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基本情報
登録情報 | データベース: EMDB / ID: EMD-25161 | |||||||||
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タイトル | 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 | |||||||||
機能・相同性 | ![]() 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 / Translocation of SLC2A4 (GLUT4) to the plasma membrane / cell periphery / RHO GTPases Activate Formins / axon guidance / peptide binding / 加水分解酵素; 酸無水物に作用; GTPに作用・細胞または細胞小器官の運動に関与 / 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 類似検索 - 分子機能 | |||||||||
生物種 | ![]() | |||||||||
手法 | らせん対称体再構成法 / クライオ電子顕微鏡法 / 解像度: 5.0 Å | |||||||||
![]() | LaFrance BJ / Greber BJ / Zhang R / McCollum C / Nogales E | |||||||||
資金援助 | ![]()
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![]() | ![]() タイトル: Structural transitions in the GTP cap visualized by cryo-electron microscopy of catalytically inactive microtubules. 著者: Benjamin J LaFrance / Johanna Roostalu / Gil Henkin / Basil J Greber / Rui Zhang / Davide Normanno / Chloe O McCollum / Thomas Surrey / Eva Nogales / ![]() ![]() ![]() 要旨: 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: ![]() タイトル: Structural transitions in the GTP cap visualized by cryo-EM of catalytically inactive microtubules 著者: LaFrance BJ / Roostalu J / Henkin G / Greber BJ / Zhang R / Normanno D / McCollum C / Surrey T / Nogales E | |||||||||
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構造ビューア | EMマップ: ![]() ![]() ![]() |
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-検証レポート
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CIF形式データ | ![]() | 7.6 KB | 表示 | |
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「今月の分子」の関連する項目 |
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注釈 | 13pf E254N microtubule decorated with EB3, from recombinant human tubulin | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
ボクセルのサイズ | X=Y=Z: 2.31 Å | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
密度 |
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対称性 | 空間群: 1 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
詳細 | EMDB XML:
CCP4マップ ヘッダ情報:
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試料の構成要素
-全体 : 13pf E254N microtubule decorated with EB3 from recombinant human ...
全体 | 名称: 13pf E254N microtubule decorated with EB3 from recombinant human tubulin |
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要素 |
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-超分子 #1: 13pf E254N microtubule decorated with EB3 from recombinant human ...
超分子 | 名称: 13pf E254N microtubule decorated with EB3 from recombinant human tubulin タイプ: complex / ID: 1 / 親要素: 0 / 含まれる分子: all |
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由来(天然) | 生物種: ![]() |
分子量 | 理論値: 102 KDa |
-分子 #1: human tubulin alpha-1B chain (TUBA1B) with E254N mutation
分子 | 名称: human tubulin alpha-1B chain (TUBA1B) with E254N mutation タイプ: protein_or_peptide / ID: 1 / 光学異性体: LEVO |
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由来(天然) | 生物種: ![]() |
組換発現 | 生物種: ![]() |
配列 | 文字列: MRECISIHVG QAGVQIGNAC WELYCLEHGI QPDGQMPSDK TIHHHHHHGG GDDSFNTFFS ETGAGKHVPR AVFVDLEPTV IDE VRTGTY RQLFHPEQLI TGKEDAANNY ARGHYTIGKE IIDLVLDRIR KLADQCTGLQ GFLVFHSFGG GTGSGFTSLL MERLSVD YG ...文字列: 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 |
-分子 #2: human tubulin beta-3 chain (TUBB3)
分子 | 名称: human tubulin beta-3 chain (TUBB3) / タイプ: protein_or_peptide / ID: 2 / 光学異性体: LEVO |
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由来(天然) | 生物種: ![]() |
組換発現 | 生物種: ![]() |
配列 | 文字列: MREIVHIQAG QCGNQIGAKF WEVISDEHGI DPSGNYVGDS DLQLERISVY YNEASSHKYV PRAILVDLEP GTMDSVRSGA FGH LFRPDN FIFGQSGAGN NWAKGHYTEG AELVDSVLDV VRKECENCDC LQGFQLTHSL GGGTGSGMGT LLISKVREEY PDRIMNT FS ...文字列: 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 |
-分子 #3: human microtubule end-binding protein 3 (EB3)
分子 | 名称: human microtubule end-binding protein 3 (EB3) / タイプ: protein_or_peptide / ID: 3 / 光学異性体: LEVO |
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由来(天然) | 生物種: ![]() |
組換発現 | 生物種: ![]() ![]() |
配列 | 文字列: MAVNVYSTSV TSENLSRHDM LAWVNDSLHL NYTKIEQLCS GAAYCQFMDM LFPGCVHLRK VKFQAKLEH EYIHNFKVLQ AAFKKMGVDK IIPVEKLVKG KFQDNFEFIQ WFKKFFDANY D GKDYNPLL ARQGQDVAPP PNPGDQIFNK SKKLIGTAVP QRTSPTGPKN ...文字列: 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 |
-実験情報
-構造解析
手法 | クライオ電子顕微鏡法 |
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![]() | らせん対称体再構成法 |
試料の集合状態 | helical array |
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試料調製
濃度 | 4.2 mg/mL |
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緩衝液 | pH: 6.8 詳細: 80 mM PIPES, 1 mM EGTA, 5 mM MgCl2, 2 mM GTP, pH 6.8 |
グリッド | モデル: C-flat-1.2/1.3 / 材質: COPPER / メッシュ: 300 / 前処理 - タイプ: GLOW DISCHARGE |
凍結 | 凍結剤: ETHANE-PROPANE |
詳細 | 42 uM [wildtype tubulin] assembled and pseudo-helical microtubules adsorbed onto the grid |
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電子顕微鏡法
顕微鏡 | FEI TITAN KRIOS |
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撮影 | フィルム・検出器のモデル: GATAN K3 BIOQUANTUM (6k x 4k) 平均電子線量: 40.0 e/Å2 |
電子線 | 加速電圧: 300 kV / 電子線源: ![]() |
電子光学系 | 照射モード: FLOOD BEAM / 撮影モード: BRIGHT FIELD / Cs: 2.6 mm / 最大 デフォーカス(公称値): 2.7 µm / 最小 デフォーカス(公称値): 1.0 µm / 倍率(公称値): 64000 |
試料ステージ | 試料ホルダーモデル: FEI TITAN KRIOS AUTOGRID HOLDER ホルダー冷却材: NITROGEN |
実験機器 | ![]() モデル: Titan Krios / 画像提供: FEI Company |
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画像解析
-原子モデル構築 1
初期モデル | PDB ID: Chain - Source name: PDB / Chain - Initial model type: experimental model |
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精密化 | 空間: REAL / プロトコル: RIGID BODY FIT / 温度因子: 100 / 当てはまり具合の基準: Correlation Coefficient |