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- PDB-9c6r: Blood cell-specific tubulin in complex with Cryptophycin-52 -

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Basic information

Entry
Database: PDB / ID: 9c6r
TitleBlood cell-specific tubulin in complex with Cryptophycin-52
Components
  • Detyrosinated tubulin alpha-1A chain
  • Tubulin beta-6 chain
KeywordsCELL CYCLE / Tubulin Beta-6 chain / TBB6_CHICK / TUBB1 / Ring C8 / Hematopoietic isotype Cryptophycin / Anticancer / GTPase / Cytoskeleton
Function / homology
Function and homology information


Intraflagellar transport / Kinesins / COPI-dependent Golgi-to-ER retrograde traffic / Aggrephagy / Resolution of Sister Chromatid Cohesion / EML4 and NUDC in mitotic spindle formation / Separation of Sister Chromatids / HSP90 chaperone cycle for steroid hormone receptors (SHR) in the presence of ligand / COPI-independent Golgi-to-ER retrograde traffic / COPI-mediated anterograde transport ...Intraflagellar transport / Kinesins / COPI-dependent Golgi-to-ER retrograde traffic / Aggrephagy / Resolution of Sister Chromatid Cohesion / EML4 and NUDC in mitotic spindle formation / Separation of Sister Chromatids / HSP90 chaperone cycle for steroid hormone receptors (SHR) in the presence of ligand / COPI-independent Golgi-to-ER retrograde traffic / COPI-mediated anterograde transport / structural constituent of cytoskeleton / microtubule cytoskeleton organization / mitotic cell cycle / Hydrolases; Acting on acid anhydrides; Acting on GTP to facilitate cellular and subcellular movement / microtubule / hydrolase activity / GTPase activity / GTP binding / metal ion binding / cytoplasm
Similarity search - Function
Tubulin-beta mRNA autoregulation signal. / Alpha tubulin / 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-beta mRNA autoregulation signal. / Alpha tubulin / 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 / Cryptophycin 52 / Tubulin alpha-1A chain / Tubulin beta-6 chain
Similarity search - Component
Biological speciesGallus gallus (chicken)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.2 Å
AuthorsMontecinos, F.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIH/NIAMS)Intramural Research United States
CitationJournal: J Biol Chem / Year: 2025
Title: Structure of blood cell-specific tubulin and demonstration of dimer spacing compaction in a single protofilament.
Authors: Felipe Montecinos / Elif Eren / Norman R Watts / Dan L Sackett / Paul T Wingfield /
Abstract: Microtubule (MT) function plasticity originates from its composition of α- and β-tubulin isotypes and the posttranslational modifications of both subunits. Aspects such as MT assembly dynamics, ...Microtubule (MT) function plasticity originates from its composition of α- and β-tubulin isotypes and the posttranslational modifications of both subunits. Aspects such as MT assembly dynamics, structure, and anticancer drug binding can be modulated by αβ-tubulin heterogeneity. However, the exact molecular mechanism regulating these aspects is only partially understood. A recent insight is the discovery of expansion and compaction of the MT lattice, which can occur via fine modulation of dimer longitudinal spacing mediated by GTP hydrolysis, taxol binding, protein binding, or isotype composition. Here, we report the first structure of the blood cell-specific α1/β1-tubulin isolated from the marginal band of chicken erythrocytes (ChET) determined to a resolution of 3.2 Å by cryo-EM. We show that ChET rings induced with cryptophycin-52 (Cp-52) are smaller in diameter than HeLa cell line tubulin (HeLaT) rings induced with Cp-52 and composed of the same number of heterodimers. We observe compacted interdimer and intradimer curved protofilament interfaces, characterized by shorter distances between ChET subunits and accompanied by conformational changes in the β-tubulin subunit. The compacted ChET interdimer interface brings more residues near the Cp-52 binding site. We measured the Cp-52 apparent binding affinities of ChET and HeLaT by mass photometry, observing small differences, and detected the intermediates of the ring assembly reaction. These findings demonstrate that compaction/expansion of dimer spacing can occur in a single protofilament context and that the subtle structural differences between tubulin isotypes can modulate tubulin small molecule binding.
History
DepositionJun 8, 2024Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jan 1, 2025Provider: repository / Type: Initial release
Revision 1.1Jan 8, 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.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _em_admin.last_update
Revision 1.2Jan 29, 2025Group: Data collection / Database references / Category: citation / em_admin / Item: _citation.journal_volume / _em_admin.last_update

