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- EMDB-2920: End of a microtubule assembled in the presence of GTP-gamma-S -

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Basic information

Entry
Database: EMDB / ID: EMD-2920
TitleEnd of a microtubule assembled in the presence of GTP-gamma-S
Map dataEnd of a microtubule assembled in the presence of GTP-gamma-S
Sample
  • Sample: Outwardly curved tubulin sheet at the end of a microtubule assembled in the presence of GMTP-gamma-S.
  • Protein or peptide: Tubulin alpha chain
  • Protein or peptide: Tubulin beta chain
KeywordsEnd-binding one protein / tubulin / microtubule / functionalized gold nanoparticles / GTP-cap / GTP-hydrolysis
Function / homology
Function and homology information


microtubule-based process / Hydrolases; Acting on acid anhydrides; Acting on GTP to facilitate cellular and subcellular movement / structural constituent of cytoskeleton / microtubule cytoskeleton organization / mitotic cell cycle / microtubule / hydrolase activity / GTPase activity / GTP binding / metal ion binding / cytoplasm
Similarity search - Function
Tubulin-beta mRNA autoregulation signal. / Alpha tubulin / Beta tubulin, autoregulation binding site / Beta tubulin / Tubulin / Tubulin / Tubulin, C-terminal / Tubulin C-terminal domain / Tubulin, conserved site / Tubulin, conserved site ...Tubulin-beta mRNA autoregulation signal. / Alpha tubulin / Beta tubulin, autoregulation binding site / Beta tubulin / Tubulin / Tubulin / Tubulin, C-terminal / Tubulin C-terminal domain / Tubulin, conserved site / 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)
Methodelectron tomography / cryo EM / negative staining
AuthorsGuesdon A / Bazile F / Buey RM / Mohan R / Monier S / Angevin M / Heichette C / Tampe R / Duchesne L / Akhmanova A ...Guesdon A / Bazile F / Buey RM / Mohan R / Monier S / Angevin M / Heichette C / Tampe R / Duchesne L / Akhmanova A / Steinmetz MO / Chretien D
CitationJournal: Nat Cell Biol / Year: 2016
Title: EB1 interacts with outwardly curved and straight regions of the microtubule lattice.
Authors: Audrey Guesdon / Franck Bazile / Rubén M Buey / Renu Mohan / Solange Monier / Ruddi Rodríguez García / Morgane Angevin / Claire Heichette / Ralph Wieneke / Robert Tampé / Laurence ...Authors: Audrey Guesdon / Franck Bazile / Rubén M Buey / Renu Mohan / Solange Monier / Ruddi Rodríguez García / Morgane Angevin / Claire Heichette / Ralph Wieneke / Robert Tampé / Laurence Duchesne / Anna Akhmanova / Michel O Steinmetz / Denis Chrétien /
Abstract: EB1 is a microtubule plus-end tracking protein that recognizes GTP-tubulin dimers in microtubules and thus represents a unique probe to investigate the architecture of the GTP cap of growing ...EB1 is a microtubule plus-end tracking protein that recognizes GTP-tubulin dimers in microtubules and thus represents a unique probe to investigate the architecture of the GTP cap of growing microtubule ends. Here, we conjugated EB1 to gold nanoparticles (EB1-gold) and imaged by cryo-electron tomography its interaction with dynamic microtubules assembled in vitro from purified tubulin. EB1-gold forms comets at the ends of microtubules assembled in the presence of GTP, and interacts with the outer surface of curved and straight tubulin sheets as well as closed regions of the microtubule lattice. Microtubules assembled in the presence of GTP, different GTP analogues or cell extracts display similarly curved sheets at their growing ends, which gradually straighten as their protofilament number increases until they close into a tube. Together, our data provide unique structural information on the interaction of EB1 with growing microtubule ends. They further offer insights into the conformational changes that tubulin dimers undergo during microtubule assembly and the architecture of the GTP-cap region.
History
DepositionFeb 24, 2015-
Header (metadata) releaseMar 11, 2015-
Map releaseSep 21, 2016-
UpdateOct 12, 2016-
Current statusOct 12, 2016Processing site: PDBe / Status: Released

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Structure visualization

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Structure viewerEM map:
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Supplemental images

emd_2920.tif

Downloads & links

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Map

FileDownload / File: emd_2920.map.gz / Format: CCP4 / Size: 33.8 MB / Type: IMAGE STORED AS SIGNED INTEGER (2 BYTES)
AnnotationEnd of a microtubule assembled in the presence of GTP-gamma-S
Voxel sizeX=Y=Z: 7.9 Å
Density
Minimum - Maximum-13960.0 - 6926.0
Average (Standard dev.)292.820190430000025 (±664.054626460000009)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin1220190225
Dimensions784185125
Spacing784185125
CellA: 1461.5 Å / B: 6193.6 Å / C: 987.5 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Integer*27
Å/pix. X/Y/Z7.97.97.9
M x/y/z185784125
origin x/y/z0.0000.0000.000
length x/y/z1461.5006193.600987.500
α/β/γ90.00090.00090.000
start NX/NY/NZ-147-147-146
NX/NY/NZ294294294
MAP C/R/S123
start NC/NR/NS1902122025
NC/NR/NS185784125
D min/max/mean-13960.0006926.000292.820

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Supplemental data

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Sample components

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Entire : Outwardly curved tubulin sheet at the end of a microtubule assemb...

