ジャーナル: Mol Cell / 年: 2016 タイトル: Cryo-EM of Mitotic Checkpoint Complex-Bound APC/C Reveals Reciprocal and Conformational Regulation of Ubiquitin Ligation. 著者: Masaya Yamaguchi / Ryan VanderLinden / Florian Weissmann / Renping Qiao / Prakash Dube / Nicholas G Brown / David Haselbach / Wei Zhang / Sachdev S Sidhu / Jan-Michael Peters / Holger Stark / ...著者: Masaya Yamaguchi / Ryan VanderLinden / Florian Weissmann / Renping Qiao / Prakash Dube / Nicholas G Brown / David Haselbach / Wei Zhang / Sachdev S Sidhu / Jan-Michael Peters / Holger Stark / Brenda A Schulman / 要旨: The mitotic checkpoint complex (MCC) coordinates proper chromosome biorientation on the spindle with ubiquitination activities of CDC20-activated anaphase-promoting complex/cyclosome (APC/C(CDC20)). ...The mitotic checkpoint complex (MCC) coordinates proper chromosome biorientation on the spindle with ubiquitination activities of CDC20-activated anaphase-promoting complex/cyclosome (APC/C(CDC20)). APC/C(CDC20) and two E2s, UBE2C and UBE2S, catalyze ubiquitination through distinct architectures for linking ubiquitin (UB) to substrates and elongating polyUB chains, respectively. MCC, which contains a second molecule of CDC20, blocks APC/C(CDC20)-UBE2C-dependent ubiquitination of Securin and Cyclins, while differentially determining or inhibiting CDC20 ubiquitination to regulate spindle surveillance, checkpoint activation, and checkpoint termination. Here electron microscopy reveals conformational variation of APC/C(CDC20)-MCC underlying this multifaceted regulation. MCC binds APC/C-bound CDC20 to inhibit substrate access. However, rotation about the CDC20-MCC assembly and conformational variability of APC/C modulate UBE2C-catalyzed ubiquitination of MCC's CDC20 molecule. Access of UBE2C is limiting for subsequent polyubiquitination by UBE2S. We propose that conformational dynamics of APC/C(CDC20)-MCC modulate E2 activation and determine distinctive ubiquitination activities as part of a response mechanism ensuring accurate sister chromatid segregation.
ダウンロード / ファイル: emd_4022.map.gz / 形式: CCP4 / 大きさ: 125 MB / タイプ: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
注釈
Human Anaphase-Promoting Complex/Cyclosome (APC/C) APC15 deletion mutant with Mitotic Checkpoint Complex (MCC) in an open conformation
ボクセルのサイズ
X
Y
Z
EMDB情報
1.57
1.57
1.57
CCP4マップ ヘッダ情報
1.57
1.57
1.57
EM Navigator ムービー #1
1.27
1.27
1.27
密度
表面レベル
登録者による: 0.04 / ムービー #1: 0.04
最小 - 最大
-0.03517601 - 0.10941491
平均 (標準偏差)
0.000709696 (±0.009250927)
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
320
320
320
Spacing
320
320
320
セル
A=B=C: 502.40002 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
1.57
1.57
1.57
M x/y/z
320
320
320
origin x/y/z
0.000
0.000
0.000
length x/y/z
502.400
502.400
502.400
α/β/γ
90.000
90.000
90.000
MAP C/R/S
1
2
3
start NC/NR/NS
0
0
0
NC/NR/NS
320
320
320
D min/max/mean
-0.035
0.109
0.001
-
添付データ
-
試料の構成要素
-
全体 : Human Anaphase-Promoting Complex/Cyclosome (APC/C) APC15 deletion...
全体
名称: Human Anaphase-Promoting Complex/Cyclosome (APC/C) APC15 deletion mutant with Mitotic Checkpoint Complex (MCC) in an open conformation.
要素
複合体: Human Anaphase-Promoting Complex/Cyclosome (APC/C) APC15 deletion mutant with Mitotic Checkpoint Complex (MCC) in an open conformation.
-
超分子 #1: Human Anaphase-Promoting Complex/Cyclosome (APC/C) APC15 deletion...
超分子
名称: Human Anaphase-Promoting Complex/Cyclosome (APC/C) APC15 deletion mutant with Mitotic Checkpoint Complex (MCC) in an open conformation. タイプ: complex / ID: 1 / 親要素: 0 詳細: Human Anaphase-Promoting Complex/Cyclosome (APC/C) APC15 deletion mutant with Mitotic Checkpoint Complex (MCC) in an open conformation.
由来(天然)
生物種: Homo sapiens (ヒト)
組換発現
生物種: Trichoplusia ni (イラクサキンウワバ) / 組換プラスミド: biGBac
分子量
理論値: 1.5 MDa
-
実験情報
-
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
-
試料調製
濃度
0.1 mg/mL
緩衝液
pH: 8 構成要素:
濃度
名称
式
50.0 mM
Hepes
200.0 mM
sodium chloride
NaCl
2.0 mM
Magnesium chloride
MgCl2
凍結
凍結剤: ETHANE / チャンバー内湿度: 100 % / チャンバー内温度: 277 K / 装置: FEI VITROBOT MARK IV
詳細
Grafix treated complex
-
電子顕微鏡法
顕微鏡
FEI TITAN KRIOS
撮影
フィルム・検出器のモデル: FEI FALCON II (4k x 4k) 検出モード: INTEGRATING / デジタル化 - 画像ごとのフレーム数: 2-17 / 撮影したグリッド数: 1 / 実像数: 5911 / 平均露光時間: 1.0 sec. / 平均電子線量: 2.35 e/Å2