ジャーナル: Nat Struct Mol Biol / 年: 2007 タイトル: Architecture of the Dam1 kinetochore ring complex and implications for microtubule-driven assembly and force-coupling mechanisms. 著者: Hong-Wei Wang / Vincent H Ramey / Stefan Westermann / Andres E Leschziner / Julie P I Welburn / Yuko Nakajima / David G Drubin / Georjana Barnes / Eva Nogales / 要旨: The Dam1 kinetochore complex is essential for chromosome segregation in budding yeast. This ten-protein complex self-assembles around microtubules, forming ring-like structures that move with ...The Dam1 kinetochore complex is essential for chromosome segregation in budding yeast. This ten-protein complex self-assembles around microtubules, forming ring-like structures that move with depolymerizing microtubule ends, a mechanism with implications for cellular function. Here we used EM-based single-particle and helical analyses to define the architecture of the Dam1 complex at 30-A resolution and the self-assembly mechanism. Ring oligomerization seems to be facilitated by a conformational change upon binding to microtubules, suggesting that the Dam1 ring is not preformed, but self-assembles around kinetochore microtubules. The C terminus of the Dam1p protein, where most of the Aurora kinase Ipl1 phosphorylation sites reside, is in a strategic location to affect oligomerization and interactions with the microtubule. One of Ipl1's roles might be to fine-tune the coupling of the microtubule interaction with the conformational change required for oligomerization, with phosphorylation resulting in ring breakdown.
ダウンロード / ファイル: emd_1373.map.gz / 形式: CCP4 / 大きさ: 8.2 MB / タイプ: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
注釈
This is the single particle reconstruction of the DeltaC Dam1 mutant complex in its dimeric form.
ボクセルのサイズ
X=Y=Z: 4 Å
密度
表面レベル
1: 2.44 / ムービー #1: 7.27815
最小 - 最大
-5.74718 - 30.2408
平均 (標準偏差)
-0.000191472 (±0.976579)
対称性
空間群: 1
詳細
EMDB XML:
マップ形状
Axis order
X
Y
Z
Origin
0
0
0
サイズ
130
130
130
Spacing
130
130
130
セル
A=B=C: 520 Å α=β=γ: 90 °
CCP4マップ ヘッダ情報:
mode
Image stored as Reals
Å/pix. X/Y/Z
4
4
4
M x/y/z
130
130
130
origin x/y/z
0.000
0.000
0.000
length x/y/z
520.000
520.000
520.000
α/β/γ
90.000
90.000
90.000
start NX/NY/NZ
-64
-64
-64
NX/NY/NZ
128
128
128
MAP C/R/S
1
2
3
start NC/NR/NS
0
0
0
NC/NR/NS
130
130
130
D min/max/mean
-5.747
30.241
-0.000
-
添付データ
-
試料の構成要素
-
全体 : Dam1 DeltC mutant complex
全体
名称: Dam1 DeltC mutant complex
要素
試料: Dam1 DeltC mutant complex
タンパク質・ペプチド: Dam1 DeltC mutant complex
-
超分子 #1000: Dam1 DeltC mutant complex
超分子
名称: Dam1 DeltC mutant complex / タイプ: sample / ID: 1000 詳細: The sample was mainly composed of dimers and little percentage of monomers and trimers. 集合状態: Dimeric decamer / Number unique components: 1
pH: 6.8 詳細: 500 mM NaCl, 20 mM sodium phosphate pH 6.8, 1 mM EDTA
染色
タイプ: NEGATIVE 詳細: The complexes were negatively stained with 2% uranyl formate with the sandwich method between two layers of thin carbon film.
グリッド
詳細: 400 mesh copper grid
凍結
凍結剤: NONE
-
電子顕微鏡法
顕微鏡
FEI TECNAI 12
アライメント法
Legacy - 非点収差: objective lens astigmatism was corrected at 120,000 magnification
詳細
The micrographs were taken at low dose mode.
撮影
カテゴリ: FILM / フィルム・検出器のモデル: KODAK SO-163 FILM / デジタル化 - スキャナー: OTHER / デジタル化 - サンプリング間隔: 12.7 µm / 実像数: 200 詳細: The micrographs were scanned on a Nikon Super Coolscan 8000 scanner Od range: 1.4 / ビット/ピクセル: 14