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- EMDB-25161: 13pf E254N microtubule from recombinant human tubulin decorated w... -
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Open data
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Basic information
Entry | Database: EMDB / ID: EMD-25161 | |||||||||
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Title | 13pf E254N microtubule from recombinant human tubulin decorated with EB3 | |||||||||
![]() | 13pf E254N microtubule decorated with EB3, from recombinant human tubulin | |||||||||
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![]() | microtubule / cell division / cytoskeleton / GTPase / STRUCTURAL PROTEIN | |||||||||
Function / homology | ![]() netrin receptor binding / Post-chaperonin tubulin folding pathway / dorsal root ganglion development / Microtubule-dependent trafficking of connexons from Golgi to the plasma membrane / Cilium Assembly / Carboxyterminal post-translational modifications of tubulin / Sealing of the nuclear envelope (NE) by ESCRT-III / Intraflagellar transport / cytoskeleton-dependent intracellular transport / Formation of tubulin folding intermediates by CCT/TriC ...netrin receptor binding / Post-chaperonin tubulin folding pathway / dorsal root ganglion development / Microtubule-dependent trafficking of connexons from Golgi to the plasma membrane / Cilium Assembly / Carboxyterminal post-translational modifications of tubulin / Sealing of the nuclear envelope (NE) by ESCRT-III / Intraflagellar transport / cytoskeleton-dependent intracellular transport / Formation of tubulin folding intermediates by CCT/TriC / Gap junction assembly / COPI-independent Golgi-to-ER retrograde traffic / Prefoldin mediated transfer of substrate to CCT/TriC / Assembly and cell surface presentation of NMDA receptors / Kinesins / COPI-dependent Golgi-to-ER retrograde traffic / intercellular bridge / Recycling pathway of L1 / cytoplasmic microtubule / microtubule-based process / RHOH GTPase cycle / 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 / Resolution of Sister Chromatid Cohesion / HSP90 chaperone cycle for steroid hormone receptors (SHR) in the presence of ligand / MHC class II antigen presentation / filopodium / cell periphery / Translocation of SLC2A4 (GLUT4) to the plasma membrane / RHO GTPases Activate Formins / peptide binding / Hydrolases; Acting on acid anhydrides; Acting on GTP to facilitate cellular and subcellular movement / axon guidance / PKR-mediated signaling / structural constituent of cytoskeleton / mitotic spindle / Aggrephagy / microtubule cytoskeleton organization / HCMV Early Events / Separation of Sister Chromatids / The role of GTSE1 in G2/M progression after G2 checkpoint / microtubule cytoskeleton / double-stranded RNA binding / lamellipodium / mitotic cell cycle / growth cone / microtubule / cell division / axon / GTPase activity / neuronal cell body / ubiquitin protein ligase binding / dendrite / GTP binding / structural molecule activity / extracellular exosome / nucleus / metal ion binding / cytoplasm Similarity search - Function | |||||||||
Biological species | ![]() | |||||||||
Method | helical reconstruction / cryo EM / Resolution: 5.0 Å | |||||||||
![]() | LaFrance BJ / Greber BJ / Zhang R / McCollum C / Nogales E | |||||||||
Funding support | ![]()
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![]() | ![]() 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: ![]() 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
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Structure viewer | EM map: ![]() ![]() ![]() |
Supplemental images |
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Downloads & links
-EMDB archive
Map data | ![]() | 42 MB | ![]() | |
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Header (meta data) | ![]() ![]() | 17.8 KB 17.8 KB | Display Display | ![]() |
Images | ![]() | 125.4 KB | ||
Filedesc metadata | ![]() | 6.6 KB | ||
Archive directory | ![]() ![]() | HTTPS FTP |
-Validation report
Summary document | ![]() | 494.3 KB | Display | ![]() |
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Full document | ![]() | 493.8 KB | Display | |
Data in XML | ![]() | 6.7 KB | Display | |
Data in CIF | ![]() | 7.6 KB | Display | |
Arichive directory | ![]() ![]() | HTTPS FTP |
-Related structure data
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Links
EMDB pages | ![]() ![]() |
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Related items in Molecule of the Month |
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Map
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Annotation | 13pf E254N microtubule decorated with EB3, from recombinant human tubulin | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 2.31 Å | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Details | EMDB XML:
CCP4 map header:
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-Supplemental data
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Sample components
-Entire : 13pf E254N microtubule decorated with EB3 from recombinant human ...
Entire | Name: 13pf E254N microtubule decorated with EB3 from recombinant human tubulin |
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Components |
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-Supramolecule #1: 13pf E254N microtubule decorated with EB3 from recombinant human ...
