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- EMDB-15733: Microtubule decorated with kinesin-motor domains, 13 protofilamen... -

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Basic information

Entry
Database: EMDB / ID: EMD-15733
TitleMicrotubule decorated with kinesin-motor domains, 13 protofilaments, 3-start helix, 1 seam
Map dataMicrotubule decorated with kinesin-motor domains, 13 protofilaments, 3-start helix, 1 seam
Sample
  • Organelle or cellular component: Microtubule assembled in Xenopus egg cytoplasmic extract and decorated with kinesin-motor domain Kif5B
    • Complex: Kinesin-motor domain Kif5B
KeywordsCytoplasmic extract / Cytoskeleton / Microtubule / kinesin / STRUCTURAL PROTEIN
Biological speciesXenopus laevis (African clawed frog) / Homo sapiens (human)
Methodsubtomogram averaging / cryo EM / Resolution: 43.3 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_15733.png

Downloads & links

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Map

FileDownload / File: emd_15733.map.gz / Format: CCP4 / Size: 762.7 KB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationMicrotubule decorated with kinesin-motor domains, 13 protofilaments, 3-start helix, 1 seam
Voxel sizeX=Y=Z: 8.89 Å
Density
Contour LevelBy AUTHOR: 157.0
Minimum - Maximum84.310010000000005 - 204.610019999999992
Average (Standard dev.)139.315300000000008 (±13.280048000000001)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions585858
Spacing585858
CellA=B=C: 515.62 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_15733_additional_1.map
AnnotationMicrotubule decorated with kinesin-motor domains, 13 protofilaments, 3-start helix, 1 seam, 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_15733_half_map_1.map
AnnotationMicrotubule decorated with kinesin-motor domains, 13 protofilaments, 3-start helix, 1 seam, 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_15733_half_map_2.map
AnnotationMicrotubule decorated with kinesin-motor domains, 13 protofilaments, 3-start helix, 1 seam, 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 in Xenopus egg cytoplasmic extract and deco...

EntireName: Microtubule assembled in Xenopus egg cytoplasmic extract and decorated with kinesin-motor domain Kif5B
Components
  • Organelle or cellular component: Microtubule assembled in Xenopus egg cytoplasmic extract and decorated with kinesin-motor domain Kif5B
    • Complex: Kinesin-motor domain Kif5B

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Supramolecule #1: Microtubule assembled in Xenopus egg cytoplasmic extract and deco...

SupramoleculeName: Microtubule assembled in Xenopus egg cytoplasmic extract and decorated with kinesin-motor domain Kif5B
type: organelle_or_cellular_component / ID: 1 / Parent: 0
Source (natural)Organism: Xenopus laevis (African clawed frog) / Tissue: Egg

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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

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
1.0 mMGTPGuanosine triphosphate
0.1 mMATPAdenosine triphosphateAdenosine triphosphate

Details: pH adjusted with KOH. Buffer used to dilute kinesin-motor domains in the presence of 60 nM gold nanoparticles.
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 4 seconds using Whatman paper number 4 from opposite side before plunging.
DetailsMicrotubule aster formation was induced by addition of 5 percent DMSO in CSF-arrested egg extract. The extract was diluted 1 to 50 in BRB80 containing kinesin-motor domains (2.5 mg/ml) and gold nanoparticles (60 nM) right before freezing.

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Electron microscopy

MicroscopeFEI TECNAI 20
Electron beamAcceleration voltage: 200 kV / Electron source: LAB6
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.0 mm / Nominal defocus max: 6.0 µm / Nominal defocus min: 4.0 µm / Nominal magnification: 25000
Sample stageSpecimen holder model: GATAN CT3500TR SINGLE TILT ROTATION LIQUID NITROGEN CRYO TRANSFER HOLDER
Cooling holder cryogen: NITROGEN
TemperatureMin: 88.15 K / Max: 93.15 K
DetailsTilt series were started at 0 degrees and acquired using a Saxton scheme
Image recordingFilm or detector model: TVIPS TEMCAM-F416 (4k x 4k) / Digitization - Dimensions - Width: 2048 pixel / Digitization - Dimensions - Height: 2048 pixel / Average exposure time: 1.0 sec. / Average electron dose: 1.0 e/Å2
Details: Camera model TVIPS XF416 Images were acquired in binning 2 Electron dose was not calibrated

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Image processing

ExtractionNumber tomograms: 1 / Number images used: 45
Final angle assignmentType: NOT APPLICABLE
Final reconstructionResolution.type: BY AUTHOR / Resolution: 43.3 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: 45
DetailsCamera model TVIPS XF416

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