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- EMDB-45265: 18-mer blood cell-specific tubulin in complex with Cryptophycin-52 -

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Basic information

Entry
Database: EMDB / ID: EMD-45265
Title18-mer blood cell-specific tubulin in complex with Cryptophycin-52
Map dataPost-processed map
Sample
  • Complex: 18-mer Hematopoietic tubulin in complex with cryptophycin-52
    • Protein or peptide: Detyrosinated tubulin alpha-1A chain
    • Protein or peptide: Tubulin beta-6 chain
  • Ligand: GUANOSINE-5'-TRIPHOSPHATE
  • Ligand: GUANOSINE-5'-DIPHOSPHATE
  • Ligand: Cryptophycin 52
KeywordsTBB6_CHICK / Cryptophycin-52 / Hematopoietic / TUBB1 / Beta-6 tubulin chain / Class VI / anticancer / CELL CYCLE
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
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
Tubulin alpha-1A chain / Tubulin beta-6 chain
Similarity search - Component
Biological speciesGallus gallus (chicken)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.52 Å
AuthorsMontecinos F
Funding support United States, 1 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIH/NIAMS)Intramural Research Program 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, 2024-
Header (metadata) releaseJan 1, 2025-
Map releaseJan 1, 2025-
UpdateMay 14, 2025-
Current statusMay 14, 2025Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_45265.png

Downloads & links

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Map

FileDownload / File: emd_45265.map.gz / Format: CCP4 / Size: 1.1 GB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationPost-processed map
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.83 Å/pix.
x 660 pix.
= 547.8 Å		0.83 Å/pix.
x 660 pix.
= 547.8 Å		0.83 Å/pix.
x 660 pix.
= 547.8 Å

Surface

Projections

Slices (1/3)

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Images are generated by Spider.

Voxel sizeX=Y=Z: 0.83 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.05
Minimum - Maximum-0.28439528 - 0.4764942
Average (Standard dev.)0.0005498232 (±0.008084251)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions660660660
Spacing660660660
CellA=B=C: 547.8 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_45265_msk_1.map
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Additional map: locally-sharpened map (DeppEMhancer)

Fileemd_45265_additional_1.map
Annotationlocally-sharpened map (DeppEMhancer)
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Half map: Half-map B

Fileemd_45265_half_map_1.map
AnnotationHalf-map B
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Half map: Half-map A

Fileemd_45265_half_map_2.map
AnnotationHalf-map A
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Sample components

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Entire : 18-mer Hematopoietic tubulin in complex with cryptophycin-52

EntireName: 18-mer Hematopoietic tubulin in complex with cryptophycin-52
Components
  • Complex: 18-mer Hematopoietic tubulin in complex with cryptophycin-52
    • Protein or peptide: Detyrosinated tubulin alpha-1A chain
    • Protein or peptide: Tubulin beta-6 chain
  • Ligand: GUANOSINE-5'-TRIPHOSPHATE
  • Ligand: GUANOSINE-5'-DIPHOSPHATE
  • Ligand: Cryptophycin 52

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Supramolecule #1: 18-mer Hematopoietic tubulin in complex with cryptophycin-52

SupramoleculeName: 18-mer Hematopoietic tubulin in complex with cryptophycin-52
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#2
Source (natural)Organism: Gallus gallus (chicken) / Tissue: Blood / Organelle: Marginal Band / Location in cell: Cytoplasm
Molecular weightTheoretical: 840 KDa

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Macromolecule #1: Detyrosinated tubulin alpha-1A chain

MacromoleculeName: Detyrosinated tubulin alpha-1A chain / type: protein_or_peptide / ID: 1 / Details: Tubulin alpha-1A chain / Number of copies: 9 / Enantiomer: LEVO
Source (natural)Organism: Gallus gallus (chicken) / Tissue: Blood
Molecular weightTheoretical: 50.188441 KDa
SequenceString: MRECISIHVG QAGVQIGNAC WELYCLEHGI QPDGQMPSDK TIGGGDDSFN TFFSETGAGK HVPRAVFVDL EPTVIDEVRT GTYRQLFHP EQLITGKEDA ANNYARGHYT IGKEIIDLVL DRIRKLADQC TGLQGFLVFH SFGGGTGSGF TSLLMERLSV D YGKKSKLE ...String:
MRECISIHVG QAGVQIGNAC WELYCLEHGI QPDGQMPSDK TIGGGDDSFN TFFSETGAGK HVPRAVFVDL EPTVIDEVRT GTYRQLFHP EQLITGKEDA ANNYARGHYT IGKEIIDLVL DRIRKLADQC TGLQGFLVFH SFGGGTGSGF TSLLMERLSV D YGKKSKLE FSIYPAPQVS TAVVEPYNSI LTTHTTLEHS DCAFMVDNEA IYDICRRNLD IERPTYTNLN RLIGQIVSSI TA SLRFDGA LNVDLTEFQT NLVPYPRIHF PLATYAPVIS AEKAYHEQLS VAEITNACFE PANQMVKCDP RHGKYMACCL LYR GDVVPK DVNAAIATIK TKRTIQFVDW CPTGFKVGIN YQPPTVVPGG DLAKVQRAVC MLSNTTAIAE AWARLDHKFD LMYA KRAFV HWYVGEGMEE GEFSEAREDM AALEKDYEEV GVDSVEGEGE EEGEEY

