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- PDB-7zcw: Cryo-EM structure of GMPCPP-microtubules in complex with VASH2-SVBP -

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Basic information

Entry
Database: PDB / ID: 7zcw
TitleCryo-EM structure of GMPCPP-microtubules in complex with VASH2-SVBP
Components
  • Small vasohibin-binding protein
  • Tubulin alpha-1B chain
  • Tubulin beta-2B chain
  • Tubulinyl-Tyr carboxypeptidase 2
KeywordsPROTEIN BINDING / Microtubule / Enzyme / Complex / Detyrosination
Function / homology
Function and homology information


cell-cell fusion / regulation of metallopeptidase activity / tubulinyl-Tyr carboxypeptidase / tubulin-tyrosine carboxypeptidase / syncytium formation by plasma membrane fusion / Post-chaperonin tubulin folding pathway / Cilium Assembly / cytoskeleton-dependent intracellular transport / Microtubule-dependent trafficking of connexons from Golgi to the plasma membrane / Carboxyterminal post-translational modifications of tubulin ...cell-cell fusion / regulation of metallopeptidase activity / tubulinyl-Tyr carboxypeptidase / tubulin-tyrosine carboxypeptidase / syncytium formation by plasma membrane fusion / Post-chaperonin tubulin folding pathway / Cilium Assembly / cytoskeleton-dependent intracellular transport / Microtubule-dependent trafficking of connexons from Golgi to the plasma membrane / Carboxyterminal post-translational modifications of tubulin / Intraflagellar transport / Sealing of the nuclear envelope (NE) by ESCRT-III / Formation of tubulin folding intermediates by CCT/TriC / embryonic brain development / Gap junction assembly / Prefoldin mediated transfer of substrate to CCT/TriC / Kinesins / positive regulation of axon guidance / COPI-independent Golgi-to-ER retrograde traffic / Assembly and cell surface presentation of NMDA receptors / negative regulation of endothelial cell migration / labyrinthine layer blood vessel development / peptidase activator activity / COPI-dependent Golgi-to-ER retrograde traffic / axon development / Recycling pathway of L1 / protein secretion / RHOH GTPase cycle / regulation of angiogenesis / RHO GTPases activate IQGAPs / microtubule-based process / Hedgehog 'off' state / intercellular bridge / COPI-mediated anterograde transport / Activation of AMPK downstream of NMDARs / cytoplasmic microtubule / metallocarboxypeptidase activity / positive regulation of endothelial cell proliferation / negative regulation of protein ubiquitination / Mitotic Prometaphase / EML4 and NUDC in mitotic spindle formation / MHC class II antigen presentation / cellular response to interleukin-4 / Recruitment of NuMA to mitotic centrosomes / HSP90 chaperone cycle for steroid hormone receptors (SHR) in the presence of ligand / Resolution of Sister Chromatid Cohesion / Translocation of SLC2A4 (GLUT4) to the plasma membrane / RHO GTPases Activate Formins / cerebral cortex development / PKR-mediated signaling / modulation of chemical synaptic transmission / structural constituent of cytoskeleton / microtubule cytoskeleton organization / Schaffer collateral - CA1 synapse / neuron migration / positive regulation of angiogenesis / apical part of cell / HCMV Early Events / Aggrephagy / The role of GTSE1 in G2/M progression after G2 checkpoint / mitotic spindle / Separation of Sister Chromatids / mitotic cell cycle / double-stranded RNA binding / actin binding / microtubule cytoskeleton / microtubule binding / Hydrolases; Acting on acid anhydrides; Acting on GTP to facilitate cellular and subcellular movement / microtubule / cytoskeleton / cilium / protein heterodimerization activity / cell division / GTPase activity / ubiquitin protein ligase binding / GTP binding / structural molecule activity / proteolysis / extracellular region / metal ion binding / nucleus / cytoplasm / cytosol
Similarity search - Function
