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- PDB-9hxc: CryoEM structure of Asgard AtubA/B2 microtubule -

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Basic information

Entry
Database: PDB / ID: 9hxc
TitleCryoEM structure of Asgard AtubA/B2 microtubule
Components
  • Asgard tubulin AtubA with residues from TEV protease cleavage site
  • Asgard tubulin AtubB2
KeywordsSTRUCTURAL PROTEIN / Asgard archaea / microtubule / cryoEM / cytomotive filaments / cytoskeleton
Function / homologyGUANOSINE-5'-DIPHOSPHATE
Function and homology information
Biological speciesCandidatus Lokiarchaeum ossiferum (archaea)
MethodELECTRON MICROSCOPY / helical reconstruction / cryo EM / Resolution: 3.3 Å
AuthorsWollweber, F. / Xu, J. / Ponce-Toledo, R.I. / Rodrigues-Oliveira, T. / Malit, J.J.L. / Kokhanovska, A. / Wieczorek, M. / Schleper, C. / Pilhofer, M.
Funding supportEuropean Union, 1items
OrganizationGrant numberCountry
European Research Council (ERC)101000232European Union
CitationJournal: Cell / Year: 2025
Title: Microtubules in Asgard archaea.
Authors: Florian Wollweber / Jingwei Xu / Rafael I Ponce-Toledo / Florina Marxer / Thiago Rodrigues-Oliveira / Anja Pössnecker / Zhen-Hao Luo / Jessie James Limlingan Malit / Anastasiia Kokhanovska ...Authors: Florian Wollweber / Jingwei Xu / Rafael I Ponce-Toledo / Florina Marxer / Thiago Rodrigues-Oliveira / Anja Pössnecker / Zhen-Hao Luo / Jessie James Limlingan Malit / Anastasiia Kokhanovska / Michal Wieczorek / Christa Schleper / Martin Pilhofer /
Abstract: Microtubules are a hallmark of eukaryotes. Archaeal and bacterial homologs of tubulins typically form homopolymers and non-tubular superstructures. The origin of heterodimeric tubulins assembling ...Microtubules are a hallmark of eukaryotes. Archaeal and bacterial homologs of tubulins typically form homopolymers and non-tubular superstructures. The origin of heterodimeric tubulins assembling into microtubules remains unclear. Here, we report the discovery of microtubule-forming tubulins in Asgard archaea, the closest known relatives of eukaryotes. These Asgard tubulins (AtubA/B) are closely related to eukaryotic α/β-tubulins and the enigmatic bacterial tubulins BtubA/B. Proteomics of Candidatus Lokiarchaeum ossiferum showed that AtubA/B were highly expressed. Cryoelectron microscopy structures demonstrate that AtubA/B form eukaryote-like heterodimers, which assembled into 5-protofilament bona fide microtubules in vitro. The additional paralog AtubB2 lacks a nucleotide-binding site and competitively displaced AtubB. These AtubA/B2 heterodimers polymerized into 7-protofilament non-canonical microtubules. In a sub-population of Ca. Lokiarchaeum ossiferum cells, cryo-tomography revealed tubular structures, while expansion microscopy identified AtubA/B cytoskeletal assemblies. Our findings suggest a pre-eukaryotic origin of microtubules and provide a framework for understanding the fundamental principles of microtubule assembly.
History
DepositionJan 7, 2025Deposition site: PDBE / Processing site: PDBE
Revision 1.0Apr 2, 2025Provider: repository / Type: Initial release
Revision 1.1May 14, 2025Group: Data collection / Database references / Category: citation / em_admin
Item: _citation.journal_volume / _citation.page_first / _em_admin.last_update

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Asgard tubulin AtubA with residues from TEV protease cleavage site
D: Asgard tubulin AtubB2
B: Asgard tubulin AtubA with residues from TEV protease cleavage site
C: Asgard tubulin AtubB2
E: Asgard tubulin AtubA with residues from TEV protease cleavage site
F: Asgard tubulin AtubB2
G: Asgard tubulin AtubA with residues from TEV protease cleavage site
H: Asgard tubulin AtubB2
I: Asgard tubulin AtubA with residues from TEV protease cleavage site
J: Asgard tubulin AtubB2
K: Asgard tubulin AtubA with residues from TEV protease cleavage site
L: Asgard tubulin AtubB2
M: Asgard tubulin AtubA with residues from TEV protease cleavage site
N: Asgard tubulin AtubB2
O: Asgard tubulin AtubA with residues from TEV protease cleavage site
P: Asgard tubulin AtubB2
Q: Asgard tubulin AtubA with residues from TEV protease cleavage site
R: Asgard tubulin AtubB2
S: Asgard tubulin AtubA with residues from TEV protease cleavage site
T: Asgard tubulin AtubB2
U: Asgard tubulin AtubA with residues from TEV protease cleavage site
V: Asgard tubulin AtubB2
W: Asgard tubulin AtubA with residues from TEV protease cleavage site
X: Asgard tubulin AtubB2
Y: Asgard tubulin AtubA with residues from TEV protease cleavage site
Z: Asgard tubulin AtubB2
a: Asgard tubulin AtubA with residues from TEV protease cleavage site
b: Asgard tubulin AtubB2
hetero molecules


Theoretical massNumber of molelcules
Total (without water)1,330,46442
Polymers1,324,25928
Non-polymers6,20514
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
Asgard tubulin AtubA with residues from TEV protease cleavage site


Mass: 46706.996 Da / Num. of mol.: 14
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Candidatus Lokiarchaeum ossiferum (archaea)
Production host: Spodoptera frugiperda (fall armyworm)
#2: Protein
Asgard tubulin AtubB2


Mass: 47882.949 Da / Num. of mol.: 14
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Candidatus Lokiarchaeum ossiferum (archaea)
Production host: Spodoptera frugiperda (fall armyworm)
#3: Chemical
ChemComp-GDP / GUANOSINE-5'-DIPHOSPHATE


Type: RNA linking / Mass: 443.201 Da / Num. of mol.: 14 / Source method: obtained synthetically / Formula: C10H15N5O11P2 / Feature type: SUBJECT OF INVESTIGATION / Comment: GDP, 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: helical reconstruction

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Sample preparation

ComponentName: microtubule structure of Asgard tubulins AtubA/B2 / Type: COMPLEX / Entity ID: #1-#2 / Source: RECOMBINANT
Source (natural)Organism: Candidatus Lokiarchaeum ossiferum (archaea)
Source (recombinant)Organism: Spodoptera frugiperda (fall armyworm)
Buffer solutionpH: 6.8
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE-PROPANE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: TFS KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 2800 nm / Nominal defocus min: 1200 nm
Image recordingElectron dose: 60 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Helical symmertyAngular rotation/subunit: 52.49 ° / Axial rise/subunit: 11.73 Å / Axial symmetry: C1
3D reconstructionResolution: 3.3 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 39838 / Symmetry type: HELICAL

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