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- EMDB-2921: End of a microtubule assembled in HeLa cytosol -

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Entry
Database: EMDB / ID: EMD-2921
TitleEnd of a microtubule assembled in HeLa cytosol
Map dataEnd of a microtubule assembled in HeLa cytosol. Centrosomes purified from KE37 cells were added to stimulate microtubule nucleation.
Sample
  • Sample: Outwardly curved tubulin sheet at the end of a microtubule assembled in HeLa cytosol.
  • Organelle or cellular component: Cytosol
KeywordsEnd-binding one protein / tubulin / microtubule / functionalized gold nanoparticles / GTP-cap / GTP-hydrolysis
Biological speciesHomo sapiens (human)
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

Movie
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  • Imaged by UCSF Chimera
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Structure viewerEM map:
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Supplemental images

emd_2921.tif

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Map

FileDownload / File: emd_2921.map.gz / Format: CCP4 / Size: 52.8 MB / Type: IMAGE STORED AS SIGNED INTEGER (2 BYTES)
AnnotationEnd of a microtubule assembled in HeLa cytosol. Centrosomes purified from KE37 cells were added to stimulate microtubule nucleation.
Voxel sizeX=Y=Z: 7.9 Å
Density
Minimum - Maximum-13985.0 - 7627.0
Average (Standard dev.)310.162689209999996 (±586.637634280000043)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-512-1966147
Dimensions908214146
Spacing908214146
CellA: 1690.6 Å / B: 7173.2 Å / C: 1153.4 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/z214908146
origin x/y/z0.0000.0000.000
length x/y/z1690.6007173.2001153.400
α/β/γ90.00090.00090.000
start NX/NY/NZ-147-147-146
NX/NY/NZ294294294
MAP C/R/S123
start NC/NR/NS-1966-512147
NC/NR/NS214908146
D min/max/mean-13985.0007627.000310.163

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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 HeLa cytosol.
Components
  • Sample: Outwardly curved tubulin sheet at the end of a microtubule assembled in HeLa cytosol.
  • Organelle or cellular component: Cytosol

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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 HeLa cytosol.
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: 1

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Supramolecule #1: Cytosol

SupramoleculeName: Cytosol / type: organelle_or_cellular_component / ID: 1 / Details: High-speed supernatant from HeLa cells. / Recombinant expression: No / Database: NCBI
Source (natural)Organism: Homo sapiens (human) / synonym: Human / Cell: HeLa / Location in cell: Cytoplasm

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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

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 ~2 min after the beginning of assembly.
Method: Assembly on the grid in the presence of purified KE37 centrosomes, 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: -58 ° / Tilt series - Axis1 - Max angle: 60 ° / Tilt series - Axis1 - Angle increment: 2 °
TemperatureAverage: 174 K
Alignment procedureLegacy - Astigmatism: Astigmatism corrected at high magnification
DetailsTilt series started from zero. Saxton acquisition scheme with 2 degrees increments. 73 images acquired in post-tracking mode. Tilt series angular range -59.3 to 57.3 after alignement. Camera used in binning mode 2, 0.79 nm pixel size.
DateSep 4, 2014
Image recordingCategory: CCD / Film or detector model: GATAN ULTRASCAN 4000 (4k x 4k) / Number real images: 73 / 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: 73
Details3D reconstruction performed using eTomo from IMOD software. Reconstruction by back projection.

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