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- EMDB-50178: 13pf mosaic 20%E254Q - 80% E254N microtubule from recombinant hum... -

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Basic information

Entry
Database: EMDB / ID: EMD-50178
Title13pf mosaic 20%E254Q - 80% E254N microtubule from recombinant human tubulin decorated with EB3
Map data
Sample
  • Organelle or cellular component: 13pf E254Q microtubule from recombinant human tubulin decorated with EB3
    • Protein or peptide: Microtubule-associated protein RP/EB family member 3
    • Protein or peptide: Detyrosinated tubulin alpha-1B chain
    • Protein or peptide: Tubulin beta-3 chain
  • Ligand: GUANOSINE-5'-TRIPHOSPHATE
  • Ligand: MAGNESIUM ION
KeywordsMicrotubule Tubulin GTP cp Cell cycle / STRUCTURAL PROTEIN
Function / homology
Function and homology information


netrin receptor binding / mitotic spindle astral microtubule end / protein localization to microtubule / Post-chaperonin tubulin folding pathway / dorsal root ganglion development / Cilium Assembly / cytoskeleton-dependent intracellular transport / Microtubule-dependent trafficking of connexons from Golgi to the plasma membrane / Carboxyterminal post-translational modifications of tubulin / microtubule plus-end ...netrin receptor binding / mitotic spindle astral microtubule end / protein localization to microtubule / Post-chaperonin tubulin folding pathway / dorsal root ganglion development / Cilium Assembly / cytoskeleton-dependent intracellular transport / Microtubule-dependent trafficking of connexons from Golgi to the plasma membrane / Carboxyterminal post-translational modifications of tubulin / microtubule plus-end / Intraflagellar transport / Sealing of the nuclear envelope (NE) by ESCRT-III / Formation of tubulin folding intermediates by CCT/TriC / Gap junction assembly / microtubule plus-end binding / Prefoldin mediated transfer of substrate to CCT/TriC / Kinesins / COPI-independent Golgi-to-ER retrograde traffic / Assembly and cell surface presentation of NMDA receptors / COPI-dependent Golgi-to-ER retrograde traffic / spindle midzone / Recycling pathway of L1 / microtubule organizing center / regulation of microtubule polymerization / RHOH GTPase cycle / regulation of microtubule polymerization or depolymerization / RHO GTPases activate IQGAPs / microtubule-based process / Hedgehog 'off' state / COPI-mediated anterograde transport / Activation of AMPK downstream of NMDARs / intercellular bridge / spindle assembly / cytoplasmic microtubule / Mitotic Prometaphase / EML4 and NUDC in mitotic spindle formation / peptide binding / MHC class II antigen presentation / cellular response to interleukin-4 / Recruitment of NuMA to mitotic centrosomes / protein serine/threonine kinase binding / axon guidance / HSP90 chaperone cycle for steroid hormone receptors (SHR) in the presence of ligand / Resolution of Sister Chromatid Cohesion / protein serine/threonine kinase activator activity / cell periphery / Translocation of SLC2A4 (GLUT4) to the plasma membrane / filopodium / RHO GTPases Activate Formins / PKR-mediated signaling / structural constituent of cytoskeleton / microtubule cytoskeleton organization / HCMV Early Events / Aggrephagy / The role of GTSE1 in G2/M progression after G2 checkpoint / mitotic spindle / Separation of Sister Chromatids / intracellular protein localization / mitotic cell cycle / lamellipodium / double-stranded RNA binding / microtubule cytoskeleton / growth cone / midbody / microtubule binding / Hydrolases; Acting on acid anhydrides; Acting on GTP to facilitate cellular and subcellular movement / microtubule / cilium / axon / cell division / neuronal cell body / GTPase activity / dendrite / ubiquitin protein ligase binding / positive regulation of DNA-templated transcription / GTP binding / perinuclear region of cytoplasm / structural molecule activity / extracellular exosome / metal ion binding / identical protein binding / nucleus / 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. / Alpha tubulin ...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. / 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-1B chain / Tubulin beta-3 chain / Microtubule-associated protein RP/EB family member 3
Similarity search - Component
Biological speciesHomo sapiens (human)
Methodhelical reconstruction / cryo EM / Resolution: 4.2 Å
AuthorsEstevez-Gallego J / Blum TB / Steinmetz MO / Surrey T
Funding support Spain, Switzerland, European Union, 5 items
OrganizationGrant numberCountry
Spanish Ministry of Science, Innovation, and UniversitiesPID2019-108415GB-I00 Spain
Swiss National Science Foundation310030_192566 Switzerland
Generalitat de CatalunyaIU16-014045 (CRYO-TEM) Spain
European Research Council (ERC)951430European Union
Spanish Ministry of Science, Innovation, and UniversitiesFJC2020-043857-I Spain
CitationJournal: Nat Commun / Year: 2025
Title: Hydrolysis-deficient mosaic microtubules as faithful mimics of the GTP cap.
Authors: Juan Estévez-Gallego / Thorsten B Blum / Felix Ruhnow / María Gili / Silvia Speroni / Raquel García-Castellanos / Michel O Steinmetz / Thomas Surrey /
Abstract: A critical feature of microtubules is their GTP cap, a stabilizing GTP-tubulin rich region at growing microtubule ends. Microtubules polymerized in the presence of GTP analogs or from GTP hydrolysis- ...A critical feature of microtubules is their GTP cap, a stabilizing GTP-tubulin rich region at growing microtubule ends. Microtubules polymerized in the presence of GTP analogs or from GTP hydrolysis-deficient tubulin mutants have been used as GTP-cap mimics for structural and biochemical studies. However, these analogs and mutants generate microtubules with diverse biochemical properties and lattice structures, leaving it unclear what is the most faithful GTP mimic and hence the structure of the GTP cap. Here, we generate a hydrolysis-deficient human tubulin mutant, αE254Q, with the smallest possible modification. We show that αE254Q-microtubules are stable, but still exhibit mild mutation-induced growth abnormalities. However, mixing two GTP hydrolysis-deficient tubulin mutants, αE254Q and αE254N, at an optimized ratio eliminates growth and lattice abnormalities, indicating that these 'mosaic microtubules' are faithful GTP cap mimics. Their cryo-electron microscopy structure reveals that longitudinal lattice expansion, but not protofilament twist, is the primary structural feature distinguishing the GTP-tubulin containing cap from the GDP-tubulin containing microtubule shaft. However, alterations in protofilament twist may be transiently needed to allow lattice compaction and GTP hydrolysis. Together, our results provide insights into the structural origin of GTP cap stability, the pathway of GTP hydrolysis and hence microtubule dynamic instability.
History
DepositionApr 25, 2024-
Header (metadata) releaseMar 19, 2025-
Map releaseMar 19, 2025-
UpdateMar 19, 2025-
Current statusMar 19, 2025Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_50178.png

