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基本情報
登録情報 | データベース: PDB / ID: 7sja | ||||||
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タイトル | Undecorated 13pf E254N microtubule from recombinant human tubulin | ||||||
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![]() | STRUCTURAL PROTEIN / microtubule / cell division / cytoskeleton / GTPase | ||||||
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 / cytoplasmic microtubule / Recycling pathway of L1 / 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 / axon guidance / cell periphery / Translocation of SLC2A4 (GLUT4) to the plasma membrane / RHO GTPases Activate Formins / 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 / peptide binding / 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 / 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 | 電子顕微鏡法 / らせん対称体再構成法 / クライオ電子顕微鏡法 / 解像度: 3.8 Å | ||||||
![]() | LaFrance, B.J. / Greber, B.J. / 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, B.J. / Roostalu, J. / Henkin, G. / Greber, B.J. / Zhang, R. / Normanno, D. / McCollum, C. / Surrey, T. / Nogales, E. | ||||||
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構造の表示
ムービー |
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構造ビューア | 分子: ![]() ![]() |
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-関連構造データ
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リンク
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集合体
登録構造単位 | ![]()
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要素
#1: タンパク質 | 分子量: 51018.312 Da / 分子数: 6 / 変異: E254N / 由来タイプ: 組換発現 / 由来: (組換発現) ![]() ![]() #2: タンパク質 | 分子量: 51276.367 Da / 分子数: 6 / 由来タイプ: 組換発現 / 由来: (組換発現) ![]() ![]() #3: 化合物 | ChemComp-GTP / #4: 化合物 | ChemComp-MG / 研究の焦点であるリガンドがあるか | Y | |
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-実験情報
-実験
実験 | 手法: 電子顕微鏡法 |
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EM実験 | 試料の集合状態: HELICAL ARRAY / 3次元再構成法: らせん対称体再構成法 |
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試料調製
構成要素 | 名称: Undecorated 13pf E254N microtubule from recombinant human tubulin タイプ: COMPLEX / Entity ID: #1-#2 / 由来: RECOMBINANT |
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分子量 | 値: 0.102 MDa / 実験値: NO |
由来(天然) | 生物種: ![]() |
由来(組換発現) | 生物種: ![]() |
緩衝液 | pH: 6.8 詳細: 80 mM PIPES, 1 mM EGTA, 5 mM MgCl2, 2 mM GTP, pH 6.8 |
試料 | 濃度: 4.2 mg/ml / 包埋: NO / シャドウイング: NO / 染色: NO / 凍結: YES 詳細: 42 uM [wildtype tubulin] assembled and pseudo-helical microtubules adsorbed onto the grid |
試料支持 | グリッドの材料: COPPER / グリッドのサイズ: 300 divisions/in. / グリッドのタイプ: C-flat-1.2/1.3 |
急速凍結 | 凍結剤: ETHANE-PROPANE |
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電子顕微鏡撮影
実験機器 | ![]() モデル: Titan Krios / 画像提供: FEI Company |
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顕微鏡 | モデル: FEI TITAN KRIOS |
電子銃 | 電子線源: ![]() |
電子レンズ | モード: BRIGHT FIELD / 倍率(公称値): 64000 X / 最大 デフォーカス(公称値): 2700 nm / 最小 デフォーカス(公称値): 1000 nm / Cs: 2.6 mm |
試料ホルダ | 凍結剤: NITROGEN 試料ホルダーモデル: FEI TITAN KRIOS AUTOGRID HOLDER |
撮影 | 電子線照射量: 40 e/Å2 フィルム・検出器のモデル: GATAN K3 BIOQUANTUM (6k x 4k) |
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解析
ソフトウェア | 名称: PHENIX / バージョン: 1.18_3845: / 分類: 精密化 | ||||||||||||||||||||||||||||||||||||||||
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EMソフトウェア |
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画像処理 | 詳細: Images collected in super-resolution mode, Motioncorr2 5x5 binning of frames | ||||||||||||||||||||||||||||||||||||||||
CTF補正 | 詳細: CTFFind4 and CTFRefine were used within the RELION3.1 framework. タイプ: PHASE FLIPPING AND AMPLITUDE CORRECTION | ||||||||||||||||||||||||||||||||||||||||
らせん対称 | 回転角度/サブユニット: 83.45 ° / 軸方向距離/サブユニット: 0.13 Å / らせん対称軸の対称性: C1 | ||||||||||||||||||||||||||||||||||||||||
粒子像の選択 | 詳細: Microtubules were picked within RELION 3.1 helical picking functionality and extracted along the filament with an 82A sliding box. | ||||||||||||||||||||||||||||||||||||||||
3次元再構成 | 解像度: 3.8 Å / 解像度の算出法: FSC 0.143 CUT-OFF / 粒子像の数: 13706 / クラス平均像の数: 1 / 対称性のタイプ: HELICAL | ||||||||||||||||||||||||||||||||||||||||
原子モデル構築 | B value: 120 / プロトコル: RIGID BODY FIT / 空間: REAL / Target criteria: Correlation Coefficient | ||||||||||||||||||||||||||||||||||||||||
原子モデル構築 | PDB-ID: 6DPU Accession code: 6DPU / Source name: PDB / タイプ: experimental model | ||||||||||||||||||||||||||||||||||||||||
拘束条件 |
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