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基本情報
登録情報 | データベース: PDB / ID: 5tre | ||||||
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タイトル | Zinc and the Iron Donor Frataxin Regulate Oligomerization of the Scaffold Protein to Form New Fe-S Cluster Assembly Centers | ||||||
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![]() | OXIDOREDUCTASE / Friedreich Ataxia / frataxin / iron-sulfur protein / mitochondria / protein complex | ||||||
機能・相同性 | ![]() Mitochondrial iron-sulfur cluster biogenesis / Mitochondrial protein import / mitochondrial electron transport, succinate to ubiquinone / tRNA wobble uridine modification / iron chaperone activity / iron-sulfur cluster assembly complex / response to iron(II) ion / iron-sulfur cluster assembly / heme biosynthetic process / ferroxidase ...Mitochondrial iron-sulfur cluster biogenesis / Mitochondrial protein import / mitochondrial electron transport, succinate to ubiquinone / tRNA wobble uridine modification / iron chaperone activity / iron-sulfur cluster assembly complex / response to iron(II) ion / iron-sulfur cluster assembly / heme biosynthetic process / ferroxidase / ATPase activator activity / ferroxidase activity / glutathione metabolic process / ferric iron binding / ferrous iron binding / mitochondrial intermembrane space / 2 iron, 2 sulfur cluster binding / iron ion transport / intracellular iron ion homeostasis / response to oxidative stress / mitochondrial inner membrane / mitochondrial matrix / iron ion binding / mitochondrion / zinc ion binding / identical protein binding / cytoplasm 類似検索 - 分子機能 | ||||||
生物種 | ![]() ![]() | ||||||
手法 | 電子顕微鏡法 / 単粒子再構成法 / ネガティブ染色法 / 解像度: 15.6 Å | ||||||
![]() | Ranatunga, W. / Gakh, O. / Galeano, B.K. / Smith IV, D.Y. / Thompson, J.R. / Isaya, G. | ||||||
資金援助 | ![]()
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![]() | ![]() タイトル: Zinc and the iron donor frataxin regulate oligomerization of the scaffold protein to form new Fe-S cluster assembly centers. 著者: B K Galeano / W Ranatunga / O Gakh / D Y Smith / J R Thompson / G Isaya / ![]() 要旨: Early studies of the bacterial Fe-S cluster assembly system provided structural details for how the scaffold protein and the cysteine desulfurase interact. This work and additional work on the yeast ...Early studies of the bacterial Fe-S cluster assembly system provided structural details for how the scaffold protein and the cysteine desulfurase interact. This work and additional work on the yeast and human systems elucidated a conserved mechanism for sulfur donation but did not provide any conclusive insights into the mechanism for iron delivery from the iron donor, frataxin, to the scaffold. We previously showed that oligomerization is a mechanism by which yeast frataxin (Yfh1) can promote assembly of the core machinery for Fe-S cluster synthesis both in vitro and in cells, in such a manner that the scaffold protein, Isu1, can bind to Yfh1 independent of the presence of the cysteine desulfurase, Nfs1. Here, in the absence of Yfh1, Isu1 was found to exist in two forms, one mostly monomeric with limited tendency to dimerize, and one with a strong propensity to oligomerize. Whereas the monomeric form is stabilized by zinc, the loss of zinc promotes formation of dimer and higher order oligomers. However, upon binding to oligomeric Yfh1, both forms take on a similar symmetrical trimeric configuration that places the Fe-S cluster coordinating residues of Isu1 in close proximity of iron-binding residues of Yfh1. This configuration is suitable for docking of Nfs1 in a manner that provides a structural context for coordinate iron and sulfur donation to the scaffold. Moreover, distinct structural features suggest that in physiological conditions the zinc-regulated abundance of monomeric vs. oligomeric Isu1 yields [Yfh1]·[Isu1] complexes with different Isu1 configurations that afford unique functional properties for Fe-S cluster assembly and delivery. | ||||||