Downloads & links

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Map

FileDownload / File: emd_50178.map.gz / Format: CCP4 / Size: 824 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
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AxesZ (Sec.)Y (Row.)X (Col.)
0.96 Å/pix.
x 600 pix.
= 573. Å		0.96 Å/pix.
x 600 pix.
= 573. Å		0.96 Å/pix.
x 600 pix.
= 573. Å

Surface

Projections

Slices (1/3)

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Images are generated by Spider.

Voxel sizeX=Y=Z: 0.955 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.018
Minimum - Maximum-0.05045585 - 0.10040231
Average (Standard dev.)0.00003954871 (±0.0037235492)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions600600600
Spacing600600600
CellA=B=C: 573.0 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #2

Fileemd_50178_half_map_1.map
Projections & Slices
AxesZYX

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Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_50178_half_map_2.map
Projections & Slices
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Histogram (log scale)

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Sample components

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Entire : 13pf E254Q microtubule from recombinant human tubulin decorated w...

EntireName: 13pf E254Q microtubule from recombinant human tubulin decorated with EB3
Components
  • Organelle or cellular component: 13pf E254Q microtubule from recombinant human tubulin decorated with EB3
    • Protein or peptide: Microtubule-associated protein RP/EB family member 3
    • Protein or peptide: Detyrosinated tubulin alpha-1B chain
    • Protein or peptide: Tubulin beta-3 chain
  • Ligand: GUANOSINE-5'-TRIPHOSPHATE
  • Ligand: MAGNESIUM ION

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Supramolecule #1: 13pf E254Q microtubule from recombinant human tubulin decorated w...

SupramoleculeName: 13pf E254Q microtubule from recombinant human tubulin decorated with EB3
type: organelle_or_cellular_component / ID: 1 / Parent: 0 / Macromolecule list: #1-#3
Source (natural)Organism: Homo sapiens (human)

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Macromolecule #1: Microtubule-associated protein RP/EB family member 3

MacromoleculeName: Microtubule-associated protein RP/EB family member 3 / type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 15.386819 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
MAVNVYSTSV TSENLSRHDM LAWVNDSLHL NYTKIEQLCS GAAYCQFMDM LFPGCVHLRK VKFQAKLEHE YIHNFKVLQA AFKKMGVDK IIPVEKLVKG KFQDNFEFIQ WFKKFFDANY DGKDYNPLLA RQ

UniProtKB: Microtubule-associated protein RP/EB family member 3

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Macromolecule #2: Detyrosinated tubulin alpha-1B chain

