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- EMDB-32033: 14pf microtubule decorated with EML1-GFP -

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Basic information

Entry
Database: EMDB / ID: EMD-32033
Title14pf microtubule decorated with EML1-GFP
Map dataRefined map of 14pf microtubule bound to EML1-GFP
Sample
  • Complex: Complex of microtubule with HEK293 cell lysate containing over expressed EML1-GFP
    • Protein or peptide: Tubulin alpha-1B chain
    • Protein or peptide: Tubulin beta chain
KeywordsCytoskeleton / Echinoderm microtubule-associated protein-like 1 (EML1) / Microtubule associated protein (MAP) / STRUCTURAL PROTEIN
Function / homology
Function and homology information


odontoblast differentiation / Post-chaperonin tubulin folding pathway / Carboxyterminal post-translational modifications of tubulin / Microtubule-dependent trafficking of connexons from Golgi to the plasma membrane / Cilium Assembly / cytoskeleton-dependent intracellular transport / Intraflagellar transport / Sealing of the nuclear envelope (NE) by ESCRT-III / Gap junction assembly / Formation of tubulin folding intermediates by CCT/TriC ...odontoblast differentiation / Post-chaperonin tubulin folding pathway / Carboxyterminal post-translational modifications of tubulin / Microtubule-dependent trafficking of connexons from Golgi to the plasma membrane / Cilium Assembly / cytoskeleton-dependent intracellular transport / Intraflagellar transport / Sealing of the nuclear envelope (NE) by ESCRT-III / Gap junction assembly / Formation of tubulin folding intermediates by CCT/TriC / natural killer cell mediated cytotoxicity / COPI-independent Golgi-to-ER retrograde traffic / Kinesins / Assembly and cell surface presentation of NMDA receptors / GTPase activating protein binding / COPI-dependent Golgi-to-ER retrograde traffic / regulation of synapse organization / intercellular bridge / nuclear envelope lumen / Recycling pathway of L1 / RHOH GTPase cycle / spindle assembly / RHO GTPases activate IQGAPs / MHC class I protein binding / Hedgehog 'off' state / cytoplasmic microtubule / microtubule-based process / COPI-mediated anterograde transport / Activation of AMPK downstream of NMDARs / Mitotic Prometaphase / 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 / HSP90 chaperone cycle for steroid hormone receptors (SHR) in the presence of ligand / Anchoring of the basal body to the plasma membrane / MHC class II antigen presentation / cellular response to interleukin-4 / AURKA Activation by TPX2 / RHO GTPases Activate Formins / Translocation of SLC2A4 (GLUT4) to the plasma membrane / Hydrolases; Acting on acid anhydrides; Acting on GTP to facilitate cellular and subcellular movement / PKR-mediated signaling / structural constituent of cytoskeleton / cytoplasmic ribonucleoprotein granule / mitotic spindle / microtubule cytoskeleton organization / Aggrephagy / HCMV Early Events / Separation of Sister Chromatids / The role of GTSE1 in G2/M progression after G2 checkpoint / microtubule cytoskeleton / azurophil granule lumen / Regulation of PLK1 Activity at G2/M Transition / double-stranded RNA binding / mitotic cell cycle / cell body / microtubule / Potential therapeutics for SARS / cytoskeleton / membrane raft / cell division / protein domain specific binding / GTPase activity / ubiquitin protein ligase binding / Neutrophil degranulation / protein-containing complex binding / GTP binding / structural molecule activity / protein-containing complex / extracellular exosome / extracellular region / metal ion binding / nucleus / cytosol / cytoplasm
Similarity search - Function
Tubulin-beta mRNA autoregulation signal. / Alpha tubulin / 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-beta mRNA autoregulation signal. / Alpha tubulin / 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 beta chain / Tubulin alpha-1B chain
Similarity search - Component
Biological speciesHomo sapiens (human)
Methodhelical reconstruction / cryo EM / Resolution: 3.7 Å
AuthorsBangera M / Sirajuddin M
Funding support3 items
OrganizationGrant numberCountry
Department of Biotechnology (DBT, India)DBT/PR12422/MED/31/287/2014
Department of Science & Technology (DST, India)CRG/2019/003246
Other governmentCEFIPRA 5703-1
CitationJournal: Nat Cell Biol / Year: 2022
Title: Lysate-based pipeline to characterize microtubule-associated proteins uncovers unique microtubule behaviours.
Authors: A S Jijumon / Satish Bodakuntla / Mariya Genova / Mamata Bangera / Violet Sackett / Laetitia Besse / Fatlinda Maksut / Veronique Henriot / Maria M Magiera / Minhajuddin Sirajuddin / Carsten Janke /
Abstract: The microtubule cytoskeleton forms complex macromolecular assemblies with a range of microtubule-associated proteins (MAPs) that have fundamental roles in cell architecture, division and motility. ...The microtubule cytoskeleton forms complex macromolecular assemblies with a range of microtubule-associated proteins (MAPs) that have fundamental roles in cell architecture, division and motility. Determining how an individual MAP modulates microtubule behaviour is an important step in understanding the physiological roles of various microtubule assemblies. To characterize how MAPs control microtubule properties and functions, we developed an approach allowing for medium-throughput analyses of MAPs in cell-free conditions using lysates of mammalian cells. Our pipeline allows for quantitative as well as ultrastructural analyses of microtubule-MAP assemblies. Analysing 45 bona fide and potential mammalian MAPs, we uncovered previously unknown activities that lead to distinct and unique microtubule behaviours such as microtubule coiling or hook formation, or liquid-liquid phase separation along the microtubule lattice that initiates microtubule branching. We have thus established a powerful tool for a thorough characterization of a wide range of MAPs and MAP variants, thus opening avenues for the determination of mechanisms underlying their physiological roles and pathological implications.
History
DepositionOct 7, 2021-
Header (metadata) releaseDec 1, 2021-
Map releaseDec 1, 2021-
UpdateDec 13, 2023-
Current statusDec 13, 2023Processing site: PDBj / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.04
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.04
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_32033.png

