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- EMDB-14634: Cryo-EM structure of GMPCPP-microtubules in complex with VASH2-SVBP -

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Basic information

Entry
Database: EMDB / ID: EMD-14634
TitleCryo-EM structure of GMPCPP-microtubules in complex with VASH2-SVBP
Map data
Sample
  • Complex: VASH2-SVBP complex bound to the microtubule
    • Protein or peptide: Tubulin alpha-1B chain
    • Protein or peptide: Tubulin beta-2B chain
    • Protein or peptide: Tubulinyl-Tyr carboxypeptidase 2
    • Protein or peptide: Small vasohibin-binding protein
  • Ligand: GUANOSINE-5'-TRIPHOSPHATEGuanosine triphosphate
  • Ligand: MAGNESIUM ION
  • Ligand: PHOSPHOMETHYLPHOSPHONIC ACID GUANYLATE ESTER
KeywordsMicrotubule / Enzyme / Complex / Detyrosination / PROTEIN BINDING
Function / homology
Function and homology information


cell-cell fusion / syncytium formation by plasma membrane fusion / regulation of metallopeptidase activity / tubulinyl-Tyr carboxypeptidase / tubulin-tyrosine carboxypeptidase / Post-chaperonin tubulin folding pathway / Carboxyterminal post-translational modifications of tubulin / Microtubule-dependent trafficking of connexons from Golgi to the plasma membrane / Cilium Assembly / peptidase activator activity ...cell-cell fusion / syncytium formation by plasma membrane fusion / regulation of metallopeptidase activity / tubulinyl-Tyr carboxypeptidase / tubulin-tyrosine carboxypeptidase / Post-chaperonin tubulin folding pathway / Carboxyterminal post-translational modifications of tubulin / Microtubule-dependent trafficking of connexons from Golgi to the plasma membrane / Cilium Assembly / peptidase activator activity / cytoskeleton-dependent intracellular transport / Intraflagellar transport / Sealing of the nuclear envelope (NE) by ESCRT-III / Gap junction assembly / embryonic brain development / Formation of tubulin folding intermediates by CCT/TriC / COPI-independent Golgi-to-ER retrograde traffic / Prefoldin mediated transfer of substrate to CCT/TriC / Kinesins / Assembly and cell surface presentation of NMDA receptors / negative regulation of endothelial cell migration / positive regulation of axon guidance / COPI-dependent Golgi-to-ER retrograde traffic / labyrinthine layer blood vessel development / intercellular bridge / axon development / Recycling pathway of L1 / RHOH GTPase cycle / protein secretion / RHO GTPases activate IQGAPs / Hedgehog 'off' state / regulation of angiogenesis / cytoplasmic microtubule / metallocarboxypeptidase activity / microtubule-based process / COPI-mediated anterograde transport / Activation of AMPK downstream of NMDARs / Mitotic Prometaphase / EML4 and NUDC in mitotic spindle formation / negative regulation of protein ubiquitination / Resolution of Sister Chromatid Cohesion / Recruitment of NuMA to mitotic centrosomes / HSP90 chaperone cycle for steroid hormone receptors (SHR) in the presence of ligand / MHC class II antigen presentation / cellular response to interleukin-4 / positive regulation of endothelial cell proliferation / RHO GTPases Activate Formins / Translocation of SLC2A4 (GLUT4) to the plasma membrane / Hydrolases; Acting on acid anhydrides; Acting on GTP to facilitate cellular and subcellular movement / neuron migration / modulation of chemical synaptic transmission / Schaffer collateral - CA1 synapse / PKR-mediated signaling / structural constituent of cytoskeleton / mitotic spindle / cerebral cortex development / microtubule cytoskeleton organization / Aggrephagy / HCMV Early Events / Separation of Sister Chromatids / positive regulation of angiogenesis / The role of GTSE1 in G2/M progression after G2 checkpoint / microtubule cytoskeleton / double-stranded RNA binding / apical part of cell / mitotic cell cycle / actin binding / microtubule binding / microtubule / cytoskeleton / protein heterodimerization activity / cell division / GTPase activity / ubiquitin protein ligase binding / GTP binding / structural molecule activity / proteolysis / extracellular region / metal ion binding / nucleus / cytosol / cytoplasm
Similarity search - Function
Tubulinyl-Tyr carboxypeptidase / Small vasohibin-binding protein / Vasohibin / Coiled-coil domain-containing protein 23 / Tubulin-beta mRNA autoregulation signal. / Alpha tubulin / Beta tubulin, autoregulation binding site / Beta tubulin / Tubulin / Tubulin, C-terminal ...Tubulinyl-Tyr carboxypeptidase / Small vasohibin-binding protein / Vasohibin / Coiled-coil domain-containing protein 23 / 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
Tubulin alpha-1B chain / Tubulinyl-Tyr carboxypeptidase 2 / Small vasohibin-binding protein / Tubulin beta-2B chain
Similarity search - Component
Biological speciesHomo sapiens (human) / human (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.6 Å
AuthorsChoi SR / Blum T / Steinmetz MO
Funding support Switzerland, 1 items
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, 2022-
Header (metadata) releaseDec 14, 2022-
Map releaseDec 14, 2022-
UpdateJun 28, 2023-
Current statusJun 28, 2023Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_14634.png