MacromoleculeName: Detyrosinated tubulin alpha-1B chain / type: protein_or_peptide / ID: 2 / Number of copies: 6 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 50.718211 KDa
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper)
SequenceString: MRECISIHVG QAGVQIGNAC WELYCLEHGI QPDGQMPSDK TIHHHHHHGG GHHHFNTFDS FNTFFSETGA GKHVPRAVFV DLEPTVIDE VRTGTYRQLF HPEQLITGKE DAANNYARGH YTIGKEIIDL VLDRIRKLAD QCTGLQGFLV FHSFGGGTGS G FTSLLMER ...String:
MRECISIHVG QAGVQIGNAC WELYCLEHGI QPDGQMPSDK TIHHHHHHGG GHHHFNTFDS FNTFFSETGA GKHVPRAVFV DLEPTVIDE VRTGTYRQLF HPEQLITGKE DAANNYARGH YTIGKEIIDL VLDRIRKLAD QCTGLQGFLV FHSFGGGTGS G FTSLLMER LSVDYGKKSK LEFSIYPAPQ VSTAVVEPYN SILTTHTTLE HSDCAFMVDN EAIYDICRRN LDIERPTYTN LN RLISQIV SSITASLRFD GALNVDLTNF QTNLVPYPRI HFPLATYAPV ISAEKAYHEQ LSVAEITNAC FEPANQMVKC DPR HGKYMA CCLLYRGDVV PKDVNAAIAT IKTKRSIQFV DWCPTGFKVG INYQPPTVVP GGDLAKVQRA VCMLSNTTAI AEAW ARLDH KFDLMYAKRA FVHWYVGEGM EEGEFSEARE DMAALEKDYE EVGVDSVE

UniProtKB: Tubulin alpha-1B chain, Tubulin alpha-1B chain

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Macromolecule #3: Tubulin beta-3 chain

MacromoleculeName: Tubulin beta-3 chain / type: protein_or_peptide / ID: 3 / Number of copies: 6 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 51.276367 KDa
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper)
SequenceString: MREIVHIQAG QCGNQIGAKF WEVISDEHGI DPSGNYVGDS DLQLERISVY YNEASSHKYV PRAILVDLEP GTMDSVRSGA FGHLFRPDN FIFGQSGAGN NWAKGHYTEG AELVDSVLDV VRKECENCDC LQGFQLTHSL GGGTGSGMGT LLISKVREEY P DRIMNTFS ...String:
MREIVHIQAG QCGNQIGAKF WEVISDEHGI DPSGNYVGDS DLQLERISVY YNEASSHKYV PRAILVDLEP GTMDSVRSGA FGHLFRPDN FIFGQSGAGN NWAKGHYTEG AELVDSVLDV VRKECENCDC LQGFQLTHSL GGGTGSGMGT LLISKVREEY P DRIMNTFS VVPSPKVSDT VVEPYNATLS IHQLVENTDE TYCIDNEALY DICFRTLKLA TPTYGDLNHL VSATMSGVTT SL RFPGQLN ADLRKLAVNM VPFPRLHFFM PGFAPLTARG SQQYRALTVP ELTQQMFDAK NMMAACDPRH GRYLTVATVF RGR MSMKEV DEQMLAIQSK NSSYFVEWIP NNVKVAVCDI PPRGLKMSST FIGNSTAIQE LFKRISEQFT AMFRRKAFLH WYTG EGMDE MEFTEAESNM NDLVSEYQQY QDATAEEEGE MYEDDEEESE AQGPKENLYF Q

UniProtKB: Tubulin beta-3 chain

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Macromolecule #4: GUANOSINE-5'-TRIPHOSPHATE

MacromoleculeName: GUANOSINE-5'-TRIPHOSPHATE / type: ligand / ID: 4 / Number of copies: 12 / Formula: GTP
Molecular weightTheoretical: 523.18 Da
Chemical component information

ChemComp-GTP:
GUANOSINE-5'-TRIPHOSPHATE / GTP, energy-carrying molecule*YM

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Macromolecule #5: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 5 / Number of copies: 12 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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Experimental details

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Structure determination

Methodcryo EM
Processinghelical reconstruction
Aggregation statehelical array

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Sample preparation

Concentration20 mg/mL
BufferpH: 6.8
GridModel: C-flat-2/2 / Support film - Material: CARBON / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 60 sec.
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 310 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeTFS GLACIOS
Image recordingFilm or detector model: FEI FALCON IV (4k x 4k) / Number grids imaged: 1 / Number real images: 5581 / Average electron dose: 7.27 e/Å2
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.5 µm / Nominal defocus min: 1.0 µm

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Image processing

Final reconstructionNumber classes used: 1
Applied symmetry - Helical parameters - Δz: 9.58598 Å
Applied symmetry - Helical parameters - Δ&Phi: -27.6855 °
Applied symmetry - Helical parameters - Axial symmetry: C13 (13 fold cyclic)
Resolution.type: BY AUTHOR / Resolution: 4.2 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: PHENIX (ver. 1.21) / Number images used: 30010
Segment selectionNumber selected: 54200 / Software - Name: RELION (ver. 3.1.2)
Startup modelType of model: NONE
Final angle assignmentType: PROJECTION MATCHING

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Atomic model buiding 1

Initial modelPDB ID:

7sj7


Chain - Source name: PDB / Chain - Initial model type: experimental model
RefinementSpace: REAL / Protocol: RIGID BODY FIT / Overall B value: 150
Output model

PDB-9f3s:
13pf mosaic 20%E254Q - 80% E254N microtubule from recombinant human tubulin decorated with EB3

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