / Format: CCP4 / Size: 73.3 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationAsymmetric reconstruction of 13-protofilament GTPgammaS microtubules decorated with Mal3 CH domain
Voxel sizeX=Y=Z: 2.2 Å
Density
Contour LevelBy AUTHOR: 0.045 / Movie #1: 0.05
Minimum - Maximum-0.05021228 - 0.14973304
Average (Standard dev.)-0.00248467 (±0.02520477)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-135-135-135
Dimensions270270270
Spacing270270270
CellA=B=C: 594.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z2.22.22.2
M x/y/z270270270
origin x/y/z0.0000.0000.000
length x/y/z594.000594.000594.000
α/β/γ90.00090.00090.000
start NX/NY/NZ-56-56-55
NX/NY/NZ112112112
MAP C/R/S123
start NC/NR/NS-135-135-135
NC/NR/NS270270270
D min/max/mean-0.0500.150-0.002

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Supplemental data

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Sample components

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Entire : 13-protofilament GTPgammaS microtubule decorated with monomeric Mal3

EntireName: 13-protofilament GTPgammaS microtubule decorated with monomeric Mal3
Components
  • Sample: 13-protofilament GTPgammaS microtubule decorated with monomeric Mal3
  • Protein or peptide: Alpha-beta tubulin dimer
  • Protein or peptide: Mal3
  • Ligand: guanosine 5'-O-(gamma-thio)triphosphate

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Supramolecule #1000: 13-protofilament GTPgammaS microtubule decorated with monomeric Mal3

SupramoleculeName: 13-protofilament GTPgammaS microtubule decorated with monomeric Mal3
type: sample / ID: 1000
Details: tubulin was mixed with GMPCPP microtubule seeds, GTPgammaS and monomeric Mal3, and incubated 3-10min at 37degC
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: Alpha-beta tubulin dimer / Oligomeric state: Dimer / Recombinant expression: No
Source (natural)Organism: Sus scrofa (pig) / synonym: Pig / Tissue: Brain / Location in cell: cytoplasmic
Molecular weightExperimental: 100 KDa / Theoretical: 100 KDa

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Macromolecule #3: Mal3

MacromoleculeName: Mal3 / type: protein_or_peptide / ID: 3 / Name.synonym: Mal3 / Oligomeric state: monomer / Recombinant expression: Yes
Source (natural)Organism: Saccharomyces pombe / synonym: fission yeast / Location in cell: cytoplasmic
Molecular weightExperimental: 16 KDa / Theoretical: 16 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)

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Macromolecule #2: guanosine 5'-O-(gamma-thio)triphosphate

MacromoleculeName: guanosine 5'-O-(gamma-thio)triphosphate / type: ligand / ID: 2 / Name.synonym: GTPgammaS / Recombinant expression: No
Source (natural)Organism: synthetic construct (others)

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

BufferpH: 6.8 / Details: 40mM Pipes, 1mM MgCl2, 1mM EGTA
GridDetails: 300 mesh lacey carbon grid
VitrificationCryogen name: ETHANE / Chamber humidity: 90 % / 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: 3.6 µm / Nominal defocus min: 0.7 µm / Nominal magnification: 68000
Sample stageSpecimen holder: Eucentric / Specimen holder model: OTHER
TemperatureMin: 88 K / Max: 98 K / Average: 93 K
Alignment procedureLegacy - Astigmatism: Objective lens astigmatism was corrected at 150,000 times magnification
Image recordingCategory: CCD / Film or detector model: GATAN ULTRASCAN 4000 (4k x 4k) / Number real images: 162 / Average electron dose: 17 e/Å2 / Details: sampling size 2.2 A per pixel
Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company

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Image processing

CTF correctionDetails: FREALIGN
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 15.0 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: SPIDER, FREALIGN
Details: C1 map calculated from approximately 11000 one-dimer-long microtubule segments

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