Downloads & links

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Map

FileDownload / File: emd_14634.map.gz / Format: CCP4 / Size: 15.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.06 Å/pix.
x 160 pix.
= 169.28 Å		1.06 Å/pix.
x 160 pix.
= 169.28 Å		1.06 Å/pix.
x 160 pix.
= 169.28 Å

Surface

Projections

Slices (1/3)

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Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.058 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.008
Minimum - Maximum-0.059908353 - 0.09423978
Average (Standard dev.)-0.00023937626 (±0.006015211)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions160160160
Spacing160160160
CellA=B=C: 169.28 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #1

Fileemd_14634_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_14634_half_map_2.map
Projections & Slices
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Histogram (log scale)

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Sample components

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Entire : VASH2-SVBP complex bound to the microtubule

EntireName: VASH2-SVBP complex bound to the microtubule
Components
  • Complex: VASH2-SVBP complex bound to the microtubule
    • Protein or peptide: Tubulin alpha-1B chain
    • Protein or peptide: Tubulin beta-2B chain
    • Protein or peptide: Tubulinyl-Tyr carboxypeptidase 2
    • Protein or peptide: Small vasohibin-binding protein
  • Ligand: GUANOSINE-5'-TRIPHOSPHATEGuanosine triphosphate
  • Ligand: MAGNESIUM ION
  • Ligand: PHOSPHOMETHYLPHOSPHONIC ACID GUANYLATE ESTER

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Supramolecule #1: VASH2-SVBP complex bound to the microtubule

SupramoleculeName: VASH2-SVBP complex bound to the microtubule / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#4
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 380 KDa

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Macromolecule #1: Tubulin alpha-1B chain

MacromoleculeName: Tubulin alpha-1B chain / type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: human (human)
Molecular weightTheoretical: 50.204445 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 RLISQIVSSI TA SLRFDGA LNVDLTEFQT NLVPYPRIHF PLATYAPVIS AEKAYHEQLS VAEITNACFE PANQMVKCDP RHGKYMACCL LYR GDVVPK DVNAAIATIK TKRSIQFVDW CPTGFKVGIN YQPPTVVPGG DLAKVQRAVC MLSNTTAIAE AWARLDHKFD LMYA KRAFV HWYVGEGMEE GEFSEAREDM AALEKDYEEV GVDSVEGEGE EEGEEY

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Macromolecule #2: Tubulin beta-2B chain

MacromoleculeName: Tubulin beta-2B chain / type: protein_or_peptide / ID: 2 / Number of copies: 4 / Enantiomer: LEVO
Source (natural)Organism: human (human)
Molecular weightTheoretical: 49.90777 KDa
SequenceString: MREIVHIQAG QCGNQIGAKF WEVISDEHGI DPTGSYHGDS DLQLERINVY YNEAAGNKYV PRAILVDLEP GTMDSVRSGP FGQIFRPDN FVFGQSGAGN NWAKGHYTEG AELVDSVLDV VRKESESCDC LQGFQLTHSL GGGTGSGMGT LLISKIREEY P DRIMNTFS ...String:
MREIVHIQAG QCGNQIGAKF WEVISDEHGI DPTGSYHGDS DLQLERINVY YNEAAGNKYV PRAILVDLEP GTMDSVRSGP FGQIFRPDN FVFGQSGAGN NWAKGHYTEG AELVDSVLDV VRKESESCDC LQGFQLTHSL GGGTGSGMGT LLISKIREEY P DRIMNTFS VVPSPKVSDT VVEPYNATLS VHQLVENTDE TYCIDNEALY DICFRTLKLT TPTYGDLNHL VSATMSGVTT CL RFPGQLN ADLRKLAVNM VPFPRLHFFM PGFAPLTSRG SQQYRALTVP ELTQQMFDAK NMMAACDPRH GRYLTVAAVF RGR MSMKEV DEQMLNVQNK NSSYFVEWIP NNVKTAVCDI PPRGLKMSAT FIGNSTAIQE LFKRISEQFT AMFRRKAFLH WYTG EGMDE MEFTEAESNM NDLVSEYQQY QDATADEQGE FEEEEGEDEA

