- EMDB-2081: Electron cryo-microscopy of microtubule-bound human kinesin-5 mot... -
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基本情報
登録情報
データベース: EMDB / ID: EMD-2081
タイトル
Electron cryo-microscopy of microtubule-bound human kinesin-5 motor domain in the AMPPNP state (gold cluster in the neck linker V365C).
マップデータ
3D reconstruction of microtubule-bound human kinesin-5 motor domain with AMPPNP bound in the nucleotide-binding site (gold cluster attached to the neck linker, V365C)
試料
試料: 13-protofilament microtubule-bound human kinesin-5 motor domain with AMPPNP bound. A gold cluster is attached to the neck linker (V365C).
タンパク質・ペプチド: alpha tubulin
タンパク質・ペプチド: beta tubulin
タンパク質・ペプチド: Kinesin-5 motor domain
キーワード
electron cryo-microscopy / kinesin / microtubule / mitosis / cancer
機能・相同性
Kinesin motor domain, conserved site / Alpha tubulin / Beta tubulin, autoregulation binding site
ジャーナル: J Biol Chem / 年: 2012 タイトル: The structural basis of force generation by the mitotic motor kinesin-5. 著者: Adeline Goulet / William M Behnke-Parks / Charles V Sindelar / Jennifer Major / Steven S Rosenfeld / Carolyn A Moores / 要旨: Kinesin-5 is required for forming the bipolar spindle during mitosis. Its motor domain, which contains nucleotide and microtubule binding sites and mechanical elements to generate force, has evolved ...Kinesin-5 is required for forming the bipolar spindle during mitosis. Its motor domain, which contains nucleotide and microtubule binding sites and mechanical elements to generate force, has evolved distinct properties for its spindle-based functions. In this study, we report subnanometer resolution cryoelectron microscopy reconstructions of microtubule-bound human kinesin-5 before and after nucleotide binding and combine this information with studies of the kinetics of nucleotide-induced neck linker and cover strand movement. These studies reveal coupled, nucleotide-dependent conformational changes that explain many of this motor's properties. We find that ATP binding induces a ratchet-like docking of the neck linker and simultaneous, parallel docking of the N-terminal cover strand. Loop L5, the binding site for allosteric inhibitors of kinesin-5, also undergoes a dramatic reorientation when ATP binds, suggesting that it is directly involved in controlling nucleotide binding. Our structures indicate that allosteric inhibitors of human kinesin-5, which are being developed as anti-cancer therapeutics, bind to a motor conformation that occurs in the course of normal function. However, due to evolutionarily defined sequence variations in L5, this conformation is not adopted by invertebrate kinesin-5s, explaining their resistance to drug inhibition. Together, our data reveal the precision with which the molecular mechanism of kinesin-5 motors has evolved for force generation.
ダウンロード / ファイル: emd_2081.map.gz / 形式: CCP4 / 大きさ: 348.6 KB / タイプ: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
注釈
3D reconstruction of microtubule-bound human kinesin-5 motor domain with AMPPNP bound in the nucleotide-binding site (gold cluster attached to the neck linker, V365C)
ボクセルのサイズ
X=Y=Z: 2.8 Å
密度
表面レベル
登録者による: 1.34 / ムービー #1: 1.34
最小 - 最大
-7.33667994 - 6.94212723
平均 (標準偏差)
0.20147304 (±1.93402481)
対称性
空間群: 1
詳細
EMDB XML:
マップ形状
Axis order
X
Y
Z
Origin
-14
-28
-42
サイズ
45
45
45
Spacing
45
45
45
セル
A=B=C: 126.0 Å α=β=γ: 90.0 °
CCP4マップ ヘッダ情報:
mode
Image stored as Reals
Å/pix. X/Y/Z
2.8
2.8
2.8
M x/y/z
45
45
45
origin x/y/z
0.000
0.000
0.000
length x/y/z
126.000
126.000
126.000
α/β/γ
90.000
90.000
90.000
start NX/NY/NZ
0
0
0
NX/NY/NZ
128
128
168
MAP C/R/S
1
2
3
start NC/NR/NS
-28
-14
-42
NC/NR/NS
45
45
45
D min/max/mean
-7.337
6.942
0.201
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添付データ
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試料の構成要素
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全体 : 13-protofilament microtubule-bound human kinesin-5 motor domain w...
全体
名称: 13-protofilament microtubule-bound human kinesin-5 motor domain with AMPPNP bound. A gold cluster is attached to the neck linker (V365C).
要素
試料: 13-protofilament microtubule-bound human kinesin-5 motor domain with AMPPNP bound. A gold cluster is attached to the neck linker (V365C).
タンパク質・ペプチド: alpha tubulin
タンパク質・ペプチド: beta tubulin
タンパク質・ペプチド: Kinesin-5 motor domain
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超分子 #1000: 13-protofilament microtubule-bound human kinesin-5 motor domain w...
超分子
名称: 13-protofilament microtubule-bound human kinesin-5 motor domain with AMPPNP bound. A gold cluster is attached to the neck linker (V365C). タイプ: sample / ID: 1000 集合状態: 13-protofilament microtubule with one kinesin-5 motor domain bound every tubulin heterodimers Number unique components: 3
InterPro: Beta tubulin, autoregulation binding site
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分子 #3: Kinesin-5 motor domain
分子
名称: Kinesin-5 motor domain / タイプ: protein_or_peptide / ID: 3 / Name.synonym: KINESIN-LIKE PROTEIN KIF11 詳細: undecagold cluster was attached to the specific cysteine residue in the neck linker (V365C) 集合状態: monomer / 組換発現: Yes
由来(天然)
生物種: Homo sapiens (ヒト) / 別称: Human
組換発現
生物種: Escherichia coli (大腸菌) / 組換プラスミド: pET21a
配列
InterPro: Kinesin motor domain, conserved site
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実験情報
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構造解析
手法
クライオ電子顕微鏡法
解析
単粒子再構成法
試料の集合状態
particle
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試料調製
緩衝液
pH: 6.8 / 詳細: 80 mM PIPES, 5 mM MgCl2, 1 mM EGTA, 5 mM AMPPNP
グリッド
詳細: 400 mesh holey carbon grids
凍結
凍結剤: ETHANE / チャンバー内湿度: 100 % / 装置: FEI VITROBOT MARK I / 手法: chamber at 24 degrees C, blot 2.5 sec
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電子顕微鏡法
顕微鏡
FEI TECNAI F20
温度
平均: 90 K
アライメント法
Legacy - 非点収差: Objective lens astigmatism was corrected at 150,000 times magnification
The particles were selected along individual microtubules.
CTF補正
詳細: FREALIGN
最終 再構成
解像度のタイプ: BY AUTHOR / 解像度: 15.0 Å / 解像度の算出法: FSC 0.5 CUT-OFF / ソフトウェア - 名称: SPIDER, FREALIGN 詳細: Approximately 21,000 asymmetric units were averaged in the final reconstruction. The deposited map is low pass filtered at 16 A. 使用した粒子像数: 1753