ジャーナル: Cell / 年: 2010 タイトル: Insights into antiparallel microtubule crosslinking by PRC1, a conserved nonmotor microtubule binding protein. 著者: Radhika Subramanian / Elizabeth M Wilson-Kubalek / Christopher P Arthur / Matthew J Bick / Elizabeth A Campbell / Seth A Darst / Ronald A Milligan / Tarun M Kapoor / 要旨: Formation of microtubule architectures, required for cell shape maintenance in yeast, directional cell expansion in plants and cytokinesis in eukaryotes, depends on antiparallel microtubule ...Formation of microtubule architectures, required for cell shape maintenance in yeast, directional cell expansion in plants and cytokinesis in eukaryotes, depends on antiparallel microtubule crosslinking by the conserved MAP65 protein family. Here, we combine structural and single molecule fluorescence methods to examine how PRC1, the human MAP65, crosslinks antiparallel microtubules. We find that PRC1's microtubule binding is mediated by a structured domain with a spectrin-fold and an unstructured Lys/Arg-rich domain. These two domains, at each end of a homodimer, are connected by a linkage that is flexible on single microtubules, but forms well-defined crossbridges between antiparallel filaments. Further, we show that PRC1 crosslinks are compliant and do not substantially resist filament sliding by motor proteins in vitro. Together, our data show how MAP65s, by combining structural flexibility and rigidity, tune microtubule associations to establish crosslinks that selectively "mark" antiparallel overlap in dynamic cytoskeletal networks.
温度: 277 K / 手法: 蒸気拡散法, ハンギングドロップ法 / pH: 9.5 詳細: 1:1 v/v protein (50 mg/ml in 80 mM PIPES, pH 6.8, 1 mM MgCl2, 1 mM EDTA, 150 mM KCl) and reservoir buffer (100 mM CHES pH 9.5, 30 % PEG 3000), VAPOR DIFFUSION, HANGING DROP, temperature 277K
解像度: 2→2.03 Å / 冗長度: 2.8 % / Rmerge(I) obs: 0.469 / Mean I/σ(I) obs: 2 / Num. unique all: 914 / Rsym value: 0.469 / % possible all: 97.3
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解析
ソフトウェア
名称
バージョン
分類
ADSC
Quantum
データ収集
SHARP
位相決定
REFMAC
5
精密化
HKL-2000
データ削減
HKL-2000
データスケーリング
精密化
構造決定の手法: 単波長異常分散 / 解像度: 2→25 Å / Cor.coef. Fo:Fc: 0.932 / Cor.coef. Fo:Fc free: 0.902 / SU B: 5.103 / SU ML: 0.142 / 交差検証法: THROUGHOUT / ESU R Free: 0.199 立体化学のターゲット値: MAXIMUM LIKELIHOOD WITH PHASES 詳細: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS
Rfactor
反射数
%反射
Selection details
Rfree
0.27378
435
4.8 %
RANDOM
Rwork
0.23788
-
-
-
all
0.239
17696
-
-
obs
0.23953
8670
99.76 %
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溶媒の処理
イオンプローブ半径: 0.8 Å / 減衰半径: 0.8 Å / VDWプローブ半径: 1.4 Å / 溶媒モデル: MASK