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Yorodumi- PDB-7sj9: 13pf E254A microtubule from recombinant human tubulin decorated w... -
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-Basic information
Entry | Database: PDB / ID: 7sj9 | ||||||
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Title | 13pf E254A microtubule from recombinant human tubulin decorated with EB3 | ||||||
Components |
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Keywords | STRUCTURAL PROTEIN / microtubule / cell division / cytoskeleton / GTPase | ||||||
Function / homology | Function and homology information netrin receptor binding / mitotic spindle astral microtubule end / Post-chaperonin tubulin folding pathway / protein localization to microtubule / dorsal root ganglion development / Microtubule-dependent trafficking of connexons from Golgi to the plasma membrane / Cilium Assembly / Carboxyterminal post-translational modifications of tubulin / microtubule plus-end / Sealing of the nuclear envelope (NE) by ESCRT-III ...netrin receptor binding / mitotic spindle astral microtubule end / Post-chaperonin tubulin folding pathway / protein localization to microtubule / dorsal root ganglion development / Microtubule-dependent trafficking of connexons from Golgi to the plasma membrane / Cilium Assembly / Carboxyterminal post-translational modifications of tubulin / microtubule plus-end / Sealing of the nuclear envelope (NE) by ESCRT-III / Intraflagellar transport / cytoskeleton-dependent intracellular transport / Formation of tubulin folding intermediates by CCT/TriC / COPI-independent Golgi-to-ER retrograde traffic / microtubule plus-end binding / Gap junction assembly / Prefoldin mediated transfer of substrate to CCT/TriC / Kinesins / Assembly and cell surface presentation of NMDA receptors / microtubule organizing center / COPI-dependent Golgi-to-ER retrograde traffic / intercellular bridge / cytoplasmic microtubule / regulation of microtubule polymerization / Recycling pathway of L1 / microtubule-based process / RHOH GTPase cycle / positive regulation of cyclin-dependent protein serine/threonine kinase activity / positive regulation of protein kinase activity / spindle midzone / spindle assembly / regulation of microtubule polymerization or depolymerization / RHO GTPases activate IQGAPs / cellular response to interleukin-4 / Hedgehog 'off' state / COPI-mediated anterograde transport / Activation of AMPK downstream of NMDARs / Mitotic Prometaphase / EML4 and NUDC in mitotic spindle formation / Recruitment of NuMA to mitotic centrosomes / HSP90 chaperone cycle for steroid hormone receptors (SHR) in the presence of ligand / MHC class II antigen presentation / Resolution of Sister Chromatid Cohesion / filopodium / axon guidance / cell periphery / Translocation of SLC2A4 (GLUT4) to the plasma membrane / RHO GTPases Activate Formins / peptide binding / PKR-mediated signaling / mitotic spindle / structural constituent of cytoskeleton / microtubule cytoskeleton organization / HCMV Early Events / Aggrephagy / Separation of Sister Chromatids / The role of GTSE1 in G2/M progression after G2 checkpoint / microtubule cytoskeleton / double-stranded RNA binding / protein localization / lamellipodium / mitotic cell cycle / growth cone / midbody / Hydrolases; Acting on acid anhydrides; Acting on GTP to facilitate cellular and subcellular movement / microtubule binding / microtubule / axon / cell division / GTPase activity / neuronal cell body / ubiquitin protein ligase binding / dendrite / GTP binding / protein kinase binding / positive regulation of DNA-templated transcription / perinuclear region of cytoplasm / structural molecule activity / extracellular exosome / identical protein binding / nucleus / metal ion binding / cytoplasm Similarity search - Function | ||||||
Biological species | Homo sapiens (human) | ||||||
Method | ELECTRON MICROSCOPY / helical reconstruction / cryo EM / Resolution: 3.8 Å | ||||||
Authors | LaFrance, B.J. / Greber, B.J. / Zhang, R. / McCollum, C. / Nogales, E. | ||||||
