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Yorodumi- EMDB-25157: 13pf wildtype microtubule from recombinant human tubulin decorate... -
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-Basic information
Entry | Database: EMDB / ID: EMD-25157 | |||||||||
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Title | 13pf wildtype microtubule from recombinant human tubulin decorated with kinesin | |||||||||
Map data | microtubules (recombinant human tubulin source) decorated with kinesin and reconstructed with pseudo-helical symmetry | |||||||||
Sample |
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Function / homology | Function and homology information netrin receptor binding / Post-chaperonin tubulin folding pathway / Carboxyterminal post-translational modifications of tubulin / dorsal root ganglion development / Microtubule-dependent trafficking of connexons from Golgi to the plasma membrane / Cilium Assembly / cytoskeleton-dependent intracellular transport / Intraflagellar transport / Sealing of the nuclear envelope (NE) by ESCRT-III / Gap junction assembly ...netrin receptor binding / Post-chaperonin tubulin folding pathway / Carboxyterminal post-translational modifications of tubulin / dorsal root ganglion development / Microtubule-dependent trafficking of connexons from Golgi to the plasma membrane / Cilium Assembly / cytoskeleton-dependent intracellular transport / Intraflagellar transport / Sealing of the nuclear envelope (NE) by ESCRT-III / Gap junction assembly / 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 / COPI-dependent Golgi-to-ER retrograde traffic / intercellular bridge / Recycling pathway of L1 / RHOH GTPase cycle / RHO GTPases activate IQGAPs / Hedgehog 'off' state / cytoplasmic microtubule / microtubule-based process / COPI-mediated anterograde transport / Activation of AMPK downstream of NMDARs / Mitotic Prometaphase / EML4 and NUDC in mitotic spindle formation / 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 / filopodium / cell periphery / RHO GTPases Activate Formins / Translocation of SLC2A4 (GLUT4) to the plasma membrane / axon guidance / peptide binding / Hydrolases; Acting on acid anhydrides; Acting on GTP to facilitate cellular and subcellular movement / PKR-mediated signaling / structural constituent of cytoskeleton / mitotic spindle / microtubule cytoskeleton organization / Aggrephagy / HCMV Early Events / Separation of Sister Chromatids / The role of GTSE1 in G2/M progression after G2 checkpoint / microtubule cytoskeleton / double-stranded RNA binding / mitotic cell cycle / lamellipodium / growth cone / microtubule / cell division / axon / GTPase activity / dendrite / neuronal cell body / ubiquitin protein ligase binding / structural molecule activity / GTP binding / extracellular exosome / metal ion binding / nucleus / cytoplasm Similarity search - Function | |||||||||
Biological species | Homo sapiens (human) | |||||||||
Method | helical reconstruction / cryo EM / Resolution: 3.6 Å | |||||||||
Authors | LaFrance BJ / Greber BJ / Zhang R / McCollum C / Nogales E | |||||||||
Funding support | United States, 1 items
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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 BJ / Roostalu J / Henkin G / Greber BJ / 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 | EM map: SurfViewMolmilJmol/JSmol |
Supplemental images |
-Downloads & links
-EMDB archive
Map data | emd_25157.map.gz | 460.1 MB | EMDB map data format | |
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Header (meta data) | emd-25157-v30.xml emd-25157.xml | 19 KB 19 KB | Display Display | EMDB header |
Images | emd_25157.png | 288.2 KB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-25157 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-25157 | HTTPS FTP |
-Related structure data
Related structure data | 7sj8MC 7sj7C 7sj9C 7sjaC C: citing same article (ref.) M: atomic model generated by this map |
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Similar structure data |
-Links
EMDB pages | EMDB (EBI/PDBe) / EMDataResource |
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Related items in Molecule of the Month |
-Map
File | Download / File: emd_25157.map.gz / Format: CCP4 / Size: 512 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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Annotation | microtubules (recombinant human tubulin source) decorated with kinesin and reconstructed with pseudo-helical symmetry | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 0.92 Å | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Details | EMDB XML:
CCP4 map header:
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-Supplemental data
-Sample components
-Entire : 13pf wildtype microtubule from recombinant human tubulin and deco...
