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- EMDB-15740: Microtubule decorated with kinesin-motor domains, 13 protofilamen... -

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Basic information

Entry
Database: EMDB / ID: EMD-15740
TitleMicrotubule decorated with kinesin-motor domains, 13 protofilaments, 3-start helix, 3 seams
Map dataMicrotubule decorated with kinesin-motor domains, 13 protofilaments, 3-start helix, 3 seams
Sample
  • Organelle or cellular component: Microtubule assembled from purified porcine brain alpha-beta tubulin and decorated with kinesin-motor domain Kif5B
    • Complex: Kinesin-motor domain Kif5B
KeywordsCytoskeleton / Microtubule / tubulin / kinesin / STRUCTURAL PROTEIN
Biological speciesSus scrofa domesticus (domestic pig) / Homo sapiens (human)
Methodsubtomogram averaging / cryo EM / Resolution: 43.5 Å
AuthorsChretien D / Guyomar C
Funding support France, Switzerland, 4 items
OrganizationGrant numberCountry
Agence Nationale de la Recherche (ANR)ANR-16-CE11-0017-01 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 21, 2022-
Map releaseSep 21, 2022-
UpdateDec 13, 2023-
Current statusDec 13, 2023Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_15740.png

Downloads & links

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Map

FileDownload / File: emd_15740.map.gz / Format: CCP4 / Size: 931.6 KB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationMicrotubule decorated with kinesin-motor domains, 13 protofilaments, 3-start helix, 3 seams
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
8.34 Å/pix.
x 62 pix.
= 517.08 Å		8.34 Å/pix.
x 62 pix.
= 517.08 Å		8.34 Å/pix.
x 62 pix.
= 517.08 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 8.34 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 138.0
Minimum - Maximum68.787000000000006 - 177.039999999999992
Average (Standard dev.)116.080414000000005 (±12.006880000000001)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions626262
Spacing626262
CellA=B=C: 517.08 Å
α=β=γ: 90.0 °

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Supplemental data

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Additional map: Microtubule decorated with kinesin-motor domains, 13 protofilaments, 3-start...

Fileemd_15740_additional_1.map
AnnotationMicrotubule decorated with kinesin-motor domains, 13 protofilaments, 3-start helix, 3 seams, reoriented
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Microtubule decorated with kinesin-motor domains, 13 protofilaments, 3-start...

Fileemd_15740_half_map_1.map
AnnotationMicrotubule decorated with kinesin-motor domains, 13 protofilaments, 3-start helix, 3 seams, odd half-map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Microtubule decorated with kinesin-motor domains, 13 protofilaments, 3-start...

Fileemd_15740_half_map_2.map
AnnotationMicrotubule decorated with kinesin-motor domains, 13 protofilaments, 3-start helix, 3 seams, even half-map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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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
    • Complex: 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: Microtubule assembled in the presence of GMPCPP
Source (natural)Organism: Sus scrofa domesticus (domestic pig) / Tissue: Brain

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Supramolecule #2: Kinesin-motor domain Kif5B

SupramoleculeName: Kinesin-motor domain Kif5B / type: complex / ID: 2 / Parent: 1
Source (natural)Organism: Homo sapiens (human)

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Experimental details

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Structure determination

Methodcryo EM
Processingsubtomogram averaging
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.
DetailsTubulin was mixed with 10 nm gold nanoparticles at 80 nM final concentration. The sample was incubated at 35 degrees for 1 hour. Kinesin-motor domains mixed with gold nanoparticles were added to the sample right before freezing.

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Electron microscopy

MicroscopeFEI TECNAI 20
TemperatureMin: 88.15 K / Max: 93.15 K
DetailsTilt series were started at 0 degrees. 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: 1.0 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 reconstructionResolution.type: BY AUTHOR / Resolution: 43.5 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: PEET
Details: The picking method involves a large overlap between sub-tomograms. The value provided by the FSC must be taken with caution (no Gold Standard). No FCS curve provided.
Number subtomograms used: 20
ExtractionNumber tomograms: 1 / Number images used: 20
Details: Sub-tomogram averages where extracted from a binned 4 version of the tomogram
Final angle assignmentType: NOT APPLICABLE

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