Funding support | United States, 1items
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Citation | Journal: Proc Natl Acad Sci U S A / Year: 2022 Title: Structural transitions in the GTP cap visualized by cryo-electron microscopy of catalytically inactive microtubules. Authors: Benjamin J LaFrance / Johanna Roostalu / Gil Henkin / Basil J Greber / Rui Zhang / Davide Normanno / Chloe O McCollum / Thomas Surrey / Eva Nogales / Abstract: Microtubules (MTs) are polymers of αβ-tubulin heterodimers that stochastically switch between growth and shrinkage phases. This dynamic instability is critically important for MT function. It is ...Microtubules (MTs) are polymers of αβ-tubulin heterodimers that stochastically switch between growth and shrinkage phases. This dynamic instability is critically important for MT function. It is believed that GTP hydrolysis within the MT lattice is accompanied by destabilizing conformational changes and that MT stability depends on a transiently existing GTP cap at the growing MT end. Here, we use cryo-electron microscopy and total internal reflection fluorescence microscopy of GTP hydrolysis-deficient MTs assembled from mutant recombinant human tubulin to investigate the structure of a GTP-bound MT lattice. We find that the GTP-MT lattice of two mutants in which the catalytically active glutamate in α-tubulin was substituted by inactive amino acids (E254A and E254N) is remarkably plastic. Undecorated E254A and E254N MTs with 13 protofilaments both have an expanded lattice but display opposite protofilament twists, making these lattices distinct from the compacted lattice of wild-type GDP-MTs. End-binding proteins of the EB family have the ability to compact both mutant GTP lattices and to stabilize a negative twist, suggesting that they promote this transition also in the GTP cap of wild-type MTs, thereby contributing to the maturation of the MT structure. We also find that the MT seam appears to be stabilized in mutant GTP-MTs and destabilized in GDP-MTs, supporting the proposal that the seam plays an important role in MT stability. Together, these structures of catalytically inactive MTs add mechanistic insight into the GTP state of MTs, the stability of the GTP- and GDP-bound lattice, and our overall understanding of MT dynamic instability. #1: Journal: BioRxiv / Year: 2021 Title: Structural transitions in the GTP cap visualized by cryo-EM of catalytically inactive microtubules Authors: LaFrance, B.J. / Roostalu, J. / Henkin, G. / Greber, B.J. / Zhang, R. / Normanno, D. / McCollum, C. / Surrey, T. / Nogales, E. | ||||||
History |
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-Structure visualization
Movie |
Movie viewer |
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Structure viewer | Molecule: MolmilJmol/JSmol |
-Downloads & links
-Download
PDBx/mmCIF format | 7sj9.cif.gz | 939.1 KB | Display | PDBx/mmCIF format |
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PDB format | pdb7sj9.ent.gz | 778.4 KB | Display | PDB format |
PDBx/mmJSON format | 7sj9.json.gz | Tree view | PDBx/mmJSON format | |
Others | Other downloads |
-Validation report
Summary document | 7sj9_validation.pdf.gz | 1.8 MB | Display | wwPDB validaton report |
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Full document | 7sj9_full_validation.pdf.gz | 1.9 MB | Display | |
Data in XML | 7sj9_validation.xml.gz | 152.7 KB | Display | |
Data in CIF | 7sj9_validation.cif.gz | 223.1 KB | Display | |
Arichive directory | https://data.pdbj.org/pub/pdb/validation_reports/sj/7sj9 ftp://data.pdbj.org/pub/pdb/validation_reports/sj/7sj9 | HTTPS FTP |
-Related structure data
Related structure data | 25159MC 7sj7C 7sj8C 7sjaC M: map data used to model this data C: citing same article (ref.) |
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Similar structure data |
-Links
-Assembly
Deposited unit |
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-Components