Tubulinyl-Tyr carboxypeptidase / Small vasohibin-binding protein / Vasohibin / Coiled-coil domain-containing protein 23 / Helix hairpin bin / Tubulin/FtsZ, C-terminal domain / Tubulin/FtsZ, GTPase domain / 60s Ribosomal Protein L30; Chain: A; / Alpha tubulin / Tubulin-beta mRNA autoregulation signal. ...Tubulinyl-Tyr carboxypeptidase / Small vasohibin-binding protein / Vasohibin / Coiled-coil domain-containing protein 23 / Helix hairpin bin / Tubulin/FtsZ, C-terminal domain / Tubulin/FtsZ, GTPase domain / 60s Ribosomal Protein L30; Chain: A; / 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 / Helix Hairpins / Rossmann fold / 2-Layer Sandwich / Orthogonal Bundle / 3-Layer(aba) Sandwich / Mainly Alpha / Alpha Beta
Similarity search - Domain/homology
PHOSPHOMETHYLPHOSPHONIC ACID GUANYLATE ESTER / GUANOSINE-5'-TRIPHOSPHATE / Tubulin alpha-1B chain / Tubulinyl-Tyr carboxypeptidase 2 / Small vasohibin-binding protein / Tubulin beta-2B chain
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.6 Å
AuthorsChoi, S.R. / Blum, T. / Steinmetz, M.O.
Funding support Switzerland, 1items
OrganizationGrant numberCountry
Swiss National Science Foundation310030_192566 Switzerland
CitationJournal: J Cell Biol / Year: 2023
Title: VASH1-SVBP and VASH2-SVBP generate different detyrosination profiles on microtubules.
Authors: Sacnicte Ramirez-Rios / Sung Ryul Choi / Chadni Sanyal / Thorsten B Blum / Christophe Bosc / Fatma Krichen / Eric Denarier / Jean-Marc Soleilhac / Béatrice Blot / Carsten Janke / Virginie ...Authors: Sacnicte Ramirez-Rios / Sung Ryul Choi / Chadni Sanyal / Thorsten B Blum / Christophe Bosc / Fatma Krichen / Eric Denarier / Jean-Marc Soleilhac / Béatrice Blot / Carsten Janke / Virginie Stoppin-Mellet / Maria M Magiera / Isabelle Arnal / Michel O Steinmetz / Marie-Jo Moutin /
Abstract: The detyrosination/tyrosination cycle of α-tubulin is critical for proper cell functioning. VASH1-SVBP and VASH2-SVBP are ubiquitous enzymes involved in microtubule detyrosination, whose mode of ...The detyrosination/tyrosination cycle of α-tubulin is critical for proper cell functioning. VASH1-SVBP and VASH2-SVBP are ubiquitous enzymes involved in microtubule detyrosination, whose mode of action is little known. Here, we show in reconstituted systems and cells that VASH1-SVBP and VASH2-SVBP drive the global and local detyrosination of microtubules, respectively. We solved the cryo-electron microscopy structure of VASH2-SVBP bound to microtubules, revealing a different microtubule-binding configuration of its central catalytic region compared to VASH1-SVBP. We show that the divergent mode of detyrosination between the two enzymes is correlated with the microtubule-binding properties of their disordered N- and C-terminal regions. Specifically, the N-terminal region is responsible for a significantly longer residence time of VASH2-SVBP on microtubules compared to VASH1-SVBP. We suggest that this VASH region is critical for microtubule detachment and diffusion of VASH-SVBP enzymes on lattices. Our results suggest a mechanism by which VASH1-SVBP and VASH2-SVBP could generate distinct microtubule subpopulations and confined areas of detyrosinated lattices to drive various microtubule-based cellular functions.
History
DepositionMar 29, 2022Deposition site: PDBE / Processing site: PDBE
Revision 1.0Dec 14, 2022Provider: repository / Type: Initial release
Revision 1.1Jun 28, 2023Group: Database references / Category: citation / citation_author
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.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year
Revision 1.2Jul 24, 2024Group: Data collection / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / em_3d_fitting_list / em_admin
Item: _em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id ..._em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id / _em_3d_fitting_list.source_name / _em_3d_fitting_list.type / _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 alpha-1B chain
B: Tubulin beta-2B chain
C: Tubulinyl-Tyr carboxypeptidase 2
D: Small vasohibin-binding protein
E: Tubulin alpha-1B chain
F: Tubulin beta-2B chain
G: Tubulin beta-2B chain
H: Tubulin beta-2B chain
hetero molecules


