National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)
P41 GM109824
米国
引用
ジャーナル: Structure / 年: 2017 タイトル: Molecular Architecture of the Major Membrane Ring Component of the Nuclear Pore Complex. 著者: Paula Upla / Seung Joong Kim / Parthasarathy Sampathkumar / Kaushik Dutta / Sean M Cahill / Ilan E Chemmama / Rosemary Williams / Jeffrey B Bonanno / William J Rice / David L Stokes / David ...著者: Paula Upla / Seung Joong Kim / Parthasarathy Sampathkumar / Kaushik Dutta / Sean M Cahill / Ilan E Chemmama / Rosemary Williams / Jeffrey B Bonanno / William J Rice / David L Stokes / David Cowburn / Steven C Almo / Andrej Sali / Michael P Rout / Javier Fernandez-Martinez / 要旨: The membrane ring that equatorially circumscribes the nuclear pore complex (NPC) in the perinuclear lumen of the nuclear envelope is composed largely of Pom152 in yeast and its ortholog Nup210 (or ...The membrane ring that equatorially circumscribes the nuclear pore complex (NPC) in the perinuclear lumen of the nuclear envelope is composed largely of Pom152 in yeast and its ortholog Nup210 (or Gp210) in vertebrates. Here, we have used a combination of negative-stain electron microscopy, nuclear magnetic resonance, and small-angle X-ray scattering methods to determine an integrative structure of the ∼120 kDa luminal domain of Pom152. Our structural analysis reveals that the luminal domain is formed by a flexible string-of-pearls arrangement of nine repetitive cadherin-like Ig-like domains, indicating an evolutionary connection between NPCs and the cell adhesion machinery. The 16 copies of Pom152 known to be present in the yeast NPC are long enough to form the observed membrane ring, suggesting how interactions between Pom152 molecules help establish and maintain the NPC architecture.
詳細: Solutions were made fresh from concentrated, and sterile filtered.
染色
タイプ: NEGATIVE / 材質: Uranyl formate 詳細: 3 ul of protein was added to grids. The excess sample was blotted and the grid washed several times with water. The sample was stained with several drops of uranyl formate, blotted between ...詳細: 3 ul of protein was added to grids. The excess sample was blotted and the grid washed several times with water. The sample was stained with several drops of uranyl formate, blotted between each stain. After the last staining (30 sec), the sample was blotted and air dried.
The endogenous sample was purified using affinity tags, then natively eluted and isolated on a 5-20% sucrose gradient. Fractions identified by SDS-PAGE and mass spectrometry.
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電子顕微鏡法
顕微鏡
JEOL 2100F
撮影
フィルム・検出器のモデル: TVIPS TEMCAM-F224 (2k x 2k) 撮影したグリッド数: 2 / 実像数: 1220 / 平均露光時間: 2.0 sec. / 平均電子線量: 15.0 e/Å2 詳細: Images were collected at tilt angles 0 and 40 degrees
タイプ: OTHER / ソフトウェア - 名称: SPARX (ver. EMAN2 2.06) 詳細: Sparx was used to generate the 2D classes of the untilted particles and to get their relative orientations to derive the Euler angles, then Spider (version 17.12) was used to do the back projection.