#1: Protein | Mass: 50975.289 Da / Num. of mol.: 6 / Mutation: E254A Source method: isolated from a genetically manipulated source Source: (gene. exp.) Homo sapiens (human) / Gene: TUBA1B / Plasmid: pFastBacDual / Cell line (production host): High Five / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: P68363 #2: Protein | Mass: 51276.367 Da / Num. of mol.: 6 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Homo sapiens (human) / Gene: TUBB3, TUBB4 / Plasmid: pFastBacDual / Cell line (production host): High Five / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: Q13509 #3: Protein | Mass: 32021.166 Da / Num. of mol.: 2 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Homo sapiens (human) / Gene: MAPRE3 / Production host: Escherichia coli BL21 (bacteria) / Strain (production host): BL21 RIL / References: UniProt: Q9UPY8 #4: Chemical | ChemComp-GTP / #5: Chemical | ChemComp-MG / Has ligand of interest | Y | |
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-Experimental details
-Experiment
Experiment | Method: ELECTRON MICROSCOPY |
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EM experiment | Aggregation state: HELICAL ARRAY / 3D reconstruction method: helical reconstruction |
-Sample preparation
Component | Name: 13pf E254A microtubule decorated with EB3 / Type: COMPLEX / Entity ID: #1-#3 / Source: MULTIPLE SOURCES |
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Molecular weight | Value: 0.102 MDa / Experimental value: NO |
Source (natural) | Organism: Homo sapiens (human) |
Source (recombinant) | Organism: Trichoplusia ni (cabbage looper) / Cell: High-Five Insect Cell / Plasmid: pFastBacDual |
Buffer solution | pH: 6.8 Details: 80 mM PIPES, 1 mM EGTA, 5 mM MgCl2, 2 mM GTP, pH 6.8 |
Specimen | Conc.: 5.5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES Details: 55 uM [wildtype tubulin] assembled and pseudo-helical microtubules adsorbed onto the grid |
Specimen support | Grid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: C-flat-1.2/1.3 |
Vitrification | Cryogen name: ETHANE-PROPANE |
-Electron microscopy imaging
Experimental equipment | Model: Titan Krios / Image courtesy: FEI Company |
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Microscopy | Model: FEI TITAN KRIOS |
Electron gun | Electron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM |
Electron lens | Mode: BRIGHT FIELD / Nominal magnification: 64000 X / Nominal defocus max: 2500 nm / Nominal defocus min: 500 nm / Cs: 2.6 mm |
Specimen holder | Cryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER |
Image recording | Electron dose: 40 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K2 SUMMIT (4k x 4k) |
-Processing
Software | Name: PHENIX / Version: 1.19_4080: / Classification: refinement | ||||||||||||||||||||||||||||||||||||||||
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EM software |
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Image processing | Details: Images collected in super-resolution mode, Motioncorr2 5x5 binning of frames | ||||||||||||||||||||||||||||||||||||||||
CTF correction | Details: CTFFind4 and CTFRefine were used within the RELION3.1 framework. Type: PHASE FLIPPING AND AMPLITUDE CORRECTION | ||||||||||||||||||||||||||||||||||||||||
Helical symmerty | Angular rotation/subunit: -0.19 ° / Axial rise/subunit: 81.64 Å / Axial symmetry: C1 | ||||||||||||||||||||||||||||||||||||||||
Particle selection | Details: Microtubules were picked within RELION 3.1 helical picking functionality and extracted along the filament with an 82A sliding box. | ||||||||||||||||||||||||||||||||||||||||
3D reconstruction | Resolution: 3.8 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 10008 / Num. of class averages: 1 / Symmetry type: HELICAL | ||||||||||||||||||||||||||||||||||||||||
Atomic model building | B value: 115 / Protocol: RIGID BODY FIT / Space: REAL / Target criteria: Correlation Coefficient | ||||||||||||||||||||||||||||||||||||||||
Atomic model building | PDB-ID: 6DPU Accession code: 6DPU / Source name: PDB / Type: experimental model | ||||||||||||||||||||||||||||||||||||||||
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