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Open data
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Basic information
Entry | Database: EMDB / ID: EMD-2919 | |||||||||
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Title | End of a microtubule assembled in the presence of GMPCPP | |||||||||
![]() | End of a microtubule assembled in the presence of the slowly-hydrolyzable analogue of GTP, GMPCPP. | |||||||||
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![]() | End-binding one protein / tubulin / microtubule / functionalized gold nanoparticles / GTP-cap / GTP-hydrolysis | |||||||||
Function / homology | ![]() 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 | |||||||||
Biological species | ![]() ![]() | |||||||||
Method | electron tomography / cryo EM / negative staining | |||||||||
![]() | Guesdon 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 | |||||||||
![]() | ![]() 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 |
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Structure visualization
Movie |
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Structure viewer | EM map: ![]() ![]() ![]() |
Supplemental images |
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Downloads & links
-EMDB archive
Map data | ![]() | 53.2 MB | ![]() | |
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Header (meta data) | ![]() ![]() | 12.8 KB 12.8 KB | Display Display | ![]() |
Images | ![]() | 136 KB | ||
Archive directory | ![]() ![]() | HTTPS FTP |
-Related structure data
Related structure data | ![]() 2912C ![]() 2915C ![]() 2916C ![]() 2918C ![]() 2920C ![]() 2921C C: citing same article ( |
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Similar structure data | Similarity search - Function & homology ![]() |
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Links
EMDB pages | ![]() ![]() |
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Related items in Molecule of the Month |
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Map
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Annotation | End of a microtubule assembled in the presence of the slowly-hydrolyzable analogue of GTP, GMPCPP. | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 7.9 Å | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Details | EMDB XML:
CCP4 map header:
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-Supplemental data
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Sample components
-Entire : Outwardly curved tubulin sheet at the end of a microtubule assemb...
Entire | Name: Outwardly curved tubulin sheet at the end of a microtubule assembled in the presence of GMPCPP. |
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Components |
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-Supramolecule #1000: Outwardly curved tubulin sheet at the end of a microtubule assemb...
Supramolecule | Name: Outwardly curved tubulin sheet at the end of a microtubule assembled in the presence of GMPCPP. type: sample / ID: 1000 / Details: The sample was monodisperse / Oligomeric state: Microtubule: polymer of tubulin. / Number unique components: 2 |
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-Macromolecule #1: Tubulin alpha chain
Macromolecule | Name: Tubulin alpha chain / type: protein_or_peptide / ID: 1 Details: Heterodimer alpha-beta polymerized into microtubules Recombinant expression: No / Database: NCBI |
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Source (natural) | Organism: ![]() ![]() |
Molecular weight | Theoretical: 500.68 KDa |
Sequence | UniProtKB: Tubulin alpha-1A chain / GO: microtubule-based process / InterPro: Tubulin |
-Macromolecule #2: Tubulin beta chain
Macromolecule | Name: Tubulin beta chain / type: protein_or_peptide / ID: 2 Details: Heterodimer alpha-beta polymerized into microtubules Recombinant expression: No / Database: NCBI |
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Source (natural) | Organism: ![]() ![]() |
Molecular weight | Theoretical: 498.61 KDa |
Sequence | UniProtKB: Tubulin beta chain / GO: microtubule-based process / InterPro: Tubulin, conserved site |
-Experimental details
-Structure determination
Method | negative staining, cryo EM |
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![]() | electron tomography |
Aggregation state | particle |
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Sample preparation
Concentration | 1 mg/mL |
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Buffer | pH: 6.8 Details: 80 mM Pipes, 1 mM MgCl2, 50 mM KCl, 1mM EGTA, 0.1 mM GMPCPP |
Staining | Type: NEGATIVE / Details: Vitrified specimen. |
Grid | Details: 300 mesh grid coated with home-made holey-carbon film. |
Vitrification | Cryogen 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 ~6 min after the beginning of assembly. Method: Assembly in a test tube at 35 degrees Celsius, deposit of a 4 microliter onto the grid, blotting for ~2 seconds and rapid plunging into liquid ethane. |
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Electron microscopy
Microscope | FEI TECNAI 20 |
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Temperature | Average: 174 K |
Alignment procedure | Legacy - Astigmatism: Astigmatism corrected at high magnification |
Details | Tilt serie started from zero. Saxton acquisition scheme with 2.1 degrees increments. 78 images acquired in post-tracking mode. Tilt series between -58.7 and 66 degrees after correction.Camera used in binning mode 2, 0.79 nm pixel size. |
Date | May 15, 2014 |
Image recording | Category: CCD / Film or detector model: GATAN ULTRASCAN 4000 (4k x 4k) / Number real images: 78 / 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 |
Electron beam | Acceleration voltage: 200 kV / Electron source: LAB6 |
Electron optics | Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.0 mm / Nominal defocus max: 1.5 µm / Nominal defocus min: 1.5 µm / Nominal magnification: 29000 |
Sample stage | Specimen holder: Liquid nitrogen cooled / Specimen holder model: GATAN LIQUID NITROGEN / Tilt series - Axis1 - Min angle: -65 ° / Tilt series - Axis1 - Max angle: 60 ° / Tilt series - Axis1 - Angle increment: 2.1 ° |
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Image processing
Details | 3D reconstruction performed using eTomo from IMOD software. Reconstruction by backprojection, using a Radial filter cutoffof 0.1 and a faloff of 0.1. |
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Final reconstruction | Algorithm: OTHER / Software - Name: eTomo, IMOD Details: Backprojection using a radial filter cutoff of 0.1 and a faloff of 0.1. Number images used: 78 |