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- PDB-7miz: Atomic structure of cortical microtubule from Toxoplasma gondii -

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Basic information

Entry
Database: PDB / ID: 7miz
TitleAtomic structure of cortical microtubule from Toxoplasma gondii
Components
  • (PDI family protein) x 2
  • Microtubule associated protein SPM1
  • Tubulin alpha chain
  • Tubulin beta chain
KeywordsSTRUCTURAL PROTEIN / cortical / parasite
Function / homology
Function and homology information


microtubule-based process / Hydrolases; Acting on acid anhydrides; Acting on GTP to facilitate cellular and subcellular movement / structural constituent of cytoskeleton / microtubule cytoskeleton organization / mitotic cell cycle / microtubule / hydrolase activity / GTPase activity / GTP binding / metal ion binding / cytoplasm
Similarity search - Function
Stabilizer of axonemal microtubules 1/2 / Thioredoxin-like / Thioredoxin-like fold / Alpha tubulin / Tubulin-beta mRNA autoregulation signal. / Beta tubulin, autoregulation binding site / Beta tubulin / Tubulin / Tubulin, C-terminal / Tubulin C-terminal domain ...Stabilizer of axonemal microtubules 1/2 / Thioredoxin-like / Thioredoxin-like fold / 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 / Thioredoxin-like superfamily
Similarity search - Domain/homology
GUANOSINE-5'-DIPHOSPHATE / GUANOSINE-5'-TRIPHOSPHATE / PDI family protein / Microtubule associated protein SPM1 / Tubulin beta chain / Tubulin alpha chain / PDI family protein
Similarity search - Component
Biological speciesToxoplasma gondii (eukaryote)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.4 Å
AuthorsWang, X. / Brown, A. / Sibley, L.D. / Zhang, R.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)AI034036 United States
CitationJournal: Nat Commun / Year: 2021
Title: Cryo-EM structure of cortical microtubules from human parasite Toxoplasma gondii identifies their microtubule inner proteins.
Authors: Xiangli Wang / Yong Fu / Wandy L Beatty / Meisheng Ma / Alan Brown / L David Sibley / Rui Zhang /
Abstract: In living cells, microtubules (MTs) play pleiotropic roles, which require very different mechanical properties. Unlike the dynamic MTs found in the cytoplasm of metazoan cells, the specialized ...In living cells, microtubules (MTs) play pleiotropic roles, which require very different mechanical properties. Unlike the dynamic MTs found in the cytoplasm of metazoan cells, the specialized cortical MTs from Toxoplasma gondii, a prevalent human pathogen, are extraordinarily stable and resistant to detergent and cold treatments. Using single-particle cryo-EM, we determine their ex vivo structure and identify three proteins (TrxL1, TrxL2 and SPM1) as bona fide microtubule inner proteins (MIPs). These three MIPs form a mesh on the luminal surface and simultaneously stabilize the tubulin lattice in both longitudinal and lateral directions. Consistent with previous observations, deletion of the identified MIPs compromises MT stability and integrity under challenges by chemical treatments. We also visualize a small molecule like density at the Taxol-binding site of β-tubulin. Our results provide the structural basis to understand the stability of cortical MTs and suggest an evolutionarily conserved mechanism of MT stabilization from the inside.
History
DepositionApr 18, 2021Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jun 2, 2021Provider: repository / Type: Initial release
Revision 1.1Jun 9, 2021Group: Database references / Category: citation / citation_author
Item: _citation.page_first / _citation.page_last ..._citation.page_first / _citation.page_last / _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID / _citation_author.name

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Structure visualization

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Structure viewerMolecule:
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Assembly