Entire | Name: 13pf wildtype microtubule from recombinant human tubulin and decorated with kinesin |
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Components |
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-Supramolecule #1: 13pf wildtype microtubule from recombinant human tubulin and deco...
Supramolecule | Name: 13pf wildtype microtubule from recombinant human tubulin and decorated with kinesin type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#2 |
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Source (natural) | Organism: Homo sapiens (human) |
Recombinant expression | Organism: Trichoplusia ni (cabbage looper) / Recombinant cell: High-Five Insect Cell / Recombinant plasmid: pFastBacDual |
Molecular weight | Theoretical: 102 KDa |
-Macromolecule #1: Tubulin alpha-1B chain
Macromolecule | Name: Tubulin alpha-1B chain / type: protein_or_peptide / ID: 1 / Number of copies: 6 / Enantiomer: LEVO |
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Source (natural) | Organism: Homo sapiens (human) |
Molecular weight | Theoretical: 51.033324 KDa |
Recombinant expression | Organism: Trichoplusia ni (cabbage looper) |
Sequence | String: MRECISIHVG QAGVQIGNAC WELYCLEHGI QPDGQMPSDK TIHHHHHHGG GDDSFNTFFS ETGAGKHVPR AVFVDLEPTV IDEVRTGTY RQLFHPEQLI TGKEDAANNY ARGHYTIGKE IIDLVLDRIR KLADQCTGLQ GFLVFHSFGG GTGSGFTSLL M ERLSVDYG ...String: MRECISIHVG QAGVQIGNAC WELYCLEHGI QPDGQMPSDK TIHHHHHHGG GDDSFNTFFS ETGAGKHVPR AVFVDLEPTV IDEVRTGTY RQLFHPEQLI TGKEDAANNY ARGHYTIGKE IIDLVLDRIR KLADQCTGLQ GFLVFHSFGG GTGSGFTSLL M ERLSVDYG KKSKLEFSIY PAPQVSTAVV EPYNSILTTH TTLEHSDCAF MVDNEAIYDI CRRNLDIERP TYTNLNRLIS QI VSSITAS LRFDGALNVD LTEFQTNLVP YPRIHFPLAT YAPVISAEKA YHEQLSVAEI TNACFEPANQ MVKCDPRHGK YMA CCLLYR GDVVPKDVNA AIATIKTKRS IQFVDWCPTG FKVGINYQPP TVVPGGDLAK VQRAVCMLSN TTAIAEAWAR LDHK FDLMY AKRAFVHWYV GEGMEEGEFS EAREDMAALE KDYEEVGVDS VEGEGEEEGE EY |
-Macromolecule #2: Tubulin beta-3 chain
Macromolecule | Name: Tubulin beta-3 chain / type: protein_or_peptide / ID: 2 / Number of copies: 6 / Enantiomer: LEVO |
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Source (natural) | Organism: Homo sapiens (human) |
Molecular weight | Theoretical: 51.276367 KDa |
Recombinant expression | Organism: Trichoplusia ni (cabbage looper) |
Sequence | String: MREIVHIQAG QCGNQIGAKF WEVISDEHGI DPSGNYVGDS DLQLERISVY YNEASSHKYV PRAILVDLEP GTMDSVRSGA FGHLFRPDN FIFGQSGAGN NWAKGHYTEG AELVDSVLDV VRKECENCDC LQGFQLTHSL GGGTGSGMGT LLISKVREEY P DRIMNTFS ...String: MREIVHIQAG QCGNQIGAKF WEVISDEHGI DPSGNYVGDS DLQLERISVY YNEASSHKYV PRAILVDLEP GTMDSVRSGA FGHLFRPDN FIFGQSGAGN NWAKGHYTEG AELVDSVLDV VRKECENCDC LQGFQLTHSL GGGTGSGMGT LLISKVREEY P DRIMNTFS VVPSPKVSDT VVEPYNATLS IHQLVENTDE TYCIDNEALY DICFRTLKLA TPTYGDLNHL VSATMSGVTT SL RFPGQLN ADLRKLAVNM VPFPRLHFFM PGFAPLTARG SQQYRALTVP ELTQQMFDAK NMMAACDPRH GRYLTVATVF RGR MSMKEV DEQMLAIQSK NSSYFVEWIP NNVKVAVCDI PPRGLKMSST FIGNSTAIQE LFKRISEQFT AMFRRKAFLH WYTG EGMDE MEFTEAESNM NDLVSEYQQY QDATAEEEGE MYEDDEEESE AQGPKENLYF Q |
-Macromolecule #3: GUANOSINE-5'-TRIPHOSPHATE
Macromolecule | Name: GUANOSINE-5'-TRIPHOSPHATE / type: ligand / ID: 3 / Number of copies: 6 / Formula: GTP |
