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- EMDB-15119: 13pf undecorated microtubule from recombinant human tubulin (alph... -

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Basic information

Entry
Database: EMDB / ID: EMD-15119
Title13pf undecorated microtubule from recombinant human tubulin (alpha1B, beta3) with spliced unmodified C-terminal tail on alpha1B.
Map dataHuman microtubules (alpha1B, beta3) with spliced unmodified C-terminal tail on alpha1B.
Sample
  • Organelle or cellular component: 13pf microtubule from recombinant human tubulin with spliced unmodified C-terminal tail on alpha1B.
    • Protein or peptide: Tubulin alpha-1B with spliced C-terminal tail
    • Protein or peptide: Tubulin beta-3
Keywordstubulin / no tail / recombinant / semisynthetic / STRUCTURAL PROTEIN
Biological speciesHomo sapiens (human)
Methodhelical reconstruction / cryo EM / Resolution: 3.74 Å
AuthorsEbberink E / Fernandes S / Hatzopoulos GN / Agashe N / Guidotti N / Reichart T / Reymond L / Velluz MC / Schneider FZ / Pourroy C ...Ebberink E / Fernandes S / Hatzopoulos GN / Agashe N / Guidotti N / Reichart T / Reymond L / Velluz MC / Schneider FZ / Pourroy C / Janke C / Gonczy P / Aumeier C / Fierz B
Funding support Switzerland, 1 items
OrganizationGrant numberCountry
Swiss National Science Foundation Switzerland
CitationJournal: Nat Chem / Year: 2023
Title: Tubulin engineering by semi-synthesis reveals that polyglutamylation directs detyrosination.
Authors: Eduard Ebberink / Simon Fernandes / Georgios Hatzopoulos / Ninad Agashe / Po-Han Chang / Nora Guidotti / Timothy M Reichart / Luc Reymond / Marie-Claire Velluz / Fabian Schneider / Cédric ...Authors: Eduard Ebberink / Simon Fernandes / Georgios Hatzopoulos / Ninad Agashe / Po-Han Chang / Nora Guidotti / Timothy M Reichart / Luc Reymond / Marie-Claire Velluz / Fabian Schneider / Cédric Pourroy / Carsten Janke / Pierre Gönczy / Beat Fierz / Charlotte Aumeier /
Abstract: Microtubules, a critical component of the cytoskeleton, carry post-translational modifications (PTMs) that are important for the regulation of key cellular processes. Long-lived microtubules, in ...Microtubules, a critical component of the cytoskeleton, carry post-translational modifications (PTMs) that are important for the regulation of key cellular processes. Long-lived microtubules, in neurons particularly, exhibit both detyrosination of α-tubulin and polyglutamylation. Dysregulation of these PTMs can result in developmental defects and neurodegeneration. Owing to a lack of tools to study the regulation and function of these PTMs, the mechanisms that govern such PTM patterns are not well understood. Here we produce fully functional tubulin carrying precisely defined PTMs within its C-terminal tail. We ligate synthetic α-tubulin tails-which are site-specifically glutamylated-to recombinant human tubulin heterodimers by applying a sortase- and intein-mediated tandem transamidation strategy. Using microtubules reconstituted with these designer tubulins, we find that α-tubulin polyglutamylation promotes its detyrosination by enhancing the activity of the tubulin tyrosine carboxypeptidase vasohibin/small vasohibin-binding protein in a manner dependent on the length of polyglutamyl chains. We also find that modulating polyglutamylation levels in cells results in corresponding changes in detyrosination, corroborating the link between the detyrosination cycle to polyglutamylation.
History
DepositionJun 8, 2022-
Header (metadata) releaseJun 21, 2023-
Map releaseJun 21, 2023-
UpdateAug 23, 2023-
Current statusAug 23, 2023Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_15119.png

Downloads & links

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Map

FileDownload / File: emd_15119.map.gz / Format: CCP4 / Size: 824 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationHuman microtubules (alpha1B, beta3) with spliced unmodified C-terminal tail on alpha1B.
Voxel sizeX=Y=Z: 0.83 Å
Density
Contour LevelBy AUTHOR: 0.054
Minimum - Maximum-0.08231881 - 0.23393734
Average (Standard dev.)0.0006047964 (±0.017694604)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions600600600
Spacing600600600
CellA=B=C: 498.0 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_15119_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_15119_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_15119_half_map_2.map
Projections & Slices
AxesZYX

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Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : 13pf microtubule from recombinant human tubulin with spliced unmo...

