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- EMDB-5896: Cryo EM density of GDP-bound dynamic microtubules -

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Basic information

Entry
Database: EMDB / ID: EMD-5896
TitleCryo EM density of GDP-bound dynamic microtubules
Map datacryo-EM reconstruction of dynamic GDP-bound microtubule
Sample
  • Sample: Dynamic GDP-bound microtubule
  • Protein or peptide: Alpha tubulin
  • Protein or peptide: Beta tubulin
  • Protein or peptide: kinesin
Keywordsmicrotubule / gdp / dynamic
Function / homology
Function and homology information


motile cilium / structural constituent of cytoskeleton / microtubule cytoskeleton organization / neuron migration / mitotic cell cycle / Hydrolases; Acting on acid anhydrides; Acting on GTP to facilitate cellular and subcellular movement / microtubule / hydrolase activity / GTPase activity / GTP binding ...motile cilium / structural constituent of cytoskeleton / microtubule cytoskeleton organization / neuron migration / mitotic cell cycle / Hydrolases; Acting on acid anhydrides; Acting on GTP to facilitate cellular and subcellular movement / microtubule / hydrolase activity / GTPase activity / GTP binding / metal ion binding / cytoplasm
Similarity search - Function
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 ...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
Similarity search - Domain/homology
Tubulin alpha-1A chain / Tubulin beta chain
Similarity search - Component
Biological speciesSus scrofa (pig) / Homo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.9 Å
AuthorsAlushin GM / Lander GC / Kellogg EH / Zhang R / Baker D / Nogales E
CitationJournal: Cell / Year: 2014
Title: High-resolution microtubule structures reveal the structural transitions in αβ-tubulin upon GTP hydrolysis.
Authors: Gregory M Alushin / Gabriel C Lander / Elizabeth H Kellogg / Rui Zhang / David Baker / Eva Nogales /
Abstract: Dynamic instability, the stochastic switching between growth and shrinkage, is essential for microtubule function. This behavior is driven by GTP hydrolysis in the microtubule lattice and is ...Dynamic instability, the stochastic switching between growth and shrinkage, is essential for microtubule function. This behavior is driven by GTP hydrolysis in the microtubule lattice and is inhibited by anticancer agents like Taxol. We provide insight into the mechanism of dynamic instability, based on high-resolution cryo-EM structures (4.7-5.6 Å) of dynamic microtubules and microtubules stabilized by GMPCPP or Taxol. We infer that hydrolysis leads to a compaction around the E-site nucleotide at longitudinal interfaces, as well as movement of the α-tubulin intermediate domain and H7 helix. Displacement of the C-terminal helices in both α- and β-tubulin subunits suggests an effect on interactions with binding partners that contact this region. Taxol inhibits most of these conformational changes, allosterically inducing a GMPCPP-like state. Lateral interactions are similar in all conditions we examined, suggesting that microtubule lattice stability is primarily modulated at longitudinal interfaces.
History
DepositionFeb 9, 2014-
Header (metadata) releaseMar 5, 2014-
Map releaseJun 4, 2014-
UpdateJun 4, 2014-
Current statusJun 4, 2014Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 3
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 3
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-3j6f
  • Surface level: 3
  • Imaged by UCSF Chimera
  • Download
  • Surface view with fitted model
  • Atomic models: PDB-3j6f
  • Surface level: 3
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

400_5896.gif

Downloads & links

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Map

FileDownload / File: emd_5896.map.gz / Format: CCP4 / Size: 4.4 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationcryo-EM reconstruction of dynamic GDP-bound microtubule
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.74 Å/pix.
x 162 pix.
= 281.88 Å		1.74 Å/pix.
x 66 pix.
= 114.84 Å		1.74 Å/pix.
x 110 pix.
= 191.4 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: 1.74 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy EMDB: 4.0 / Movie #1: 3
Minimum - Maximum-4.67229128 - 11.280469890000001
Average (Standard dev.)0.52844977 (±1.9256407)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-33-55-81
Dimensions66110162
Spacing66110162
CellA: 191.4 Å / B: 114.840004 Å / C: 281.88 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.741.741.74
M x/y/z11066162
origin x/y/z0.0000.0000.000
length x/y/z191.400114.840281.880
α/β/γ90.00090.00090.000
start NX/NY/NZ-95-75153
NX/NY/NZ200200200
MAP C/R/S123
start NC/NR/NS-55-33-81
NC/NR/NS11066162
D min/max/mean-4.67211.2800.528

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Supplemental data

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Sample components

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Entire : Dynamic GDP-bound microtubule