Downloads & links

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Map

FileDownload / File: emd_32033.map.gz / Format: CCP4 / Size: 325 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationRefined map of 14pf microtubule bound to EML1-GFP
Voxel sizeX=Y=Z: 1.38 Å
Density
Contour LevelBy AUTHOR: 0.04 / Movie #1: 0.04
Minimum - Maximum-0.045934133 - 0.1330614
Average (Standard dev.)0.0018054614 (±0.009389645)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions440440440
Spacing440440440
CellA=B=C: 607.2 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.381.381.38
M x/y/z440440440
origin x/y/z0.0000.0000.000
length x/y/z607.200607.200607.200
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ400400400
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS440440440
D min/max/mean-0.0460.1330.002

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Supplemental data

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Mask #1

Fileemd_32033_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: 15 Angstroms low pass filtered map of 14pf...

Fileemd_32033_additional_1.map
Annotation15 Angstroms low pass filtered map of 14pf microtubule bound to EML1-GFP
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: 14pf microtubule bound to EML1-GFP-half map 1 (unfiltered)

Fileemd_32033_half_map_1.map
Annotation14pf microtubule bound to EML1-GFP-half map 1 (unfiltered)
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: 14pf microtubule bound to EML1-GFP-half map 2 (unfiltered)

Fileemd_32033_half_map_2.map
Annotation14pf microtubule bound to EML1-GFP-half map 2 (unfiltered)
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Complex of microtubule with HEK293 cell lysate containing over ex...

EntireName: Complex of microtubule with HEK293 cell lysate containing over expressed EML1-GFP
Components
  • Complex: Complex of microtubule with HEK293 cell lysate containing over expressed EML1-GFP
    • Protein or peptide: Tubulin alpha-1B chain
    • Protein or peptide: Tubulin beta chain

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Supramolecule #1: Complex of microtubule with HEK293 cell lysate containing over ex...

SupramoleculeName: Complex of microtubule with HEK293 cell lysate containing over expressed EML1-GFP
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Homo sapiens (human)

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Macromolecule #1: Tubulin alpha-1B chain

MacromoleculeName: Tubulin alpha-1B chain / type: protein_or_peptide / ID: 1
Details: The reconstruction contains a mix of tubulin from goat brain (nucleating seeds) and tubulin from HEK293 cellular lysate so will have a mixture of both sequences.
Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
SequenceString: MRECISIHVG QAGVQIGNAC WELYCLEHGI QPDGQMPSDK TIGGGDDSFN TFFSETGAGK HVPRAVFVD LEPTVIDEVR TGTYRQLFHP EQLITGKEDA ANNYARGHYT IGKEIIDLVL D RIRKLADQ CTGLQGFLVF HSFGGGTGSG FTSLLMERLS VDYGKKSKLE ...String:
MRECISIHVG QAGVQIGNAC WELYCLEHGI QPDGQMPSDK TIGGGDDSFN TFFSETGAGK HVPRAVFVD LEPTVIDEVR TGTYRQLFHP EQLITGKEDA ANNYARGHYT IGKEIIDLVL D RIRKLADQ CTGLQGFLVF HSFGGGTGSG FTSLLMERLS VDYGKKSKLE FSIYPAPQVS TA VVEPYNS ILTTHTTLEH SDCAFMVDNE AIYDICRRNL DIERPTYTNL NRLISQIVSS ITA SLRFDG ALNVDLTEFQ TNLVPYPRIH FPLATYAPVI SAEKAYHEQL SVAEITNACF EPAN QMVKC DPRHGKYMAC CLLYRGDVVP KDVNAAIATI KTKRSIQFVD WCPTGFKVGI NYQPP TVVP GGDLAKVQRA VCMLSNTTAI AEAWARLDHK FDLMYAKRAF VHWYVGEGME EGEFSE ARE DMAALEKDYE EVGVDSVEGE GEEEGEEY