EntireName: 13pf microtubule from recombinant human tubulin with spliced unmodified C-terminal tail on alpha1B.
Components
  • Organelle or cellular component: 13pf microtubule from recombinant human tubulin with spliced unmodified C-terminal tail on alpha1B.
    • Protein or peptide: Tubulin alpha-1B with spliced C-terminal tail
    • Protein or peptide: Tubulin beta-3

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Supramolecule #1: 13pf microtubule from recombinant human tubulin with spliced unmo...

SupramoleculeName: 13pf microtubule from recombinant human tubulin with spliced unmodified C-terminal tail on alpha1B.
type: organelle_or_cellular_component / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Homo sapiens (human)

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Macromolecule #1: Tubulin alpha-1B with spliced C-terminal tail

MacromoleculeName: Tubulin alpha-1B with spliced C-terminal tail / type: protein_or_peptide / ID: 1
Details: internal 6xHis tag in 40-loop; Spliced C-terminal tail without glutamylation, splicing scar: InsC440 and V441F
Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: MRECISIHVG QAGVQIGNA C WELYCLEH GI QPDGQMP SDK TIHHHH HHGGGDDSFN TFFSETGAG K HVPRAVFV DL EPTVIDE VRT GTYRQL FHPE QLITG KEDAA NNYA RGHYTI GKE IIDLVLD RI RKLADQCT G LQGFLVFHS FGGGTGSGFT ...String:
MRECISIHVG QAGVQIGNA C WELYCLEH GI QPDGQMP SDK TIHHHH HHGGGDDSFN TFFSETGAG K HVPRAVFV DL EPTVIDE VRT GTYRQL FHPE QLITG KEDAA NNYA RGHYTI GKE IIDLVLD RI RKLADQCT G LQGFLVFHS FGGGTGSGFT SLLMERLSV D YGKKSKLE FS IYPAPQV STA VVEPYN SILT THTTL EHSDC AFMV DNEAIY DIC RRNLDIE RP TYTNLNRL I SQIVSSITA SLRFDGALNV DLTEFQTNL V PYPRIHFP LA TYAPVIS AEK AYHEQL SVAE ITNAC FEPAN QMVK CDPRHG KYM ACCLLYR GD VVPKDVNA A IATIKTKRS IQFVDWCPTG FKVGINYQP P TVVPGGDL AK VQRAVCM LSN TTAIAE AWAR LDHKF DLMYA KRAF VHWYVG EGM EEGEFSE AR EDMAALEK D YEEVGVDSC FEGEGEEEGE EY

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Macromolecule #2: Tubulin beta-3

MacromoleculeName: Tubulin beta-3 / type: protein_or_peptide / ID: 2 / Details: C-terminal FLAG-tag / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: MREIVHIQAG QCGNQIGAKF WEVISDEHGI DPSGNYVGDS DLQLERISVY YNEASSHKYV PRAILVDLEP GTMDSVRSGA FGHLFRPDNF IFGQSGAGNN WAKGHYTEGA ELVDSVLDVV RKECENCDCL QGFQLTHSLG GGTGSGMGTL LISKVREEYP DRIMNTFSVV ...String:
MREIVHIQAG QCGNQIGAKF WEVISDEHGI DPSGNYVGDS DLQLERISVY YNEASSHKYV PRAILVDLEP GTMDSVRSGA FGHLFRPDNF IFGQSGAGNN WAKGHYTEGA ELVDSVLDVV RKECENCDCL QGFQLTHSLG GGTGSGMGTL LISKVREEYP DRIMNTFSVV PSPKVSDTVV EPYNATLSIH QLVENTDETY CIDNEALYDI CFRTLKLATP TYGDLNHLVS ATMSGVTTSL RFPGQLNADL RKLAVNMVPF PRLHFFMPGF APLTARGSQQ YRALTVPELT QQMFDAKNMM AACDPRHGRY LTVATVFRGR MSMKEVDEQM LAIQSKNSSY FVEWIPNNVK VAVCDIPPRG LKMSSTFIGN STAIQELFKR ISEQFTAMFR RKAFLHWYTG EGMDEMEFTE AESNMNDLVS EYQQYQDATA EEEGEMYEDD EEESEAQGPK ENLYFQSSGG DYKDDDDK

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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

BufferpH: 6.8
VitrificationCryogen name: ETHANE

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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 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 2.0 µm / Nominal defocus min: 0.6 µm
Image recordingFilm or detector model: FEI FALCON IV (4k x 4k) / Average electron dose: 50.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: INSILICO MODEL
Final angle assignmentType: NOT APPLICABLE
Final reconstructionApplied symmetry - Helical parameters - Δz: 9.46 Å
Applied symmetry - Helical parameters - Δ&Phi: -27.67 °
Applied symmetry - Helical parameters - Axial symmetry: C1 (asymmetric)
Resolution.type: BY AUTHOR / Resolution: 3.74 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 115123
FSC plot (resolution estimation)

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