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- EMDB-41167: GMPCPP-microtubule C1 reconstruction -

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Basic information

Entry
Database: EMDB / ID: EMD-41167
TitleGMPCPP-microtubule C1 reconstruction
Map dataGMPCPP-Microtubule C1 reconstruction
Sample
  • Complex: GMPCPP-microtubules
KeywordsMicrotubule / PROTEIN FIBRIL
Biological speciesSus scrofa (pig)
Methodhelical reconstruction / cryo EM / Resolution: 4.0 Å
AuthorsLyu W / Duan D / Chai P
Funding support United States, 6 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of Neurological Disorders and Stroke (NIH/NINDS)NS112121 United States
National Institutes of Health/National Institute of Mental Health (NIH/NIMH)MH115939 United States
National Institutes of Health/National Institute of Neurological Disorders and Stroke (NIH/NINDS)NS105640 United States
National Institutes of Health/National Institute of Mental Health (NIH/NIMH)R56MH122449 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R35GM142959 United States
National Institutes of Health/Office of the DirectorS10OD023603 United States
CitationJournal: Curr Biol / Year: 2023
Title: Abl2 repairs microtubules and phase separates with tubulin to promote microtubule nucleation.
Authors: Daisy Duan / Wanqing Lyu / Pengxin Chai / Shaojie Ma / Kuanlin Wu / Chunxiang Wu / Yong Xiong / Nenad Sestan / Kai Zhang / Anthony J Koleske /
Abstract: Abl family kinases are evolutionarily conserved regulators of cell migration and morphogenesis. Genetic experiments in Drosophila suggest that Abl family kinases interact functionally with ...Abl family kinases are evolutionarily conserved regulators of cell migration and morphogenesis. Genetic experiments in Drosophila suggest that Abl family kinases interact functionally with microtubules to regulate axon guidance and neuronal morphogenesis. Vertebrate Abl2 binds to microtubules and promotes their plus-end elongation, both in vitro and in cells, but the molecular mechanisms by which Abl2 regulates microtubule (MT) dynamics are unclear. We report here that Abl2 regulates MT assembly via condensation and direct interactions with both the MT lattice and tubulin dimers. We find that Abl2 promotes MT nucleation, which is further facilitated by the ability of the Abl2 C-terminal half to undergo liquid-liquid phase separation (LLPS) and form co-condensates with tubulin. Abl2 binds to regions adjacent to MT damage, facilitates MT repair via fresh tubulin recruitment, and increases MT rescue frequency and lifetime. Cryo-EM analyses strongly support a model in which Abl2 engages tubulin C-terminal tails along an extended MT lattice conformation at damage sites to facilitate repair via fresh tubulin recruitment. Abl2Δ688-790, which closely mimics a naturally occurring splice isoform, retains binding to the MT lattice but does not bind tubulin, promote MT nucleation, or increase rescue frequency. In COS-7 cells, MT reassembly after nocodazole treatment is greatly slowed in Abl2 knockout COS-7 cells compared with wild-type cells, and these defects are rescued by re-expression of Abl2, but not Abl2Δ688-790. We propose that Abl2 locally concentrates tubulin to promote MT nucleation and recruits it to defects in the MT lattice to enable repair and rescue.
History
DepositionJul 3, 2023-
Header (metadata) releaseNov 8, 2023-
Map releaseNov 8, 2023-
UpdateNov 22, 2023-
Current statusNov 22, 2023Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_41167.png

Downloads & links

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Map

FileDownload / File: emd_41167.map.gz / Format: CCP4 / Size: 178 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationGMPCPP-Microtubule C1 reconstruction
Voxel sizeX=Y=Z: 1.628 Å
Density
Contour LevelBy AUTHOR: 0.15
Minimum - Maximum-0.30111277 - 0.82151896
Average (Standard dev.)0.0020961354 (±0.0693078)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions360360360
Spacing360360360
CellA=B=C: 586.08 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_41167_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map A of GMPCPP-Microtubule C1 reconstruction

Fileemd_41167_half_map_1.map
AnnotationHalf map A of GMPCPP-Microtubule C1 reconstruction
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map B of GMPCPP-Microtubule C1 reconstruction

Fileemd_41167_half_map_2.map
AnnotationHalf map B of GMPCPP-Microtubule C1 reconstruction
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : GMPCPP-microtubules

EntireName: GMPCPP-microtubules
Components
  • Complex: GMPCPP-microtubules

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Supramolecule #1: GMPCPP-microtubules

SupramoleculeName: GMPCPP-microtubules / type: complex / ID: 1 / Parent: 0
Source (natural)Organism: Sus scrofa (pig) / Organ: brain
Molecular weightTheoretical: 0.5 kDa/nm

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Experimental details

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Structure determination

Methodcryo EM
Processinghelical reconstruction
Aggregation statefilament

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Sample preparation

Concentration1 mg/mL
BufferpH: 6.8
Details: 80 mM PIPES, pH 6.8, 1 mM MgCl2, 1 mM EGTA, 1 mM GMPCPP
VitrificationCryogen name: ETHANE / Chamber humidity: 95 % / Chamber temperature: 298 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeTFS GLACIOS
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Average electron dose: 40.0 e/Å2
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Calibrated defocus max: 2.5 µm / Calibrated defocus min: 1.2 µm / Calibrated magnification: 36000 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.5 µm / Nominal defocus min: 1.2 µm / Nominal magnification: 36000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN

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Image processing

Final reconstructionNumber classes used: 1
Applied symmetry - Helical parameters - Δz: 82.0 Å
Applied symmetry - Helical parameters - Δ&Phi: 0 °
Applied symmetry - Helical parameters - Axial symmetry: C1 (asymmetric)
Algorithm: BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 4.0 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 2) / Number images used: 24485
Startup modelType of model: NONE
Final angle assignmentType: NOT APPLICABLE / Software - Name: cryoSPARC (ver. 2)
FSC plot (resolution estimation)

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