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基本情報
登録情報 | データベース: PDB / ID: 5m5i | |||||||||
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タイトル | Pseudo-atomic model of microtubule-bound S.pombe kinesin-5 motor domain in the AMPPNP state (based on cryo-electron microscopy experiment): the N-terminus conformation allows formation of a cover neck bundle. | |||||||||
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![]() | MOTOR PROTEIN / microtubule-bound S.pombe kinesin-5 / motor domain / AMPPNP bound state | |||||||||
機能・相同性 | ![]() mitotic spindle formation (spindle phase one) / mitotic spindle elongation (spindle phase three) / Kinesins / initial mitotic spindle pole body separation / microtubule plus-end directed mitotic chromosome migration / meiotic spindle pole / meiotic spindle assembly / mitotic spindle midzone assembly / mitotic spindle midzone / mitotic spindle pole body ...mitotic spindle formation (spindle phase one) / mitotic spindle elongation (spindle phase three) / Kinesins / initial mitotic spindle pole body separation / microtubule plus-end directed mitotic chromosome migration / meiotic spindle pole / meiotic spindle assembly / mitotic spindle midzone assembly / mitotic spindle midzone / mitotic spindle pole body / spindle elongation / polar microtubule / minus-end-directed microtubule motor activity / plus-end-directed microtubule motor activity / meiotic spindle / positive regulation of axon guidance / microtubule associated complex / microtubule motor activity / microtubule-based process / mitotic spindle assembly / spindle microtubule / 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 / structural constituent of cytoskeleton / mitotic spindle / kinetochore / microtubule cytoskeleton organization / microtubule cytoskeleton / mitotic cell cycle / nervous system development / microtubule binding / microtubule / hydrolase activity / protein heterodimerization activity / cell division / GTPase activity / GTP binding / ATP hydrolysis activity / ATP binding / nucleus / metal ion binding / cytoplasm 類似検索 - 分子機能 | |||||||||
生物種 | ![]() ![]() ![]() ![]() | |||||||||
手法 | 電子顕微鏡法 / 単粒子再構成法 / クライオ電子顕微鏡法 / 解像度: 9.3 Å | |||||||||
![]() | Goulet, A. / Moores, C.A. / Cross, R.A. | |||||||||
資金援助 | ![]()
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![]() | ![]() タイトル: Schizosaccharomyces pombe kinesin-5 switches direction using a steric blocking mechanism. 著者: Mishan Britto / Adeline Goulet / Syeda Rizvi / Ottilie von Loeffelholz / Carolyn A Moores / Robert A Cross / ![]() 要旨: Cut7, the sole kinesin-5 in Schizosaccharomyces pombe, is essential for mitosis. Like other yeast kinesin-5 motors, Cut7 can reverse its stepping direction, by mechanisms that are currently unclear. ...Cut7, the sole kinesin-5 in Schizosaccharomyces pombe, is essential for mitosis. Like other yeast kinesin-5 motors, Cut7 can reverse its stepping direction, by mechanisms that are currently unclear. Here we show that for full-length Cut7, the key determinant of stepping direction is the degree of motor crowding on the microtubule lattice, with greater crowding converting the motor from minus end-directed to plus end-directed stepping. To explain how high Cut7 occupancy causes this reversal, we postulate a simple proximity sensing mechanism that operates via steric blocking. We propose that the minus end-directed stepping action of Cut7 is selectively inhibited by collisions with neighbors under crowded conditions, whereas its plus end-directed action, being less space-hungry, is not. In support of this idea, we show that the direction of Cut7-driven microtubule sliding can be reversed by crowding it with non-Cut7 proteins. Thus, crowding by either dynein microtubule binding domain or Klp2, a kinesin-14, converts Cut7 from net minus end-directed to net plus end-directed stepping. Biochemical assays confirm that the Cut7 N terminus increases Cut7 occupancy by binding directly to microtubules. Direct observation by cryoEM reveals that this occupancy-enhancing N-terminal domain is partially ordered. Overall, our data point to a steric blocking mechanism for directional reversal through which collisions of Cut7 motor domains with their neighbors inhibit their minus end-directed stepping action, but not their plus end-directed stepping action. Our model can potentially reconcile a number of previous, apparently conflicting, observations and proposals for the reversal mechanism of yeast kinesins-5. | |||||||||
