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- EMDB-2912: Cryo-electron tomograms of EB1 conjugated to gold nanoparticles, ... -

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Basic information

Entry
Database: EMDB / ID: EMD-2912
TitleCryo-electron tomograms of EB1 conjugated to gold nanoparticles, in interaction with growing microtubule ends
Map dataCryo-electron tomogram of EB1 conjugated to 6.5 nm gold nanoparticles, in interaction with a microtubule growing end
Sample
  • Sample: EB1 conjugated to 6.5 nm gold nanoparticles, in interaction with a microtubule growing end
  • Protein or peptide: End-binding protein one
  • 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


protein localization to astral microtubule / cortical microtubule cytoskeleton / mitotic spindle astral microtubule end / protein localization to microtubule / microtubule plus-end / cell projection membrane / attachment of mitotic spindle microtubules to kinetochore / microtubule plus-end binding / non-motile cilium assembly / microtubule bundle formation ...protein localization to astral microtubule / cortical microtubule cytoskeleton / mitotic spindle astral microtubule end / protein localization to microtubule / microtubule plus-end / cell projection membrane / attachment of mitotic spindle microtubules to kinetochore / microtubule plus-end binding / non-motile cilium assembly / microtubule bundle formation / protein localization to centrosome / microtubule organizing center / negative regulation of microtubule polymerization / mitotic spindle pole / microtubule polymerization / establishment of mitotic spindle orientation / regulation of microtubule polymerization or depolymerization / spindle midzone / Amplification of signal from unattached kinetochores via a MAD2 inhibitory signal / cytoplasmic microtubule / microtubule-based process / Mitotic Prometaphase / spindle assembly / EML4 and NUDC in mitotic spindle formation / Loss of Nlp from mitotic centrosomes / Loss of proteins required for interphase microtubule organization from the centrosome / Recruitment of mitotic centrosome proteins and complexes / Resolution of Sister Chromatid Cohesion / Recruitment of NuMA to mitotic centrosomes / Anchoring of the basal body to the plasma membrane / positive regulation of microtubule polymerization / AURKA Activation by TPX2 / ciliary basal body / RHO GTPases Activate Formins / Hydrolases; Acting on acid anhydrides; Acting on GTP to facilitate cellular and subcellular movement / protein localization / structural constituent of cytoskeleton / microtubule cytoskeleton organization / Separation of Sister Chromatids / The role of GTSE1 in G2/M progression after G2 checkpoint / Regulation of PLK1 Activity at G2/M Transition / cell migration / mitotic cell cycle / microtubule / molecular adaptor activity / hydrolase activity / cadherin binding / cell division / focal adhesion / GTPase activity / centrosome / GTP binding / protein kinase binding / Golgi apparatus / RNA binding / metal ion binding / identical protein binding / cytosol / cytoplasm
Similarity search - Function
EB1, C-terminal / Microtubule-associated protein RP/EB / EB1, C-terminal domain superfamily / EB1-C terminal (EB1-C) domain profile. / EB1-like C-terminal motif / Calponin homology (CH) domain / Calponin homology domain / CH domain superfamily / Calponin homology (CH) domain profile. / Tubulin-beta mRNA autoregulation signal. ...EB1, C-terminal / Microtubule-associated protein RP/EB / EB1, C-terminal domain superfamily / EB1-C terminal (EB1-C) domain profile. / EB1-like C-terminal motif / Calponin homology (CH) domain / Calponin homology domain / CH domain superfamily / Calponin homology (CH) domain profile. / 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 / Microtubule-associated protein RP/EB family member 1
Similarity search - Component
Biological speciesHomo sapiens (human) / Sus 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 20, 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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  • Solid view (volume rendering)
  • Imaged by UCSF Chimera
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  • Simplified surface model
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Structure viewerEM map:
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Supplemental images