Deposited unit
0: Microtubule associated protein SPM1
1: Microtubule associated protein SPM1
2: Microtubule associated protein SPM1
3: Microtubule associated protein SPM1
4: Microtubule associated protein SPM1
5: Microtubule associated protein SPM1
6: Microtubule associated protein SPM1
7: Microtubule associated protein SPM1
8: Microtubule associated protein SPM1
9: Microtubule associated protein SPM1
10: Microtubule associated protein SPM1
11: Microtubule associated protein SPM1
12: Microtubule associated protein SPM1
13: Microtubule associated protein SPM1
14: Microtubule associated protein SPM1
15: Microtubule associated protein SPM1
16: Microtubule associated protein SPM1
17: Microtubule associated protein SPM1
18: Microtubule associated protein SPM1
19: Microtubule associated protein SPM1
20: Microtubule associated protein SPM1
21: Microtubule associated protein SPM1
A0: Tubulin alpha chain
A1: Tubulin beta chain
A2: Tubulin alpha chain
A3: Tubulin beta chain
A4: Tubulin alpha chain
A5: Tubulin beta chain
A6: Tubulin alpha chain
A7: Tubulin beta chain
A8: Tubulin alpha chain
A9: Tubulin beta chain
B0: Tubulin alpha chain
B1: Tubulin beta chain
B2: Tubulin alpha chain
B3: Tubulin beta chain
B4: Tubulin alpha chain
B5: Tubulin beta chain
B6: Tubulin alpha chain
B7: Tubulin beta chain
B8: Tubulin alpha chain
B9: Tubulin beta chain
C0: Tubulin alpha chain
C1: Tubulin beta chain
C2: Tubulin alpha chain
C3: Tubulin beta chain
C4: Tubulin alpha chain
C5: Tubulin beta chain
C6: Tubulin alpha chain
C7: Tubulin beta chain
C8: Tubulin alpha chain
C9: Tubulin beta chain
D0: Tubulin alpha chain
D1: Tubulin beta chain
D2: Tubulin alpha chain
D3: Tubulin beta chain
D4: Tubulin alpha chain
D5: Tubulin beta chain
D6: Tubulin alpha chain
D7: Tubulin beta chain
D8: Tubulin alpha chain
D9: Tubulin beta chain
E0: Tubulin alpha chain
E1: Tubulin beta chain
E2: Tubulin alpha chain
E3: Tubulin beta chain
E4: Tubulin alpha chain
E5: Tubulin beta chain
E6: Tubulin alpha chain
E7: Tubulin beta chain
E8: Tubulin alpha chain
E9: Tubulin beta chain
F0: Tubulin alpha chain
F1: Tubulin beta chain
a: PDI family protein
b: PDI family protein
c: PDI family protein
d: PDI family protein
e: PDI family protein
f: PDI family protein
g: PDI family protein
h: PDI family protein
i: PDI family protein
j: PDI family protein
k: PDI family protein
l: PDI family protein
o: PDI family protein
p: PDI family protein
q: PDI family protein
r: PDI family protein
s: PDI family protein
t: PDI family protein
u: PDI family protein
v: PDI family protein
w: PDI family protein
x: PDI family protein
22: Microtubule associated protein SPM1
23: Microtubule associated protein SPM1
n: PDI family protein
m: PDI family protein
hetero molecules


Theoretical massNumber of molelcules
Total (without water)4,151,024178
Polymers4,125,266100
Non-polymers25,75878
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: microscopy
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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Protein , 5 types, 100 molecules 01234567891011121314151617181920212223A0A2A4A6A8B0...

