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- EMDB-2697: Cryo Electron Microscopy of GMPCPP-microtubules structure -

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Basic information

Entry
Database: EMDB / ID: EMD-2697
TitleCryo Electron Microscopy of GMPCPP-microtubules structure
Map data3D structure of GMPCPP-microtubules
Sample
  • Sample: GMPCPP-bound (GTP-state) microtubule
  • Protein or peptide: tubulin alpha chain
  • Protein or peptide: tubulin beta chain
KeywordsMicrotubule / tubuiln / GMPCPP / GTP-state structure / cryo-EM / microtubule stabilization / microtubule polymerization
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)
Methodsingle particle reconstruction / cryo EM / Resolution: 8.9 Å
AuthorsYajima H / Ogura T / Nitta R / Okada Y / Sato C / Hirokawa N
CitationJournal: J Cell Biol / Year: 2012
Title: Conformational changes in tubulin in GMPCPP and GDP-taxol microtubules observed by cryoelectron microscopy.
Authors: Hiroaki Yajima / Toshihiko Ogura / Ryo Nitta / Yasushi Okada / Chikara Sato / Nobutaka Hirokawa /
Abstract: Microtubules are dynamic polymers that stochastically switch between growing and shrinking phases. Microtubule dynamics are regulated by guanosine triphosphate (GTP) hydrolysis by β-tubulin, but the ...Microtubules are dynamic polymers that stochastically switch between growing and shrinking phases. Microtubule dynamics are regulated by guanosine triphosphate (GTP) hydrolysis by β-tubulin, but the mechanism of this regulation remains elusive because high-resolution microtubule structures have only been revealed for the guanosine diphosphate (GDP) state. In this paper, we solved the cryoelectron microscopy (cryo-EM) structure of microtubule stabilized with a GTP analogue, guanylyl 5'-α,β-methylenediphosphonate (GMPCPP), at 8.8-Å resolution by developing a novel cryo-EM image reconstruction algorithm. In contrast to the crystal structures of GTP-bound tubulin relatives such as γ-tubulin and bacterial tubulins, significant changes were detected between GMPCPP and GDP-taxol microtubules at the contacts between tubulins both along the protofilament and between neighboring protofilaments, contributing to the stability of the microtubule. These findings are consistent with the structural plasticity or lattice model and suggest the structural basis not only for the regulatory mechanism of microtubule dynamics but also for the recognition of the nucleotide state of the microtubule by several microtubule-binding proteins, such as EB1 or kinesin.
History
DepositionJun 28, 2014-
Header (metadata) releaseAug 6, 2014-
Map releaseDec 10, 2014-
UpdateDec 10, 2014-
Current statusDec 10, 2014Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 156
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 156
  • Imaged by UCSF Chimera
  • Download
  • Surface view with fitted model
  • Atomic models: PDB-3j7i
  • Surface level: 156
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

EMD-2697_ima...

Downloads & links

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Map

FileDownload / File: emd_2697.map.gz / Format: CCP4 / Size: 631.8 KB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotation3D structure of GMPCPP-microtubules
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
2.5 Å/pix.
x 43 pix.
= 107.5 Å		2.5 Å/pix.
x 63 pix.
= 157.5 Å		2.5 Å/pix.
x 61 pix.
= 152.5 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: 2.5 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 156.0 / Movie #1: 156
Minimum - Maximum-8.307334900000001 - 287.504180910000002
Average (Standard dev.)26.396753310000001 (±58.260581969999997)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin10510063
Dimensions636143
Spacing636143
CellA: 152.5 Å / B: 157.5 Å / C: 107.5 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z2.52.52.5
M x/y/z616343
origin x/y/z0.0000.0000.000
length x/y/z152.500157.500107.500
α/β/γ90.00090.00090.000
start NX/NY/NZ-180-180-179
NX/NY/NZ360360360
MAP C/R/S123
start NC/NR/NS10010563
NC/NR/NS616343
D min/max/mean-8.307287.50426.397

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Supplemental data

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Sample components

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Entire : GMPCPP-bound (GTP-state) microtubule

EntireName: GMPCPP-bound (GTP-state) microtubule
Components
  • Sample: GMPCPP-bound (GTP-state) microtubule
  • Protein or peptide: tubulin alpha chain
  • Protein or peptide: tubulin beta chain