UniProtKB: Tubulin alpha-1B chain

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Macromolecule #2: Tubulin beta chain

MacromoleculeName: Tubulin beta chain / type: protein_or_peptide / ID: 2
Details: The reconstruction contains a mix of tubulin from goat brain (nucleating seeds) and tubulin from HEK293 cellular lysate so will have a mixture of both sequences. Sequence of goat brain beta ...Details: The reconstruction contains a mix of tubulin from goat brain (nucleating seeds) and tubulin from HEK293 cellular lysate so will have a mixture of both sequences. Sequence of goat brain beta tubulin can be accessed using Uniprot ID A0A452G3J7
Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
SequenceString: MREIVHIQAG QCGNQIGAKF WEVISDEHGI DPTGTYHGDS DLQLDRISVY YNEATGGKYV PRAILVDLE PGTMDSVRSG PFGQIFRPDN FVFGQSGAGN NWAKGHYTEG AELVDSVLDV V RKEAESCD CLQGFQLTHS LGGGTGSGMG TLLISKIREE YPDRIMNTFS ...String:
MREIVHIQAG QCGNQIGAKF WEVISDEHGI DPTGTYHGDS DLQLDRISVY YNEATGGKYV PRAILVDLE PGTMDSVRSG PFGQIFRPDN FVFGQSGAGN NWAKGHYTEG AELVDSVLDV V RKEAESCD CLQGFQLTHS LGGGTGSGMG TLLISKIREE YPDRIMNTFS VVPSPKVSDT VV EPYNATL SVHQLVENTD ETYCIDNEAL YDICFRTLKL TTPTYGDLNH LVSATMSGVT TCL RFPGQL NADLRKLAVN MVPFPRLHFF MPGFAPLTSR GSQQYRALTV PELTQQVFDA KNMM AACDP RHGRYLTVAA VFRGRMSMKE VDEQMLNVQN KNSSYFVEWI PNNVKTAVCD IPPRG LKMA VTFIGNSTAI QELFKRISEQ FTAMFRRKAF LHWYTGEGMD EMEFTEAESN MNDLVS EYQ QYQDATAEEE EDFGEEAEEE A

UniProtKB: Tubulin beta chain

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Experimental details

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Structure determination

Methodcryo EM
Processinghelical reconstruction
Aggregation statefilament

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Sample preparation

Concentration5 mg/mL
BufferpH: 6.8 / Component - Formula: BRB80
Details: 80mM PIPES, 2mM MgCl2, 1mM EGTA supplemented with 1mM GMPCPP
GridModel: Quantifoil R1.2/1.3 / Material: GOLD / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 90 sec. / Pretreatment - Atmosphere: AIR / Pretreatment - Pressure: 0.025 kPa
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 298 K / Instrument: FEI VITROBOT MARK IV
Details: GMPCPP MT seeds in warm BRB80 applied to the grid followed by second application of pre-warmed lysate containing over expressed EML1-GFP. After an incubation time of 20 seconds, grid blotted ...Details: GMPCPP MT seeds in warm BRB80 applied to the grid followed by second application of pre-warmed lysate containing over expressed EML1-GFP. After an incubation time of 20 seconds, grid blotted for 3 seconds before plunging into liquid ethane..
DetailsGoat brain tubulin was polymerized with 1mM GMPCPP at 310K for 2 hours, spun on warm 50 percent BRB80 sucrose cushion and resuspended in twice the volume of warm BRB80 buffer

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Calibrated magnification: 101449 / Illumination mode: OTHER / Imaging mode: OTHER / Cs: 2.7 mm / Nominal magnification: 59000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: FEI FALCON III (4k x 4k) / Detector mode: INTEGRATING / Digitization - Dimensions - Width: 4096 pixel / Digitization - Dimensions - Height: 4096 pixel / Number grids imaged: 1 / Number real images: 1143 / Average exposure time: 1.5 sec. / Average electron dose: 44.17 e/Å2 / Details: 30 frames
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Segment selectionNumber selected: 33436 / Software - Name: RELION (ver. 3.0)
Startup modelType of model: INSILICO MODEL
In silico model: Low pass filtered insilico density map generated from tubulin PDBs fit into GMPCPP microtubule model
Final angle assignmentType: NOT APPLICABLE / Software - Name: RELION (ver. 3.0) / Details: Maximum Likelihood
Final reconstructionApplied symmetry - Helical parameters - Δz: 8.85 Å
Applied symmetry - Helical parameters - Δ&Phi: -25.75 °
Applied symmetry - Helical parameters - Axial symmetry: C1 (asymmetric)
Resolution.type: BY AUTHOR / Resolution: 3.7 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.0) / Number images used: 29372
FSC plot (resolution estimation)

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