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Macromolecule #3: Tubulinyl-Tyr carboxypeptidase 2

MacromoleculeName: Tubulinyl-Tyr carboxypeptidase 2 / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO / EC number: tubulinyl-Tyr carboxypeptidase
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 40.488914 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MTGSAADTHR CPHPKGAKGT RSRSSHARPV SLATSGGSEE EDKDGGVLFH VNKSGFPIDS HTWERMWMHV AKVHPKGGEM VGAIRNAAF LAKPSIPQVP NYRLSMTIPD WLQAIQNYMK TLQYNHTGTQ FFEIRKMRPL SGLMETAKEM TRESLPIKAL E AVILGIYL ...String:
MTGSAADTHR CPHPKGAKGT RSRSSHARPV SLATSGGSEE EDKDGGVLFH VNKSGFPIDS HTWERMWMHV AKVHPKGGEM VGAIRNAAF LAKPSIPQVP NYRLSMTIPD WLQAIQNYMK TLQYNHTGTQ FFEIRKMRPL SGLMETAKEM TRESLPIKAL E AVILGIYL TNGQPSIERF PISFKTYFSG NYFHHVVLGI YCNGRYGSLG MSRRAELMDK PLTFRTLSDL IFDFEDSYKK YL HTVKKVK IGLYVPHEPH SFQPIEWKQL VLNVSKMLRA DIRKELEKYA RDMRMKILKP ASAHSPTQVR SRGKSLSPRR RQA SPPRRL GRREKSPALP EKKVADLSTL NEVGYQIRI

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Macromolecule #4: Small vasohibin-binding protein

MacromoleculeName: Small vasohibin-binding protein / type: protein_or_peptide / ID: 4 / Details: Recombinant C-terminal his6-tag / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 8.78 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
MDPPARKEKT KVKESVSRVE KAKQKSAQQE LKQRQRAEIY ALNRVMTELE QQQFDEFCKQ MQPPGEKHHH HHH

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Macromolecule #5: GUANOSINE-5'-TRIPHOSPHATE

MacromoleculeName: GUANOSINE-5'-TRIPHOSPHATE / type: ligand / ID: 5 / Number of copies: 2 / Formula: GTP
Molecular weightTheoretical: 523.18 Da
Chemical component information

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

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Macromolecule #6: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 6 / Number of copies: 6 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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Macromolecule #7: PHOSPHOMETHYLPHOSPHONIC ACID GUANYLATE ESTER

MacromoleculeName: PHOSPHOMETHYLPHOSPHONIC ACID GUANYLATE ESTER / type: ligand / ID: 7 / Number of copies: 4 / Formula: G2P
Molecular weightTheoretical: 521.208 Da
Chemical component information

ChemComp-G2P:
PHOSPHOMETHYLPHOSPHONIC ACID GUANYLATE ESTER / GMP-CPP, energy-carrying molecule analogue*YM

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation statehelical array

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Sample preparation

Concentration2 mg/mL
BufferpH: 7.5
Component:
ConcentrationFormulaName
150.0 mMNaClSodium chlorideSodium Chloride
20.0 mMC4H11NO3Tris buffer
1.0 mMC14H9Cl5Dichlorodiphenyltrichloroethane
GridModel: Quantifoil R2/1 / Material: COPPER / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY / Support film - Film thickness: 12 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 90 sec.
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 298 K / Instrument: FEI VITROBOT MARK III
DetailsMixture of VASH2-SVBP and microtubules

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 2.2 µm / Nominal defocus min: 0.6 µm
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Number grids imaged: 1 / Number real images: 3345 / Average exposure time: 0.2 sec. / Average electron dose: 1.4 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 153041
Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

6qby


Details: 6QBY used for VASH2-SVBP complex 3JAL used for microtubules
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.0.8)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.0.8)
Final reconstructionNumber classes used: 1 / Resolution.type: BY AUTHOR / Resolution: 3.6 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.0.8) / Number images used: 96922
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial model
PDB IDChain

6qby


3jal

chain_id: H

3jal

chain_id: G

3jal

chain_id: A

3jal

chain_id: E

3jal

chain_id: B

3jal

chain_id: F
RefinementProtocol: RIGID BODY FIT / Overall B value: 54.76
Output model

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

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