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Molecular weight | Theoretical: 523.18 Da |
Chemical component information | ChemComp-GTP: |
-Macromolecule #4: MAGNESIUM ION
Macromolecule | Name: MAGNESIUM ION / type: ligand / ID: 4 / Number of copies: 6 / Formula: MG |
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Molecular weight | Theoretical: 24.305 Da |
-Macromolecule #5: GUANOSINE-5'-DIPHOSPHATE
Macromolecule | Name: GUANOSINE-5'-DIPHOSPHATE / type: ligand / ID: 5 / Number of copies: 6 / Formula: GDP |
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Molecular weight | Theoretical: 443.201 Da |
Chemical component information | ChemComp-GDP: |
-Experimental details
-Structure determination
Method | cryo EM |
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Processing | helical reconstruction |
Aggregation state | helical array |
-Sample preparation
Concentration | 4.4 mg/mL |
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Buffer | pH: 6.8 Details: 80 mM PIPES, 1 mM EGTA, 5 mM MgCl2, 2 mM GTP, pH 6.8 |
Grid | Model: C-flat-1.2/1.3 / Material: COPPER / Mesh: 300 / Pretreatment - Type: GLOW DISCHARGE |
Vitrification | Cryogen name: ETHANE-PROPANE Details: 30 second adsorption time onto grid. blot force 15, 4 second blot time, 37 degrees, and 100 percent humidity. 0.5 second drain time.. |
Details | 44 uM [wildtype tubulin] assembled and pseudo-helical microtubules adsorbed onto the grid |
-Electron microscopy
Microscope | FEI TITAN KRIOS |
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Electron beam | Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN |
Electron optics | Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.6 mm / Nominal defocus max: 2.4 µm / Nominal defocus min: 0.7000000000000001 µm / Nominal magnification: 64000 |
Sample stage | Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN |
Image recording | Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: SUPER-RESOLUTION / Average electron dose: 40.0 e/Å2 |
Experimental equipment | Model: Titan Krios / Image courtesy: FEI Company |
-Image processing
Segment selection | Details: Microtubules were picked within RELION 3.1 helical picking functionality and extracted along the filament with an 82A sliding box. |
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CTF correction | Software: (Name: CTFFIND (ver. 4), RELION (ver. 3.1)) Details: CTFFind4 and CTFRefine were used within the RELION3.1 framework. |
Startup model | Type of model: EMDB MAP EMDB ID: Details: For the 13pf model, EMD-7973 was low pass filtered to 50A to serve as an initial model. In house reconstructions of 12-15pf at 50A resolution were used to separate by PF. |
Final angle assignment | Type: NOT APPLICABLE / Software - Name: FREALIGN (ver. 9.11) |
Final reconstruction | Number classes used: 1 Applied symmetry - Helical parameters - Δz: 81.69 Å Applied symmetry - Helical parameters - Δ&Phi: -0.09 ° Applied symmetry - Helical parameters - Axial symmetry: C1 (asymmetric) Resolution.type: BY AUTHOR / Resolution: 3.6 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: FREALIGN (ver. 9.11) / Number images used: 23364 |
Details | Images collected in super-resolution mode, Motioncorr2 5x5 binning of frames |