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- PDB-9g0s: Xenopus tropicalis undecorated microtubule - 14 protofilament, 3-... -

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Basic information

Entry
Database: PDB / ID: 9g0s
TitleXenopus tropicalis undecorated microtubule - 14 protofilament, 3-start helix
Components
  • Tubulin alpha chain
  • Tubulin beta chain
KeywordsPROTEIN FIBRIL / Microtubule Tubulin Cytoskeleton Filament
Function / homology
Function and homology information


microtubule-based process / structural constituent of cytoskeleton / microtubule / hydrolase activity / GTPase activity / GTP binding / cytoplasm
Similarity search - Function
Alpha tubulin / Tubulin-beta mRNA autoregulation signal. / Beta tubulin, autoregulation binding site / Beta tubulin / Tubulin / Tubulin, C-terminal / Tubulin C-terminal domain / Tubulin, conserved site / Tubulin subunits alpha, beta, and gamma signature. / Tubulin/FtsZ family, C-terminal domain ...Alpha tubulin / Tubulin-beta mRNA autoregulation signal. / Beta tubulin, autoregulation binding site / Beta tubulin / Tubulin / Tubulin, C-terminal / Tubulin C-terminal domain / Tubulin, conserved site / Tubulin subunits alpha, beta, and gamma signature. / Tubulin/FtsZ family, C-terminal domain / Tubulin/FtsZ-like, C-terminal domain / Tubulin/FtsZ, C-terminal / Tubulin/FtsZ, 2-layer sandwich domain / Tubulin/FtsZ family, GTPase domain / Tubulin/FtsZ family, GTPase domain / Tubulin/FtsZ, GTPase domain / Tubulin/FtsZ, GTPase domain superfamily
Similarity search - Domain/homology
GUANOSINE-5'-DIPHOSPHATE / GUANOSINE-5'-TRIPHOSPHATE / Tubulin beta chain / Tubulin alpha chain
Similarity search - Component
Biological speciesXenopus tropicalis (tropical clawed frog)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.6 Å
AuthorsTroman, L.A. / Moores, C.A.
Funding support United Kingdom, 4items
OrganizationGrant numberCountry
Medical Research Council (MRC, United Kingdom)MR/R000352/1 United Kingdom
Medical Research Council (MRC, United Kingdom)MR/Y000633/1 United Kingdom
Wellcome Trust202679/Z/16/Z United Kingdom
Wellcome Trust206166/Z/17/Z United Kingdom
CitationJournal: Curr Biol / Year: 2025
Title: Mechanistic basis of temperature adaptation in microtubule dynamics across frog species.
Authors: Luca Troman / Ella de Gaulejac / Abin Biswas / Jennifer Stiens / Benno Kuropka / Carolyn A Moores / Simone Reber /
Abstract: Cellular processes are remarkably effective across diverse temperature ranges, even with highly conserved proteins. In the context of the microtubule cytoskeleton, which is critically involved in a ...Cellular processes are remarkably effective across diverse temperature ranges, even with highly conserved proteins. In the context of the microtubule cytoskeleton, which is critically involved in a wide range of cellular activities, this is particularly striking, as tubulin is one of the most conserved proteins while microtubule dynamic instability is highly temperature sensitive. Here, we leverage the diversity of natural tubulin variants from three closely related frog species that live at different temperatures. We determine the microtubule structure across all three species at between 3.0 and 3.6 Å resolution by cryo-electron microscopy and find small differences at the β-tubulin lateral interactions. Using in vitro reconstitution assays and quantitative biochemistry, we show that tubulin's free energy scales inversely with temperature. The observed weakening of lateral contacts and the low apparent activation energy for tubulin incorporation provide an explanation for the overall stability and higher growth rates of microtubules in cold-adapted frog species. This study thus broadens our conceptual framework for understanding microtubule dynamics and provides insights into how conserved cellular processes are tailored to different ecological niches.
History
DepositionJul 8, 2024Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jan 15, 2025Provider: repository / Type: Initial release
Revision 1.1Jan 22, 2025Group: Data collection / Database references / Category: citation / citation_author / em_admin
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_ASTM / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_PubMed / _citation.title / _em_admin.last_update
Revision 1.2Feb 12, 2025Group: Data collection / Database references / Category: citation / em_admin
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _em_admin.last_update