#1: Protein ...
Microtubule associated protein SPM1


Mass: 38845.750 Da / Num. of mol.: 24 / Source method: isolated from a natural source / Source: (natural) Toxoplasma gondii (eukaryote) / References: UniProt: A0A7J6K285
#2: Protein ...
Tubulin alpha chain / Alpha-tubulin


Mass: 50166.645 Da / Num. of mol.: 26 / Source method: isolated from a natural source / Source: (natural) Toxoplasma gondii (eukaryote) / References: UniProt: P10873
#3: Protein ...
Tubulin beta chain


Mass: 50119.121 Da / Num. of mol.: 26 / Source method: isolated from a natural source / Source: (natural) Toxoplasma gondii (eukaryote) / References: UniProt: I7BFC9
#4: Protein
PDI family protein / Putative PDI-like protein


Mass: 24939.332 Da / Num. of mol.: 20 / Source method: isolated from a natural source / Source: (natural) Toxoplasma gondii (eukaryote) / References: UniProt: Q8MPF4
#5: Protein
PDI family protein / TrxL2


Mass: 21687.934 Da / Num. of mol.: 4 / Source method: isolated from a natural source / Source: (natural) Toxoplasma gondii (eukaryote) / References: UniProt: A0A7J6K232

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Non-polymers , 3 types, 78 molecules

#6: Chemical...
ChemComp-GTP / GUANOSINE-5'-TRIPHOSPHATE


Mass: 523.180 Da / Num. of mol.: 26 / Source method: obtained synthetically / Formula: C10H16N5O14P3 / Comment: GTP, energy-carrying molecule*YM
#7: Chemical...
ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 26 / Source method: obtained synthetically / Formula: Mg
#8: Chemical...
ChemComp-GDP / GUANOSINE-5'-DIPHOSPHATE


Type: RNA linking / Mass: 443.201 Da / Num. of mol.: 26 / Source method: obtained synthetically / Formula: C10H15N5O11P2 / Comment: GDP, energy-carrying molecule*YM

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Details

Has ligand of interestN

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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: cortical microtubule with associated MIPs / Type: COMPLEX / Entity ID: #1-#5 / Source: NATURAL
Molecular weightExperimental value: NO
Source (natural)Organism: Toxoplasma gondii (eukaryote)
Buffer solutionpH: 7.4
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R2/2
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 95 % / Chamber temperature: 289 K / Details: blot for 4 seconds with a blot force of -15

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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: 105000 X / Nominal defocus max: 3500 nm / Calibrated defocus min: 1000 nm / Cs: 0.01 mm / Alignment procedure: ZEMLIN TABLEAU
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 9 sec. / Electron dose: 63.7 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Num. of grids imaged: 2 / Num. of real images: 9231
Details: Images were collected in counting mode, with an exposure rate of 8.5 electrons/pixel/s on the detector camera.
Image scansWidth: 3838 / Height: 3710

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Processing

EM software
IDNameVersionCategory
1Appion3.2particle selection
2EPU1.9.0.30RELimage acquisition
4Gctf1.18CTF correction
5RELION3.1CTF correction
6cryoSPARC2.15CTF correction
9Coot0.8model fitting
11PHENIX1.18.2-387464model refinement
12EMAN1.9initial Euler assignment
13RELION3.1final Euler assignment
14RELION3.1classification
15RELION3.13D reconstruction
16cryoSPARC2.153D reconstruction
CTF correctionDetails: CTF amplitude correction was performed as part of the 3D reconstruction.
Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 536132
Details: Cortical MTs were manually picked from the motion-corrected micrographs using the APPION image-processing suite. The selected MT segments were computationally cut into overlapping boxes ...Details: Cortical MTs were manually picked from the motion-corrected micrographs using the APPION image-processing suite. The selected MT segments were computationally cut into overlapping boxes (512x512 pixels) with an 8-nm non-overlapping region (step size) between adjacent boxes (corresponding to the length of a tubulin heterodimer). We refer to these boxed images as 8-nm MT particles.
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.4 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 220139 / Algorithm: FOURIER SPACE / Symmetry type: POINT
Atomic model buildingProtocol: AB INITIO MODEL
Details: Secondary structure, Ramachandran, and rotamer restraints were applied during refinement. The nonbonded_weight value was set to 500 to reduce the clashes between atoms.
Atomic model building
IDPDB-ID 3D fitting-ID
14FYU

4fyu

1
26U42

6u42

1
33JAS

3jas

1

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