Theoretical massNumber of molelcules
Total (without water)352,58620
Polymers349,3098
Non-polymers3,27712
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: immunoprecipitation, Results of microtubule co-pelleting assays with VASH2-SVBP indicate an interaction with the core microtubule lattice.
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area25060 Å2
ΔGint-158 kcal/mol
Surface area102810 Å2

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Components

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Protein , 4 types, 8 molecules AEBFGHCD

#1: Protein Tubulin alpha-1B chain / Alpha-tubulin ubiquitous / Tubulin K-alpha-1 / Tubulin alpha-ubiquitous chain


Mass: 50204.445 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) Homo sapiens (human) / Cell line: Hela / References: UniProt: P68363
#2: Protein
Tubulin beta-2B chain


Mass: 49907.770 Da / Num. of mol.: 4 / Source method: isolated from a natural source / Source: (natural) Homo sapiens (human) / Cell line: HELA / References: UniProt: Q9BVA1
#3: Protein Tubulinyl-Tyr carboxypeptidase 2 / Vasohibin-2 / Vasohibin-like protein


Mass: 40488.914 Da / Num. of mol.: 1 / Mutation: C158A
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: VASH2, VASHL / Production host: Escherichia coli (E. coli) / References: UniProt: Q86V25, tubulinyl-Tyr carboxypeptidase
#4: Protein Small vasohibin-binding protein / Coiled coil domain-containing protein 23


Mass: 8780.000 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: Recombinant C-terminal his6-tag / Source: (gene. exp.) Homo sapiens (human) / Gene: SVBP, CCDC23 / Production host: Escherichia coli (E. coli) / References: UniProt: Q8N300

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Non-polymers , 3 types, 12 molecules

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


Mass: 523.180 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C10H16N5O14P3 / Comment: GTP, energy-carrying molecule*YM
#6: Chemical
ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 6 / Source method: obtained synthetically / Formula: Mg
#7: Chemical
ChemComp-G2P / PHOSPHOMETHYLPHOSPHONIC ACID GUANYLATE ESTER


Mass: 521.208 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C11H18N5O13P3 / Comment: GMP-CPP, energy-carrying molecule analogue*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: HELICAL ARRAY / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: VASH2-SVBP complex bound to the microtubule / Type: COMPLEX / Entity ID: #1-#4 / Source: RECOMBINANT
Molecular weightValue: 0.38 MDa / Experimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7.5
Buffer component
IDConc.NameFormulaBuffer-ID
1150 mMSodium ChlorideNaCl1
220 mMTris bufferC4H11NO31
31 mMDichlorodiphenyltrichloroethaneC14H9Cl51
SpecimenConc.: 2 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: Mixture of VASH2-SVBP and microtubules
Specimen supportGrid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R2/1
VitrificationInstrument: FEI VITROBOT MARK III / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 298 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 defocus max: 2200 nm / Nominal defocus min: 600 nm
Image recordingAverage exposure time: 0.2 sec. / Electron dose: 1.4 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 3345

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Processing

EM software
IDNameVersionCategory
1RELION3.0.8particle selection
2SerialEMimage acquisition
4GctfCTF correction
7UCSF Chimera1.16model fitting
8Coot0.9.7model fitting
10PHENIX1.20.1-4487model refinement
11Coot0.9.7model refinement
12RELION3.0.8initial Euler assignment
13RELION3.0.8final Euler assignment
15RELION3.0.83D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 153041
3D reconstructionResolution: 3.6 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 96922 / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingB value: 54.76 / Protocol: RIGID BODY FIT
Atomic model building

3D fitting-ID: 1 / Source name: PDB / Type: experimental model

IDPDB-IDPdb chain-IDAccession codeInitial refinement model-ID
16QBY

6qby

6QBY1
23JAL

3jal

H3JAL2
33JAL

3jal

G3JAL2
43JAL

3jal

A3JAL2
53JAL

3jal

E3JAL2
63JAL

3jal

B3JAL2
73JAL

3jal

F3JAL2

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