Supramolecule | Name: 13pf E254N microtubule decorated with EB3 from recombinant human tubulin type: complex / ID: 1 / Parent: 0 / Macromolecule list: all |
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Source (natural) | Organism: ![]() |
Molecular weight | Theoretical: 102 KDa |
-Macromolecule #1: human tubulin alpha-1B chain (TUBA1B) with E254N mutation
Macromolecule | Name: human tubulin alpha-1B chain (TUBA1B) with E254N mutation type: protein_or_peptide / ID: 1 / Enantiomer: LEVO |
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Source (natural) | Organism: ![]() |
Recombinant expression | Organism: ![]() |
Sequence | String: MRECISIHVG QAGVQIGNAC WELYCLEHGI QPDGQMPSDK TIHHHHHHGG GDDSFNTFFS ETGAGKHVPR AVFVDLEPTV IDE VRTGTY RQLFHPEQLI TGKEDAANNY ARGHYTIGKE IIDLVLDRIR KLADQCTGLQ GFLVFHSFGG GTGSGFTSLL MERLSVD YG ...String: MRECISIHVG QAGVQIGNAC WELYCLEHGI QPDGQMPSDK TIHHHHHHGG GDDSFNTFFS ETGAGKHVPR AVFVDLEPTV IDE VRTGTY RQLFHPEQLI TGKEDAANNY ARGHYTIGKE IIDLVLDRIR KLADQCTGLQ GFLVFHSFGG GTGSGFTSLL MERLSVD YG KKSKLEFSIY PAPQVSTAVV EPYNSILTTH TTLEHSDCAF MVDNEAIYDI CRRNLDIERP TYTNLNRLIS QIVSSITASL R FDGALNVD LTNFQTNLVP YPRIHFPLAT YAPVISAEKA YHEQLSVAEI TNACFEPANQ MVKCDPRHGK YMACCLLYRG DVVPK DVNA AIATIKTKRS IQFVDWCPTG FKVGINYQPP TVVPGGDLAK VQRAVCMLSN TTAIAEAWAR LDHKFDLMYA KRAFVHWYV GEGMEEGEFS EAREDMAALE KDYEEVGVDS VEGEGEEEGE EY |
-Macromolecule #2: human tubulin beta-3 chain (TUBB3)
Macromolecule | Name: human tubulin beta-3 chain (TUBB3) / type: protein_or_peptide / ID: 2 / Enantiomer: LEVO |
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Source (natural) | Organism: ![]() |
Recombinant expression | Organism: ![]() |
Sequence | String: MREIVHIQAG QCGNQIGAKF WEVISDEHGI DPSGNYVGDS DLQLERISVY YNEASSHKYV PRAILVDLEP GTMDSVRSGA FGH LFRPDN FIFGQSGAGN NWAKGHYTEG AELVDSVLDV VRKECENCDC LQGFQLTHSL GGGTGSGMGT LLISKVREEY PDRIMNT FS ...String: MREIVHIQAG QCGNQIGAKF WEVISDEHGI DPSGNYVGDS DLQLERISVY YNEASSHKYV PRAILVDLEP GTMDSVRSGA FGH LFRPDN FIFGQSGAGN NWAKGHYTEG AELVDSVLDV VRKECENCDC LQGFQLTHSL GGGTGSGMGT LLISKVREEY PDRIMNT FS VVPSPKVSDT VVEPYNATLS IHQLVENTDE TYCIDNEALY DICFRTLKLA TPTYGDLNHL VSATMSGVTT SLRFPGQLNA D LRKLAVNM VPFPRLHFFM PGFAPLTARG SQQYRALTVP ELTQQMFDAK NMMAACDPRH GRYLTVATVF RGRMSMKEVD EQMLA IQSK NSSYFVEWIP NNVKVAVCDI PPRGLKMSST FIGNSTAIQE LFKRISEQFT AMFRRKAFLH WYTGEGMDEM EFTEAESNM NDLVSEYQQY QDATAEEEGE MYEDDEEESE AQGPKENLYF Q |
-Macromolecule #3: human microtubule end-binding protein 3 (EB3)
Macromolecule | Name: human microtubule end-binding protein 3 (EB3) / type: protein_or_peptide / ID: 3 / Enantiomer: LEVO |
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Source (natural) | Organism: ![]() |
Recombinant expression | Organism: ![]() ![]() |
Sequence | String: MAVNVYSTSV TSENLSRHDM LAWVNDSLHL NYTKIEQLCS GAAYCQFMDM LFPGCVHLRK VKFQAKLEH EYIHNFKVLQ AAFKKMGVDK IIPVEKLVKG KFQDNFEFIQ WFKKFFDANY D GKDYNPLL ARQGQDVAPP PNPGDQIFNK SKKLIGTAVP QRTSPTGPKN ...String: MAVNVYSTSV TSENLSRHDM LAWVNDSLHL NYTKIEQLCS GAAYCQFMDM LFPGCVHLRK VKFQAKLEH EYIHNFKVLQ AAFKKMGVDK IIPVEKLVKG KFQDNFEFIQ WFKKFFDANY D GKDYNPLL ARQGQDVAPP PNPGDQIFNK SKKLIGTAVP QRTSPTGPKN MQTSGRLSNV AP PCILRKN PPSARNGGHE TDAQILELNQ QLVDLKLTVD GLEKERDFYF SKLRDIELIC QEH ESENSP VISGIIGILY ATEEGFAPPE DDEIEEHQQE DQDEY |
-Experimental details
-Structure determination
Method | cryo EM |
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![]() | helical reconstruction |
Aggregation state | helical array |
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Sample preparation
Concentration | 4.2 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 | 42 uM [wildtype tubulin] assembled and pseudo-helical microtubules adsorbed onto the grid |
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Electron microscopy
Microscope | FEI TITAN KRIOS |
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Image recording | Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average electron dose: 40.0 e/Å2 |
Electron beam | Acceleration voltage: 300 kV / Electron source: ![]() |
Electron optics | Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.6 mm / Nominal defocus max: 2.7 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 64000 |
Sample stage | Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN |
Experimental equipment | ![]() Model: Titan Krios / Image courtesy: FEI Company |
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Image processing
-Atomic model buiding 1
Initial model | PDB ID: Chain - Source name: PDB / Chain - Initial model type: experimental model |
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Refinement | Space: REAL / Protocol: RIGID BODY FIT / Overall B value: 100 / Target criteria: Correlation Coefficient |