Downloads & links

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Map

FileDownload / File: emd_2912.map.gz / Format: CCP4 / Size: 20.9 MB / Type: IMAGE STORED AS SIGNED INTEGER (2 BYTES)
AnnotationCryo-electron tomogram of EB1 conjugated to 6.5 nm gold nanoparticles, in interaction with a microtubule growing end
Voxel sizeX=Y=Z: 8.8 Å
Density
Minimum - Maximum-12743.0 - 6116.0
Average (Standard dev.)498.681823730000019 (±438.751342770000008)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin93-707
Dimensions681131126
Spacing681131126
CellA: 1152.8 Å / B: 5992.8003 Å / C: 1108.8 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Integer*27
Å/pix. X/Y/Z8.88.88.8
M x/y/z131681126
origin x/y/z0.0000.0000.000
length x/y/z1152.8005992.8001108.800
α/β/γ90.00090.00090.000
start NX/NY/NZ-147-147-146
NX/NY/NZ294294294
MAP C/R/S123
start NC/NR/NS-70937
NC/NR/NS131681126
D min/max/mean-12743.0006116.000498.682

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Supplemental data

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Sample components

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Entire : EB1 conjugated to 6.5 nm gold nanoparticles, in interaction with ...

EntireName: EB1 conjugated to 6.5 nm gold nanoparticles, in interaction with a microtubule growing end
Components
  • Sample: EB1 conjugated to 6.5 nm gold nanoparticles, in interaction with a microtubule growing end
  • Protein or peptide: End-binding protein one
  • Protein or peptide: Tubulin alpha chain
  • Protein or peptide: Tubulin beta chain

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Supramolecule #1000: EB1 conjugated to 6.5 nm gold nanoparticles, in interaction with ...

SupramoleculeName: EB1 conjugated to 6.5 nm gold nanoparticles, in interaction with a microtubule growing end
type: sample / ID: 1000 / Details: The sample was monodisperse
Oligomeric state: EB1: homodimer. Microtubule: polymer of tubulin.
Number unique components: 3

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Macromolecule #1: End-binding protein one

MacromoleculeName: End-binding protein one / type: protein_or_peptide / ID: 1 / Name.synonym: EB1
Details: EB1 with a haxa-histidine tag inserted into its C-terminal extremity was conjugated to 6.5 nanometer gold nanoparticles functionalized with tris-NiNTA groups
Oligomeric state: Homodimer / Recombinant expression: Yes
Source (natural)Organism: Homo sapiens (human) / synonym: Human / Location in cell: Cytoplasm
Molecular weightExperimental: 30 KDa / Theoretical: 30 KDa
Recombinant expressionOrganism: Escherichia coli BL21 (bacteria) / Recombinant plasmid: pET-3D
SequenceUniProtKB: Microtubule-associated protein RP/EB family member 1

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Macromolecule #2: Tubulin alpha chain

MacromoleculeName: Tubulin alpha 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: 500.68 KDa
SequenceUniProtKB: Tubulin alpha-1A chain / GO: microtubule-based process / InterPro: Tubulin

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Macromolecule #3: Tubulin beta chain

MacromoleculeName: Tubulin beta chain / type: protein_or_peptide / ID: 3
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

BufferpH: 6.8
Details: 80 mM Pipes, 1 mM MgCl2, 50 mM KCl, 1mM EGTA, 1 mM GTP
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 ~3 min after the beginning of assembly.
Method: Assembly in a test tube at 35 degrees celcius for ~3 min, deposit of a 4 microliter 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 opticsCalibrated magnification: 31818 / Illumination 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: 25000
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. Tilt series between -56.44 and 62.43 degrees after correction.Camera used in binning mode 2, 0.88 nm pixel size.
DateApr 4, 2012
Image recordingCategory: CCD / Film or detector model: GATAN ULTRASCAN 1000 (2k x 2k) / Number real images: 75 / Average electron dose: 0.3 e/Å2
Details: Camera used in binning mode 2, at a final pixel size of 0.88 nm.
Bits/pixel: 16

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Image processing

Final reconstructionAlgorithm: OTHER / Software - Name: eTomo, from, IMOD, software
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 backprojection, using a Radial filter cutoffof 0.15 and a faloff of 0.05.

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