UniProtKB: Tubulin alpha-1A chain

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Macromolecule #2: Tubulin beta-6 chain

MacromoleculeName: Tubulin beta-6 chain / type: protein_or_peptide / ID: 2 / Number of copies: 9 / Enantiomer: LEVO
Source (natural)Organism: Gallus gallus (chicken) / Tissue: Blood
Molecular weightTheoretical: 50.201469 KDa
SequenceString: MREIVHLQIG QCGNQIGAKF WEVISDEHGI DIAGNYCGNA SLQLERINVY FNEAYSHKYV PRSILVDLEP GTMDSVRSSK IGPLFRPDN FIHGNSGAGN NWAKGHYTEG AELIENVMDV VRNECESCDC LQGFQLIHSL GGGTGSGMGT LLINKIREEY P DRIMNTFS ...String:
MREIVHLQIG QCGNQIGAKF WEVISDEHGI DIAGNYCGNA SLQLERINVY FNEAYSHKYV PRSILVDLEP GTMDSVRSSK IGPLFRPDN FIHGNSGAGN NWAKGHYTEG AELIENVMDV VRNECESCDC LQGFQLIHSL GGGTGSGMGT LLINKIREEY P DRIMNTFS VVPSPKVSDT VVEPYNAILS IHQLIENTDE TFCIDNEALY DICFRTLKLT NPTYGDLNHL VSLTMSGVTT SL RFPGQLN ADLRKLAVNM VPFPRLHFFM PGFAPLTARG SQQYRALSVP ELTQQMFDAR NMMAACDPRR GRYLTVACIF RGR MSTREV DEQLLSVQTK NSSYFVEWIP NNVKVAVCDI PPRGLKMAAT FIGNNTAIQE LFIRVSEQFS AMFRRKAFLH WYTG EGMDE MEFSEAEGNT NDLVSEYQQY QDATADVEEY EEAEASPEKE T

UniProtKB: Tubulin beta-6 chain

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Macromolecule #3: GUANOSINE-5'-TRIPHOSPHATE

MacromoleculeName: GUANOSINE-5'-TRIPHOSPHATE / type: ligand / ID: 3 / Number of copies: 9 / Formula: GTP
Molecular weightTheoretical: 523.18 Da
Chemical component information

ChemComp-GTP:
GUANOSINE-5'-TRIPHOSPHATE / GTP, energy-carrying molecule*YM

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Macromolecule #4: GUANOSINE-5'-DIPHOSPHATE

MacromoleculeName: GUANOSINE-5'-DIPHOSPHATE / type: ligand / ID: 4 / Number of copies: 9 / Formula: GDP
Molecular weightTheoretical: 443.201 Da
Chemical component information

ChemComp-GDP:
GUANOSINE-5'-DIPHOSPHATE / GDP, energy-carrying molecule*YM

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Macromolecule #5: Cryptophycin 52

MacromoleculeName: Cryptophycin 52 / type: ligand / ID: 5 / Number of copies: 9 / Formula: YGY
Molecular weightTheoretical: 669.204 Da
Chemical component information

ChemComp-YGY:
Cryptophycin 52

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

BufferpH: 7
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 400 / Support film - Material: CARBON / Support film - topology: CONTINUOUS / Support film - Film thickness: 2 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 15 sec. / Pretreatment - Atmosphere: AIR
VitrificationCryogen name: ETHANE / Chamber humidity: 95 % / Chamber temperature: 277 K

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average exposure time: 2.88 sec. / Average electron dose: 50.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 1.8 µm / Nominal defocus min: 0.8 µm / Nominal magnification: 105000
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Startup modelType of model: INSILICO MODEL
In silico model: model built from the sequence using swiss-model server
Final reconstructionApplied symmetry - Point group: C9 (9 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 3.52 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 64200
Initial angle assignmentType: OTHER
Final angle assignmentType: OTHER
FSC plot (resolution estimation)

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