National Institutes of Health/Office of the Director
DP5 OD021396-01
米国
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)
AI064326
米国
National Institutes of Health/National Cancer Institute (NIH/NCI)
CA141722
米国
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)
GM071862
米国
引用
ジャーナル: Cell Rep / 年: 2016 タイトル: Structural Basis for the Activation of IKK1/α. 著者: Smarajit Polley / Dario Oliveira Passos / De-Bin Huang / Maria Carmen Mulero / Anup Mazumder / Tapan Biswas / Inder M Verma / Dmitry Lyumkis / Gourisankar Ghosh / 要旨: Distinct signaling pathways activate the NF-κB family of transcription factors. The canonical NF-κB-signaling pathway is mediated by IκB kinase 2/β (IKK2/β), while the non-canonical pathway ...Distinct signaling pathways activate the NF-κB family of transcription factors. The canonical NF-κB-signaling pathway is mediated by IκB kinase 2/β (IKK2/β), while the non-canonical pathway depends on IKK1/α. The structural and biochemical bases for distinct signaling by these otherwise highly similar IKKs are unclear. We report single-particle cryoelectron microscopy (cryo-EM) and X-ray crystal structures of human IKK1 in dimeric (∼150 kDa) and hexameric (∼450 kDa) forms. The hexamer, which is the representative form in the crystal but comprises only ∼2% of the particles in solution by cryo-EM, is a trimer of IKK1 dimers. While IKK1 hexamers are not detectable in cells, the surface that supports hexamer formation is critical for IKK1-dependent cellular processing of p100 to p52, the hallmark of non-canonical NF-κB signaling. Comparison of this surface to that in IKK2 indicates significant divergence, and it suggests a fundamental role for this surface in signaling by these kinases through distinct pathways.
凍結剤: ETHANE / チャンバー内湿度: 80 % / チャンバー内温度: 277 K / 装置: HOMEMADE PLUNGER 詳細: Sample containing IKK1 dimers in SEC buffer was applied onto freshly plasma-treated (6 seconds, Gatan Solarus plasma cleaner) holey carbon C-flat grids (Protochips), adsorbed for 30 seconds, ...詳細: Sample containing IKK1 dimers in SEC buffer was applied onto freshly plasma-treated (6 seconds, Gatan Solarus plasma cleaner) holey carbon C-flat grids (Protochips), adsorbed for 30 seconds, and then plunged into liquid ethane using a manual cryo-plunger in an ambient environment of 4 degrees C..
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電子顕微鏡法
顕微鏡
FEI TITAN KRIOS
温度
最低: 70.0 K / 最高: 70.0 K
撮影
フィルム・検出器のモデル: GATAN K2 SUMMIT (4k x 4k) 検出モード: COUNTING / デジタル化 - サイズ - 横: 3838 pixel / デジタル化 - サイズ - 縦: 3710 pixel / デジタル化 - 画像ごとのフレーム数: 1-50 / 撮影したグリッド数: 1 / 実像数: 2918 / 平均露光時間: 10.0 sec. / 平均電子線量: 40.0 e/Å2 詳細: The dose was fractionated over 50 raw frames collected over a 10 second exposure time (200 ms per frame) on the Gatan K2 Summit direct detection device, with each frame receiving a dose of ~6. ...詳細: The dose was fractionated over 50 raw frames collected over a 10 second exposure time (200 ms per frame) on the Gatan K2 Summit direct detection device, with each frame receiving a dose of ~6.5 e-/pixel/sec. 2918 movies were collected and recorded at a nominal magnification of 22,500, corresponding to a pixel size of 1.31 A at the specimen level. The individual frames were gain-corrected, then aligned and summed using a GPU-enabled whole frame alignment program (Li et al., 2013), and exposure-filtered (Grant and Grigorieff, 2015).
クラス数: 3 / ソフトウェア - 名称: FREALIGN (ver. 9.11) 詳細: Three conformational states were captured using a four-model classification, with the last class corresponding to particles that produced a low-resolution reconstruction (increasing this ...詳細: Three conformational states were captured using a four-model classification, with the last class corresponding to particles that produced a low-resolution reconstruction (increasing this number to 5 or 6 classes did not increase the number of recovered conformational states). Although three conformational states were recovered during classification, it is likely these states are not discrete, and a continuum of movement about the central hinge exists, which is indicated by the relatively homogeneous distribution of particles within the classes.
FSC曲線 (解像度の算出)
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原子モデル構築 1
精密化
空間: REAL / プロトコル: FLEXIBLE FIT / 温度因子: 340 / 当てはまり具合の基準: FSC 0.5
得られたモデル
PDB-5tqx: CryoEM reconstruction of human IKK1, intermediate conformation 2