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Detyrosinated tubulin alpha-1A chain
B: Tubulin beta-6 chain
F: Detyrosinated tubulin alpha-1A chain
G: Tubulin beta-6 chain
K: Detyrosinated tubulin alpha-1A chain
L: Tubulin beta-6 chain
P: Detyrosinated tubulin alpha-1A chain
Q: Tubulin beta-6 chain
U: Detyrosinated tubulin alpha-1A chain
V: Tubulin beta-6 chain
Z: Detyrosinated tubulin alpha-1A chain
a: Tubulin beta-6 chain
e: Detyrosinated tubulin alpha-1A chain
f: Tubulin beta-6 chain
j: Detyrosinated tubulin alpha-1A chain
k: Tubulin beta-6 chain
hetero molecules


Theoretical massNumber of molelcules
Total (without water)816,20440
Polymers803,11916
Non-polymers13,08524
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
Detyrosinated tubulin alpha-1A chain


Mass: 50188.441 Da / Num. of mol.: 8 / Source method: isolated from a natural source
Details: Tubulin alpha-1A chain TUBA1A,P02552,TBA1A_CHICK, NP_001292201.2
Source: (natural) Gallus gallus (chicken) / Plasmid details: Washed red blood cells / Tissue: Blood / References: UniProt: P02552
#2: Protein
Tubulin beta-6 chain / Beta-tubulin class-VI


Mass: 50201.469 Da / Num. of mol.: 8 / Source method: isolated from a natural source
Details: Tubulin beta-6 chain, P09207, TBB6_CHICK, TUBB1, Hematopoietic beta-tubulin, Beta-tubulin class-VI
Source: (natural) Gallus gallus (chicken) / Plasmid details: Washed red blood cells / Tissue: Blood / References: UniProt: P09207
#3: Chemical
ChemComp-GTP / GUANOSINE-5'-TRIPHOSPHATE


Mass: 523.180 Da / Num. of mol.: 8 / Source method: obtained synthetically / Formula: C10H16N5O14P3 / Feature type: SUBJECT OF INVESTIGATION / Comment: GTP, energy-carrying molecule*YM
#4: Chemical
ChemComp-YGY / Cryptophycin 52 / (3S,10S,13Z,16S)-10-[(3-chloro-4-methoxyphenyl)methyl]-6,6-dimethyl-3-(2-methylpropyl)-16-{(1S)-1-[(2R,3R)-3-phenyloxiran-2-yl]ethyl}-1,4-dioxa-8,11-diazacyclohexadec-13-ene-2,5,9,12-tetrone


Mass: 669.204 Da / Num. of mol.: 8 / Source method: obtained synthetically / Formula: C36H45ClN2O8 / Feature type: SUBJECT OF INVESTIGATION
#5: Chemical
ChemComp-GDP / GUANOSINE-5'-DIPHOSPHATE


Type: RNA linking / Mass: 443.201 Da / Num. of mol.: 8 / 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: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: Chicken erythrocytes tubulin in complex with cryptophycin-52
Type: COMPLEX / Entity ID: #1-#2 / Source: NATURAL
Molecular weightValue: 0.734 MDa / Experimental value: NO
Source (natural)Organism: Gallus gallus (chicken) / Cellular location: Cytoskeleton / Organelle: Marginal band / Tissue: Blood
Buffer solutionpH: 7 / Details: 0.1 M PIPES-KOH pH=7.0,1mM MgCL2
SpecimenConc.: 0.5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 400 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: LEICA EM GP / Cryogen name: ETHANE / Humidity: 95 % / Chamber temperature: 277 K

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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: 1800 nm / Nominal defocus min: 800 nm / Cs: 2.7 mm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 2.88 sec. / Electron dose: 50 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)
EM imaging opticsEnergyfilter name: GIF Bioquantum / Energyfilter slit width: 10 eV

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Processing

EM software
IDNameVersionCategory
12cryoSPARC4.3.13D reconstruction
13PHENIXmodel refinement
14Cootmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C8 (8 fold cyclic)
3D reconstructionResolution: 3.2 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 70192 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00556616
ELECTRON MICROSCOPYf_angle_d0.80576920
ELECTRON MICROSCOPYf_dihedral_angle_d10.2967840
ELECTRON MICROSCOPYf_chiral_restr0.0548408
ELECTRON MICROSCOPYf_plane_restr0.00610016

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