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Tubulin beta chain
B: Tubulin beta chain
C: Tubulin beta chain
D: Tubulin beta chain
E: Tubulin beta chain
F: Tubulin beta chain
a: Tubulin alpha chain
b: Tubulin alpha chain
c: Tubulin alpha chain
d: Tubulin alpha chain
e: Tubulin alpha chain
f: Tubulin alpha chain
hetero molecules


Theoretical massNumber of molelcules
Total (without water)605,90230
Polymers599,95812
Non-polymers5,94418
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

#1: Protein
Tubulin beta chain


Mass: 49878.770 Da / Num. of mol.: 6 / Source method: isolated from a natural source / Source: (natural) Xenopus tropicalis (tropical clawed frog) / Tissue: oocyte / References: UniProt: Q0IIR4
#2: Protein
Tubulin alpha chain


Mass: 50114.223 Da / Num. of mol.: 6 / Source method: isolated from a natural source / Source: (natural) Xenopus tropicalis (tropical clawed frog) / Tissue: oocyte / References: UniProt: Q5EB23
#3: Chemical
ChemComp-GDP / GUANOSINE-5'-DIPHOSPHATE


Type: RNA linking / Mass: 443.201 Da / Num. of mol.: 6 / Source method: obtained synthetically / Formula: C10H15N5O11P2 / Feature type: SUBJECT OF INVESTIGATION / Comment: GDP, energy-carrying molecule*YM
#4: Chemical
ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 6 / Source method: obtained synthetically / Formula: Mg / Feature type: SUBJECT OF INVESTIGATION
#5: Chemical
ChemComp-GTP / GUANOSINE-5'-TRIPHOSPHATE


Mass: 523.180 Da / Num. of mol.: 6 / Source method: obtained synthetically / Formula: C10H16N5O14P3 / Feature type: SUBJECT OF INVESTIGATION / Comment: GTP, energy-carrying molecule*YM
Has ligand of interestY
Has protein modificationN

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: FILAMENT / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: Undecorated tubulin lattice following polymerisation with GTP (no stabilising ligands).
Type: COMPLEX
Details: The native tubulin was isolated from the Xenopus eggs using TOG-affinity chromatography. Microtubules were polymerised on the cryo-EM grid.
Entity ID: #1-#2 / Source: NATURAL
Molecular weightExperimental value: NO
Source (natural)Organism: Xenopus tropicalis (tropical clawed frog) / Cellular location: cytoplasm / Tissue: Oocyte
Buffer solutionpH: 6.8 / Details: BRB80 + 1 mM GTP
Buffer component
IDConc.NameFormulaBuffer-ID
180 mMPIPES1
21 mMEGTA1
31 mMMagnesium chlorideMgCl21
41 mMGuanosine triphosphateGTP1
SpecimenConc.: 1.6 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid type: C-flat-2/2
VitrificationInstrument: LEICA EM GP / Cryogen name: ETHANE / Humidity: 95 % / Chamber temperature: 303 K
Details: The sample was incubated on the grid for 10 minutes within the Leica chamber to allow for tubulin polymerisation.

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 81000 X / Nominal defocus max: 2100 nm / Nominal defocus min: 600 nm / C2 aperture diameter: 70 µm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 3.4 sec. / Electron dose: 50 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 6200

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Processing

EM software
IDNameVersionCategoryDetails
1crYOLOv1.7.6.1particle selection
2EPUimage acquisition
4CTFFIND4CTF correction
7ISOLDEmodel fitting
12RELION3.13D reconstructionAutoRefine
13PHENIXmodel refinementreal_space_refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 192000
3D reconstructionResolution: 3.6 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 274847
Details: These are pseudo-particles following the symmetry expansion of ~29,000 particles
Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL
Details: AlphaFoldv2_multimer generated the. model for one tubulin dimer. This was docked into the density within ChimeraX. Molecular dynamics force field-based model fitting was carried out by ...Details: AlphaFoldv2_multimer generated the. model for one tubulin dimer. This was docked into the density within ChimeraX. Molecular dynamics force field-based model fitting was carried out by ISOLDE using unsharpened and deepEMhancer processed density. The model was refined through iterations of ISOLDE and Phenix real_space_refinement softwares.
Atomic model buildingDetails: AlphaFoldv2_multimer generated the dimer model / Source name: AlphaFold / Type: in silico model

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