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Yorodumi- EMDB-25159: 13pf E254A microtubule from recombinant human tubulin decorated w... -
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-Basic information
Entry | Database: EMDB / ID: EMD-25159 | |||||||||
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Title | 13pf E254A microtubule from recombinant human tubulin decorated with EB3 | |||||||||
Map data | 13pf E254A Microtubule decorated with EB3 from human recombinant tubulin | |||||||||
Sample |
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Keywords | microtubule / cell division / cytoskeleton / GTPase / STRUCTURAL PROTEIN | |||||||||
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 / Gap junction assembly / COPI-independent Golgi-to-ER retrograde traffic / microtubule plus-end binding / Prefoldin mediated transfer of substrate to CCT/TriC / Assembly and cell surface presentation of NMDA receptors / Kinesins / positive regulation of cyclin-dependent protein serine/threonine kinase activity / 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 protein kinase activity / RHO GTPases activate IQGAPs / regulation of microtubule polymerization or depolymerization / spindle assembly / spindle midzone / 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 / peptide binding / filopodium / axon guidance / cell periphery / Translocation of SLC2A4 (GLUT4) to the plasma membrane / RHO GTPases Activate Formins / Hydrolases; Acting on acid anhydrides; Acting on GTP to facilitate cellular and subcellular movement / HCMV Early Events / PKR-mediated signaling / structural constituent of cytoskeleton / mitotic spindle / microtubule cytoskeleton organization / 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 / midbody / growth cone / microtubule binding / microtubule / cell division / axon / GTPase activity / neuronal cell body / ubiquitin protein ligase binding / dendrite / positive regulation of DNA-templated transcription / GTP binding / protein kinase binding / 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 | helical reconstruction / cryo EM / Resolution: 3.8 Å | |||||||||
Authors | LaFrance BJ / Greber BJ | |||||||||
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_25159.map.gz | 298.7 MB | EMDB map data format | |
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Header (meta data) | emd-25159-v30.xml emd-25159.xml | 18.8 KB 18.8 KB | Display Display | EMDB header |
Images | emd_25159.png | 224.7 KB | ||
Filedesc metadata | emd-25159.cif.gz | 7.3 KB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-25159 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-25159 | HTTPS FTP |
-Validation report
Summary document | emd_25159_validation.pdf.gz | 627.5 KB | Display | EMDB validaton report |
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Full document | emd_25159_full_validation.pdf.gz | 627.1 KB | Display | |
Data in XML | emd_25159_validation.xml.gz | 8.3 KB | Display | |
Data in CIF | emd_25159_validation.cif.gz | 9.6 KB | Display | |
Arichive directory | https://ftp.pdbj.org/pub/emdb/validation_reports/EMD-25159 ftp://ftp.pdbj.org/pub/emdb/validation_reports/EMD-25159 | HTTPS FTP |
-Related structure data
Related structure data | 7sj9MC 7sj7C 7sj8C 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_25159.map.gz / Format: CCP4 / Size: 512 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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Annotation | 13pf E254A Microtubule decorated with EB3 from human recombinant tubulin | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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 E254A microtubule decorated with EB3
Entire | Name: 13pf E254A microtubule decorated with EB3 |
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Components |
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-Supramolecule #1: 13pf E254A microtubule decorated with EB3
Supramolecule | Name: 13pf E254A microtubule decorated with EB3 / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#3 |
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Source (natural) | Organism: Homo sapiens (human) |
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: 50.975289 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 LTAFQTNLVP YPRIHFPLAT YAPVISAEKA YHEQLSVAEI TNACFEPANQ MVKCDPRHGK YMA CCLLYR GDVVPKDVNA AIATIKTKRS IQFVDWCPTG FKVGINYQPP TVVPGGDLAK VQRAVCMLSN TTAIAEAWAR LDHK FDLMY AKRAFVHWYV GEGMEEGEFS EAREDMAALE KDYEEVGVDS VEGEGEEEGE EY UniProtKB: Tubulin alpha-1B chain |
-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 UniProtKB: Tubulin beta-3 chain |
-Macromolecule #3: Microtubule-associated protein RP/EB family member 3
Macromolecule | Name: Microtubule-associated protein RP/EB family member 3 / type: protein_or_peptide / ID: 3 / Number of copies: 2 / Enantiomer: LEVO |
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Source (natural) | Organism: Homo sapiens (human) |
Molecular weight | Theoretical: 32.021166 KDa |
Recombinant expression | Organism: Escherichia coli BL21 (bacteria) |
Sequence | String: MAVNVYSTSV TSENLSRHDM LAWVNDSLHL NYTKIEQLCS GAAYCQFMDM LFPGCVHLRK VKFQAKLEHE YIHNFKVLQA AFKKMGVDK IIPVEKLVKG KFQDNFEFIQ WFKKFFDANY DGKDYNPLLA RQGQDVAPPP NPGDQIFNKS KKLIGTAVPQ R TSPTGPKN ...String: MAVNVYSTSV TSENLSRHDM LAWVNDSLHL NYTKIEQLCS GAAYCQFMDM LFPGCVHLRK VKFQAKLEHE YIHNFKVLQA AFKKMGVDK IIPVEKLVKG KFQDNFEFIQ WFKKFFDANY DGKDYNPLLA RQGQDVAPPP NPGDQIFNKS KKLIGTAVPQ R TSPTGPKN MQTSGRLSNV APPCILRKNP PSARNGGHET DAQILELNQQ LVDLKLTVDG LEKERDFYFS KLRDIELICQ EH ESENSPV ISGIIGILYA TEEGFAPPED DEIEEHQQED QDEY UniProtKB: Microtubule-associated protein RP/EB family member 3 |
-Macromolecule #4: GUANOSINE-5'-TRIPHOSPHATE
Macromolecule | Name: GUANOSINE-5'-TRIPHOSPHATE / type: ligand / ID: 4 / Number of copies: 12 / Formula: GTP |
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Molecular weight | Theoretical: 523.18 Da |
Chemical component information | ChemComp-GTP: |
-Macromolecule #5: MAGNESIUM ION
Macromolecule | Name: MAGNESIUM ION / type: ligand / ID: 5 / Number of copies: 12 / Formula: MG |
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Molecular weight | Theoretical: 24.305 Da |
-Experimental details
-Structure determination
Method | cryo EM |
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Processing | helical reconstruction |
Aggregation state | helical array |
-Sample preparation
Concentration | 5.5 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 | 55 uM [wildtype tubulin] assembled and pseudo-helical microtubules adsorbed onto the grid |
-Electron microscopy
Microscope | FEI TITAN KRIOS |
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Image recording | Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: SUPER-RESOLUTION / Average electron dose: 40.0 e/Å2 |
Electron beam | Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN |
Electron optics | Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.6 mm / Nominal defocus max: 2.5 µm / Nominal defocus min: 0.5 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 |
+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: 115 / Target criteria: Correlation Coefficient |
Output model | PDB-7sj9: |