ジャーナル: Elife / 年: 2016 タイトル: Atomic structure of the 26S proteasome lid reveals the mechanism of deubiquitinase inhibition. 著者: Corey M Dambacher / Evan J Worden / Mark A Herzik / Andreas Martin / Gabriel C Lander / 要旨: The 26S proteasome is responsible for the selective, ATP-dependent degradation of polyubiquitinated cellular proteins. Removal of ubiquitin chains from targeted substrates at the proteasome is a ...The 26S proteasome is responsible for the selective, ATP-dependent degradation of polyubiquitinated cellular proteins. Removal of ubiquitin chains from targeted substrates at the proteasome is a prerequisite for substrate processing and is accomplished by Rpn11, a deubiquitinase within the 'lid' sub-complex. Prior to the lid's incorporation into the proteasome, Rpn11 deubiquitinase activity is inhibited to prevent unwarranted deubiquitination of polyubiquitinated proteins. Here we present the atomic model of the isolated lid sub-complex, as determined by cryo-electron microscopy at 3.5 Å resolution, revealing how Rpn11 is inhibited through its interaction with a neighboring lid subunit, Rpn5. Through mutagenesis of specific residues, we describe the network of interactions that are required to stabilize this inhibited state. These results provide significant insight into the intricate mechanisms of proteasome assembly, outlining the substantial conformational rearrangements that occur during incorporation of the lid into the 26S holoenzyme, which ultimately activates the deubiquitinase for substrate degradation.
超分子 #1000: Recombinant yeast 26S proteasome lid complex
超分子
名称: Recombinant yeast 26S proteasome lid complex / タイプ: sample / ID: 1000 / 詳細: The sample was monodisperse. / 集合状態: 9 subunits / Number unique components: 1
分子量
実験値: 370 KDa / 理論値: 370 KDa / 手法: SDS protein gels and size exclusion chromatography
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分子 #1: 26S proteasome lid sub-complex
分子
名称: 26S proteasome lid sub-complex / タイプ: protein_or_peptide / ID: 1 / Name.synonym: lid 詳細: Lid complex was recombinantly expressed in E. coli and purified by size exclusion chromatography. コピー数: 1 / 集合状態: Heterononamer / 組換発現: Yes
GO: proteasome regulatory particle, lid subcomplex / InterPro: Proteasome/cyclosome repeat
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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
濃度
2.5 mg/mL
緩衝液
pH: 7.5 / 詳細: 50 mM HEPES, 100 mM NaCl, 100 mM KCl, 1 mM TCEP
グリッド
詳細: Sample was applied directly to plasma-cleaned holey carbon C-flat grids (400 mesh, 1.2 micrometer holes).
凍結
凍結剤: ETHANE / チャンバー内湿度: 88 % / チャンバー内温度: 85 K / 装置: HOMEMADE PLUNGER / 詳細: Manual plunging was performed in a cold room. 手法: 4 microliters of sample was applied to the grid, blotted for 2 seconds, and plunged into liquid ethane.
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電子顕微鏡法
顕微鏡
FEI TITAN KRIOS
温度
最低: 85 K / 最高: 90 K / 平均: 87.5 K
アライメント法
Legacy - 非点収差: Objective lens astigmatism was corrected at a nominal magnification of 22,500.
詳細
Micrographs were collected in super-resolution mode with a total frame count of 38 and total exposure time of 7.6 seconds.
日付
2015年2月10日
撮影
カテゴリ: CCD / フィルム・検出器のモデル: GATAN K2 (4k x 4k) / 実像数: 3432 / 平均電子線量: 43.8 e/Å2 詳細: Micrographs were collected as movies using super-resolution mode with the Gatan K2 Summit direct electron detector
Image pre-processing was performed using Appion. 3D classification and reconstruction was performed with RELION.
CTF補正
詳細: whole micrograph
最終 再構成
アルゴリズム: OTHER / 解像度のタイプ: BY AUTHOR / 解像度: 3.5 Å / 解像度の算出法: OTHER ソフトウェア - 名称: Appion, CTFFIND3, FindEM, RELION 詳細: 3D classification was performed to identify the best 109,396 particles from an initial data set of 254,112. 使用した粒子像数: 109396