履歴 |
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構造の表示
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構造ビューア | 分子: ![]() ![]() |
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PDBx/mmCIF形式 | ![]() | 67 KB | 表示 | ![]() |
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PDB形式 | ![]() | 35.9 KB | 表示 | ![]() |
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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
-タンパク質 , 3種, 3分子 ABC
#1: タンパク質 | 分子量: 50107.238 Da / 分子数: 1 / 由来タイプ: 天然 / 由来: (天然) ![]() ![]() |
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#2: タンパク質 | 分子量: 49907.770 Da / 分子数: 1 / 由来タイプ: 天然 / 由来: (天然) ![]() ![]() |
#3: タンパク質 | 分子量: 40737.527 Da / 分子数: 1 / 由来タイプ: 組換発現 由来: (組換発現) ![]() ![]() 遺伝子: cut7, SPAC25G10.07c / 発現宿主: ![]() ![]() |
-非ポリマー , 5種, 6分子 ![](data/chem/img/MG.gif)
![](data/chem/img/GTP.gif)
![](data/chem/img/GDP.gif)
![](data/chem/img/TA1.gif)
![](data/chem/img/ANP.gif)
![](data/chem/img/GTP.gif)
![](data/chem/img/GDP.gif)
![](data/chem/img/TA1.gif)
![](data/chem/img/ANP.gif)
#4: 化合物 | #5: 化合物 | ChemComp-GTP / | #6: 化合物 | ChemComp-GDP / | #7: 化合物 | ChemComp-TA1 / | #8: 化合物 | ChemComp-ANP / | |
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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: 単粒子再構成法 |
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試料調製
構成要素 | 名称: microtubule-bound S.pombe kinesin-5 motor domain in the AMPPNP state タイプ: COMPLEX / Entity ID: #1-#3 / 由来: MULTIPLE SOURCES | ||||||||||||||||||||||||
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分子量 | 値: 0.14 MDa / 実験値: NO | ||||||||||||||||||||||||
緩衝液 | pH: 6.8 | ||||||||||||||||||||||||
緩衝液成分 |
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試料 | 包埋: NO / シャドウイング: NO / 染色: NO / 凍結: YES | ||||||||||||||||||||||||
試料支持 | グリッドの材料: COPPER / グリッドのサイズ: 400 divisions/in. / グリッドのタイプ: C-flat-2/2 | ||||||||||||||||||||||||
急速凍結 | 装置: FEI VITROBOT MARK I / 凍結剤: ETHANE / 湿度: 100 % / 凍結前の試料温度: 297 K |
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電子顕微鏡撮影
実験機器 | ![]() モデル: Tecnai F20 / 画像提供: FEI Company |
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顕微鏡 | モデル: FEI TECNAI F20 |
電子銃 | 電子線源: ![]() |
電子レンズ | モード: BRIGHT FIELD / 倍率(補正後): 68000 X / 最小 デフォーカス(公称値): 700 nm / Cs: 2 mm / C2レンズ絞り径: 70 µm |
試料ホルダ | 凍結剤: NITROGEN 試料ホルダーモデル: GATAN CT3500 SINGLE TILT LIQUID NITROGEN CRYO TRANSFER HOLDER |
撮影 | 電子線照射量: 20 e/Å2 フィルム・検出器のモデル: GATAN ULTRASCAN 4000 (4k x 4k) |
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解析
EMソフトウェア |
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CTF補正 | タイプ: PHASE FLIPPING AND AMPLITUDE CORRECTION | ||||||||||||||||||||||||||||||||||||
3次元再構成 | 解像度: 9.3 Å / 解像度の算出法: FSC 0.5 CUT-OFF / 粒子像の数: 144300 / 対称性のタイプ: POINT | ||||||||||||||||||||||||||||||||||||
原子モデル構築 | プロトコル: FLEXIBLE FIT / 空間: REAL / Target criteria: Cross-correlation coefficient 詳細: An initial homology model of S. pombe cut7 kinesin-5 motor domain based on human kinesin-5 structure (PDB 3HQD) was prepared using Modeller. The coordinates of motor bound to an alpha-beta ...詳細: An initial homology model of S. pombe cut7 kinesin-5 motor domain based on human kinesin-5 structure (PDB 3HQD) was prepared using Modeller. The coordinates of motor bound to an alpha-beta tubulin dimer (PDB 1JFF) were rigidly fitted into the cryo-EM map using Chimera and refined by flexible fitting using Flex-EM. Structural models of loop5 and loop10 were generated using Modeller. The conformation of the neck-linker and the N-terminus were calculated using a conjugate-gradient energy minimization approach. |