EntireName: Dynamic GDP-bound microtubule
Components
  • Sample: Dynamic GDP-bound microtubule
  • Protein or peptide: Alpha tubulin
  • Protein or peptide: Beta tubulin
  • Protein or peptide: kinesin

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Supramolecule #1000: Dynamic GDP-bound microtubule

SupramoleculeName: Dynamic GDP-bound microtubule / type: sample / ID: 1000 / Number unique components: 3

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Macromolecule #1: Alpha tubulin

MacromoleculeName: Alpha tubulin / type: protein_or_peptide / ID: 1 / Oligomeric state: heterodimer / Recombinant expression: No / Database: NCBI
Source (natural)Organism: Sus scrofa (pig) / synonym: porcine / Tissue: brain / Organelle: Cytoplasm / Location in cell: Cytoskeleton
Molecular weightTheoretical: 55 KDa

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Macromolecule #2: Beta tubulin

MacromoleculeName: Beta tubulin / type: protein_or_peptide / ID: 2 / Oligomeric state: heterodimer / Recombinant expression: No / Database: NCBI
Source (natural)Organism: Sus scrofa (pig) / synonym: porcine / Tissue: brain / Organelle: Cytoplasm / Location in cell: Cytoskeleton
Molecular weightTheoretical: 55 KDa

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Macromolecule #3: kinesin

MacromoleculeName: kinesin / type: protein_or_peptide / ID: 3
Details: Human monomeric kinesin K349 cys-lite described in: Rice, S., Lin, A.W., Safer, D., Hart, C.L., Naber, N., Carragher, B.O., Cain, S.M., Pechatnikova, E., Wilson-Kubalek, E.M., Whittaker, M., ...Details: Human monomeric kinesin K349 cys-lite described in: Rice, S., Lin, A.W., Safer, D., Hart, C.L., Naber, N., Carragher, B.O., Cain, S.M., Pechatnikova, E., Wilson-Kubalek, E.M., Whittaker, M., et al. (1999). A structural change in the kinesin motor protein that drives motility. Nature 402, 778-784.
Oligomeric state: monomer / Recombinant expression: Yes
Source (natural)Organism: Homo sapiens (human) / synonym: human / Organelle: Cytoplasm / Location in cell: Cytoskeleton
Molecular weightTheoretical: 36 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant strain: BL21

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation statehelical array

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Sample preparation

Concentration0.25 mg/mL
BufferpH: 6.8
Details: 80mM PIPES, 1mM EGTA, 1mM MgCl2, 1mM DTT, 0.05% Nonidet P-40
GridDetails: 400 mesh C-flat 1.2/1.3, glow discharged in Edwards carbon evaporator
VitrificationCryogen name: ETHANE / Chamber humidity: 90 % / Chamber temperature: 90.4 K / Instrument: FEI VITROBOT MARK II / Method: The grid was blotted for 2 seconds before plunging.

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Electron microscopy

MicroscopeFEI TITAN
Alignment procedureLegacy - Astigmatism: Objective lens astigmatism was corrected at 100,000 times magnifacation.
DateJun 15, 2012
Image recordingCategory: FILM / Film or detector model: KODAK SO-163 FILM / Digitization - Scanner: OTHER / Digitization - Sampling interval: 6.35 µm / Number real images: 219 / Average electron dose: 25.0 e/Å2 / Bits/pixel: 16
Tilt angle min0
Tilt angle max0
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 72000 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 3.5 µm / Nominal defocus min: 1.4 µm / Nominal magnification: 72000
Sample stageSpecimen holder: Gatan 626 holder / Specimen holder model: GATAN LIQUID NITROGEN

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Image processing

DetailsInitial alignments performed with EMAN2/SPARX, including refinement of helical parameters with the IHRSR programs of Egelman. Final alignment and reconstruction performed with FREALIGN.
CTF correctionDetails: ctftilt
Final reconstructionApplied symmetry - Helical parameters - Δz: 8.82 Å
Applied symmetry - Helical parameters - Δ&Phi: 25.76 °
Applied symmetry - Helical parameters - Axial symmetry: C1 (asymmetric)
Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 4.9 Å / Resolution method: OTHER / Software - Name: FREALIGN / Number images used: 54445

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Atomic model buiding 1

Initial modelPDB ID:

1jff

SoftwareName: ROSETTA
DetailsStructure represents the minimized average structure of the 1% lowest energy structures from the refinement run.
RefinementSpace: REAL / Protocol: FLEXIBLE FIT
Output model

PDB-3j6f:
Minimized average structure of GDP-bound dynamic microtubules

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