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- EMDB-15750: Microtubule decorated with kinesin-motor domains, fully embedded ... -

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Entry
Database: EMDB / ID: EMD-15750
TitleMicrotubule decorated with kinesin-motor domains, fully embedded in ice
Map dataMicrotubule decorated with kinesin-motor domains, fully embedded in ice
Sample
  • Organelle or cellular component: Microtubule assembled from purified porcine brain alpha-beta tubulin and decorated with kinesin-motor domain Kif5B
Biological speciesSus scrofa domesticus (domestic pig)
Methodelectron tomography / cryo EM
AuthorsChretien D
Funding support France, Switzerland, 4 items
OrganizationGrant numberCountry
Agence Nationale de la Recherche (ANR)ANR-18-CE13-0001-01 France
Agence Nationale de la Recherche (ANR)ANR-18-CE13-0001-01 France
Human Frontier Science Program (HFSP)CDA00019/2019-C France
Swiss National Science Foundation310030_192566 Switzerland
Citation
Journal: Elife / Year: 2022
Title: Changes in seam number and location induce holes within microtubules assembled from porcine brain tubulin and in egg cytoplasmic extracts.
Authors: Charlotte Guyomar / Clément Bousquet / Siou Ku / John M Heumann / Gabriel Guilloux / Natacha Gaillard / Claire Heichette / Laurence Duchesne / Michel O Steinmetz / Romain Gibeaux / Denis Chrétien /
Abstract: Microtubules are tubes of about 25 nm in diameter that are critically involved in a variety of cellular functions, including motility, compartmentalization, and division. They are considered as ...Microtubules are tubes of about 25 nm in diameter that are critically involved in a variety of cellular functions, including motility, compartmentalization, and division. They are considered as pseudo-helical polymers whose constituent αβ-tubulin heterodimers share lateral homotypic interactions, except at one unique region called the seam. Here, we used a segmented sub-tomogram averaging strategy to reassess this paradigm and analyze the organization of the αβ-tubulin heterodimers in microtubules assembled from purified porcine brain tubulin in the presence of GTP and GMPCPP, and in egg cytoplasmic extracts. We find that in almost all conditions, microtubules incorporate variable protofilament and/or tubulin subunit helical-start numbers, as well as variable numbers of seams. Strikingly, the seam number and location vary along individual microtubules, generating holes of one to a few subunits in size within their lattices. Together, our results reveal that the formation of mixed and discontinuous microtubule lattices is an intrinsic property of tubulin that requires the formation of unique lateral interactions without longitudinal ones. They further suggest that microtubule assembly is tightly regulated in a cytoplasmic environment.
#1: Journal: bioRxiv / Year: 2022
Title: Changes in seam number and location induce holes within microtubules assembled from porcine brain tubulin and in Xenopus egg cytoplasmic extracts
Authors: Guyomar C / Bousquet C / Ku S / Heumann J / Guilloux G / Gaillard N / Heichette C / Duchesne L / Steinmetz MO / Gibeaux R / Chretien D
History
DepositionSep 5, 2022-
Header (metadata) releaseSep 14, 2022-
Map releaseSep 14, 2022-
UpdateJan 18, 2023-
Current statusJan 18, 2023Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_15750.png

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Map

FileDownload / File: emd_15750.map.gz / Format: CCP4 / Size: 116.7 MB / Type: IMAGE STORED AS SIGNED BYTE
AnnotationMicrotubule decorated with kinesin-motor domains, fully embedded in ice
Voxel sizeX=Y=Z: 2.09 Å
Density
Minimum - Maximum-128.0 - 127.0
Average (Standard dev.)12.03625 (±28.396124)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin1772757-21
Dimensions2361892274
Spacing1892236274
CellA: 3954.2798 Å / B: 493.24 Å / C: 572.66 Å
α=β=γ: 90.0 °

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Supplemental data

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Sample components

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Entire : Microtubule assembled from purified porcine brain alpha-beta tubu...

EntireName: Microtubule assembled from purified porcine brain alpha-beta tubulin and decorated with kinesin-motor domain Kif5B
Components
  • Organelle or cellular component: Microtubule assembled from purified porcine brain alpha-beta tubulin and decorated with kinesin-motor domain Kif5B

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Supramolecule #1: Microtubule assembled from purified porcine brain alpha-beta tubu...

SupramoleculeName: Microtubule assembled from purified porcine brain alpha-beta tubulin and decorated with kinesin-motor domain Kif5B
type: organelle_or_cellular_component / ID: 1 / Parent: 0 / Details: Microtubules assembled in the presence of GMPCPP
Source (natural)Organism: Sus scrofa domesticus (domestic pig) / Organ: Brain / Tissue: Nerve cells / Organelle: Microtubules / Location in cell: Cytosolic

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Experimental details

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Structure determination

Methodcryo EM
Processingelectron tomography
Aggregation statehelical array

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Sample preparation

Concentration1 mg/mL
BufferpH: 6.8
Component:
ConcentrationFormulaName
80.0 mMPipes1,4-Piperazinediethanesulfonic acid
1.0 mMMgCl2Magnesium chloride
1.0 mMEGTAEthylene glycol-bis(2-aminoethylether)-N,N,N',N'-tetraacetic acid
0.1 mMGMPCPPGuanosine-5'-((alpha,beta)-methyleno)triphosphate

Details: pH adjusted with KOH
GridModel: Quantifoil R2/2 / Material: COPPER / Mesh: 200 / Support film - Material: CARBON / Support film - topology: HOLEY ARRAY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 20 sec. / Pretreatment - Atmosphere: AIR
VitrificationCryogen name: ETHANE / Chamber humidity: 95 % / Chamber temperature: 308.15 K / Instrument: LEICA EM GP
Details: Blot for 2 seconds using Whatman paper number 1 before plunging.
SectioningOther: NO SECTIONING
Fiducial markerManufacturer: Nanoprobes / Diameter: 10 nm

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Electron microscopy

MicroscopeFEI TECNAI 20
TemperatureMin: 88.15 K / Max: 93.15 K
DetailsTilt series were started at 0 degrees and acquired using a Saxton scheme. Grids were loaded onto a Simple Origin model 205 dual-grid cryo-holder.
Image recordingFilm or detector model: TVIPS TEMCAM-F416 (4k x 4k) / Digitization - Dimensions - Width: 4096 pixel / Digitization - Dimensions - Height: 4096 pixel / Average exposure time: 39.0 sec. / Average electron dose: 1.0 e/Å2
Details: Camera model TVIPS XF416 Electron dose was not calibrated
Electron beamAcceleration voltage: 200 kV / Electron source: LAB6
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.0 mm / Nominal defocus max: 6.0 µm / Nominal defocus min: 4.0 µm / Nominal magnification: 50000
Sample stageSpecimen holder model: OTHER / Cooling holder cryogen: NITROGEN

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Image processing

DetailsCamera model TVIPS XF416
Final reconstructionAlgorithm: BACK PROJECTION / Software - Name: eTomo / Details: Tilt series were started from 0 degrees / Number images used: 39

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