EntireName: Outwardly curved tubulin sheet at the end of a microtubule assembled in the presence of GMTP-gamma-S.
Components
  • Sample: Outwardly curved tubulin sheet at the end of a microtubule assembled in the presence of GMTP-gamma-S.
  • Protein or peptide: Tubulin alpha chain
  • Protein or peptide: Tubulin beta chain

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Supramolecule #1000: Outwardly curved tubulin sheet at the end of a microtubule assemb...

SupramoleculeName: Outwardly curved tubulin sheet at the end of a microtubule assembled in the presence of GMTP-gamma-S.
type: sample / ID: 1000
Details: The sample was mono disperse. Centrosomes isolated from KE37 cells were added to stimulate microtubule nucleation.
Oligomeric state: Microtubule: polymer of tubulin. / Number unique components: 2

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Macromolecule #1: Tubulin alpha chain

MacromoleculeName: Tubulin alpha chain / type: protein_or_peptide / ID: 1
Details: Heterodimer alpha-beta polymerized into microtubules
Recombinant expression: No / Database: NCBI
Source (natural)Organism: Sus scrofa (pig) / synonym: Pig / Tissue: Brain / Cell: Neurons / Location in cell: Cytoplasm
Molecular weightTheoretical: 500.68 KDa
SequenceUniProtKB: Tubulin alpha-1A chain / GO: microtubule-based process / InterPro: Tubulin

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Macromolecule #2: Tubulin beta chain

MacromoleculeName: Tubulin beta chain / type: protein_or_peptide / ID: 2
Details: Heterodimer alpha-beta polymerized into microtubules
Recombinant expression: No / Database: NCBI
Source (natural)Organism: Sus scrofa (pig) / synonym: Pig / Tissue: Brain / Cell: Neurons / Location in cell: Cytoplasm
Molecular weightTheoretical: 498.61 KDa
SequenceUniProtKB: Tubulin beta chain / GO: microtubule-based process / InterPro: Tubulin, conserved site

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Experimental details

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Structure determination

Methodnegative staining, cryo EM
Processingelectron tomography
Aggregation stateparticle

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Sample preparation

Concentration7.5 mg/mL
BufferpH: 6.8
Details: 80 mM Pipes, 1 mM MgCl2, 50 mM KCl, 1mM EGTA, 1 mM GTP-gamma-S
StainingType: NEGATIVE / Details: Vitrified specimen.
GridDetails: 300 mesh grid coated with home-made holey-carbon film.
VitrificationCryogen name: ETHANE / Chamber humidity: 90 % / Chamber temperature: 93 K / Instrument: HOMEMADE PLUNGER
Details: Specimen maintained at 35 degrees celcius in saturated humidity conditions before vitrification.
Timed resolved state: Specimen frozen ~30 min after the beginning of assembly.
Method: Assembly in a test tube at 35 degrees Celsius in the presence of purified KE37 centrosomes, deposit of a 4 microliter sample onto the grid, blotting for ~2 seconds and rapid plunging into liquid ethane.

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Electron microscopy

MicroscopeFEI TECNAI 20
Electron beamAcceleration voltage: 200 kV / Electron source: LAB6
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.0 mm / Nominal defocus max: 1.5 µm / Nominal defocus min: 1.5 µm / Nominal magnification: 29000
Sample stageSpecimen holder: Liquid nitrogen cooled / Specimen holder model: GATAN LIQUID NITROGEN / Tilt series - Axis1 - Min angle: -60 ° / Tilt series - Axis1 - Max angle: 60 ° / Tilt series - Axis1 - Angle increment: 2 °
TemperatureAverage: 174 K
Alignment procedureLegacy - Astigmatism: Astigmatism corrected at high magnification
DetailsTilt serie started from zero. Saxton acquisition scheme with 2 degrees increments. 75 images acquired in post-tracking mode. Camera used in binning mode 2, 0.79 nm pixel size.
DateMay 16, 2014
Image recordingCategory: CCD / Film or detector model: GATAN ULTRASCAN 4000 (4k x 4k) / Number real images: 75 / Average electron dose: 0.3 e/Å2
Details: Camera used in binning mode 2, at a final pixel size of 0.79 nm.
Bits/pixel: 16

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Image processing

Final reconstructionAlgorithm: OTHER / Software - Name: eTomo, IMOD
Details: Backprojection using a radial filter cutoff of 0.15 and a faloff of 0.05.
Number images used: 75
Details3D reconstruction performed using eTomo from IMOD software. Reconstruction by back projection.

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