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Supramolecule #1000: GMPCPP-bound (GTP-state) microtubule

SupramoleculeName: GMPCPP-bound (GTP-state) microtubule / type: sample / ID: 1000 / Oligomeric state: Alpha- and beta-tubulin heterodimer / Number unique components: 2
Molecular weightExperimental: 110 KDa / Theoretical: 110 KDa

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Macromolecule #1: tubulin alpha chain

MacromoleculeName: tubulin alpha chain / type: protein_or_peptide / ID: 1 / Name.synonym: alpha tubulin
Details: GTP binds on intra-tubulin dimer (between alpha- and beta-tubulin).
Number of copies: 1 / Oligomeric state: hetero dimer / Recombinant expression: No
Source (natural)Organism: Sus scrofa (pig) / synonym: Domestic Pig / Tissue: Brain
Molecular weightExperimental: 55 KDa / Theoretical: 55 KDa
SequenceUniProtKB: Tubulin alpha-1A chain

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Macromolecule #2: tubulin beta chain

MacromoleculeName: tubulin beta chain / type: protein_or_peptide / ID: 2 / Name.synonym: beta tubulin
Details: almost every beta-tubulin is bound to GMPCPP (>90% occupancy). without taxol.
Number of copies: 1 / Oligomeric state: hetero dimer / Recombinant expression: No
Source (natural)Organism: Sus scrofa (pig) / synonym: Domestic Pig / Tissue: Brain
Molecular weightExperimental: 55 KDa / Theoretical: 55 KDa
SequenceUniProtKB: Tubulin beta chain

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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

Concentration0.25 mg/mL
BufferpH: 6.8
Details: 100 mM Pipes, pH 6.8, adjusted by KOH, 1 mM EGTA, 1 mM MgCl2, 0.6 mM GMPCPP, 5% DMSO
GridDetails: 300 mesh copper grid with thin holey carbon support, glow discharged in amylamine atmosphere
VitrificationCryogen name: ETHANE / Chamber temperature: 88 K / Instrument: LEICA KF80
Method: A 5-uL drop of the polymerized microtubules was placed onto a glow-discharged holey carbon film on a copper mesh grid. After 30 seconds, this solution was absorbed by filter paper and quickly ...Method: A 5-uL drop of the polymerized microtubules was placed onto a glow-discharged holey carbon film on a copper mesh grid. After 30 seconds, this solution was absorbed by filter paper and quickly replaced with an 8-uL drop of the same buffer without microtubules. Immediately after blotting this drop with filter papers from both grid sides for 5 seconds, the grid was plunge frozen in liquid ethane.

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Electron microscopy

MicroscopeJEOL 2010F
TemperatureMin: 90 K / Max: 95 K / Average: 93 K
Alignment procedureLegacy - Astigmatism: Objective lens astigmatism was corrected at 100,000 times magnification
DateJun 12, 2010
Image recordingCategory: FILM / Film or detector model: KODAK SO-163 FILM / Digitization - Scanner: OTHER / Digitization - Sampling interval: 10 µm / Number real images: 350 / Average electron dose: 10.00 e/Å2 / Bits/pixel: 16
Tilt angle min0
Tilt angle max0
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 40000 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 3.3 mm / Nominal defocus max: 2.8 µm / Nominal defocus min: 1.3 µm / Nominal magnification: 40000
Sample stageSpecimen holder: Gatan 626 Single Tilt Liquid Nitrogen Cryo Transfer Holder
Specimen holder model: GATAN LIQUID NITROGEN

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Image processing

DetailsThe particles were selected along individual microtubules using an automatic selection program.
CTF correctionDetails: Each filament
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 8.9 Å / Resolution method: OTHER / Software - Name: MATLAB, ImageV / Number images used: 320000
Final two d classificationNumber classes: 18

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Atomic model buiding 1

Initial modelPDB ID:

1jff


Chain - #0 - Chain ID: A / Chain - #1 - Chain ID: B
SoftwareName: Chimera
DetailsInitial local fitting was done before the domains were separately fitted by using Chimera.
RefinementSpace: REAL / Protocol: RIGID BODY FIT / Target criteria: Cross-correlation, Average map value
Output model

PDB-3j7i:
Structure of alpha- and beta- tubulin in GMPCPP-microtubules

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