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-関連構造データ
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集合体
登録構造単位 | ![]()
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要素
#1: タンパク質 | 分子量: 15383.872 Da / 分子数: 24 / 由来タイプ: 組換発現 由来: (組換発現) ![]() ![]() 遺伝子: ISU1, NUA1, YPL135W / プラスミド: pET28b / 発現宿主: ![]() ![]() #2: タンパク質 | 分子量: 13455.976 Da / 分子数: 24 / 由来タイプ: 組換発現 由来: (組換発現) ![]() ![]() 遺伝子: YFH1, YDL120W / プラスミド: pET24a / 発現宿主: ![]() ![]() |
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-実験情報
-実験
実験 | 手法: 電子顕微鏡法 |
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EM実験 | 試料の集合状態: PARTICLE / 3次元再構成法: 単粒子再構成法 |
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試料調製
構成要素 | 名称: Yfh1-Isu1 / タイプ: COMPLEX 詳細: Macromolecular complex comprising 24-mer of Yfh1 and 24-mer of Isu1 Entity ID: all / 由来: RECOMBINANT | |||||||||||||||
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分子量 | 値: 0.7 MDa / 実験値: NO | |||||||||||||||
由来(天然) | 生物種: ![]() ![]() | |||||||||||||||
由来(組換発現) | 生物種: ![]() ![]() | |||||||||||||||
緩衝液 | pH: 7.3 | |||||||||||||||
緩衝液成分 |
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試料 | 濃度: 0.11 mg/ml / 包埋: NO / シャドウイング: NO / 染色: YES / 凍結: NO 詳細: The protein complex was prepared by incubating Yfh1 and Isu1 (1:1.5 molar ratio) in HN100 buffer (10 mM HEPES-KOH, pH 7.3, 100 mM NaCl) and purified using Sephacryl S300 gel filtration chromatography. | |||||||||||||||
染色 | タイプ: NEGATIVE 詳細: Pre-incubated in HN100 buffer, the grid was placed on an 11 microliter drop of protein sample for 1 minute. Excess protein sample was blotted and washed for 3 seconds by placing the grid on a ...詳細: Pre-incubated in HN100 buffer, the grid was placed on an 11 microliter drop of protein sample for 1 minute. Excess protein sample was blotted and washed for 3 seconds by placing the grid on a drop of sterile water. After excess water was blotted, the grid was stained with 1% w/v uranyl acetate for 1 second and 30 seconds by successively placing it on two separate drops of uranyl acetate, with excess stain drawn off after each step. 染色剤: uranyl acetate | |||||||||||||||
試料支持 | 詳細: DV-502A instrument, Denton Vacuum Inc. / グリッドの材料: COPPER / グリッドのサイズ: 400 divisions/in. / グリッドのタイプ: carbon-coated, EMS |
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電子顕微鏡撮影
実験機器 | ![]() モデル: Tecnai F30 / 画像提供: FEI Company |
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顕微鏡 | モデル: FEI TECNAI F30 |
電子銃 | 電子線源: ![]() |
電子レンズ | モード: BRIGHT FIELD / 倍率(公称値): 115000 X / 倍率(補正後): 115000 X / 最大 デフォーカス(公称値): 2800 nm / 最小 デフォーカス(公称値): 210 nm / Calibrated defocus min: 210 nm / 最大 デフォーカス(補正後): 2800 nm / Cs: 2 mm / アライメント法: BASIC |
試料ホルダ | 凍結剤: NITROGEN / 試料ホルダーモデル: SIDE ENTRY, EUCENTRIC |
撮影 | 電子線照射量: 30 e/Å2 フィルム・検出器のモデル: GATAN ULTRASCAN 4000 (4k x 4k) 撮影したグリッド数: 1 / 実像数: 475 |
画像スキャン | 横: 4096 / 縦: 4096 |
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解析
EMソフトウェア |
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画像処理 | 詳細: 432 symmetry applied for reconstruction | ||||||||||||||||||||||||||||||||||||
CTF補正 | 詳細: The ctf.auto function from EMAN2 was applied. / タイプ: PHASE FLIPPING ONLY | ||||||||||||||||||||||||||||||||||||
粒子像の選択 | 選択した粒子像数: 4218 | ||||||||||||||||||||||||||||||||||||
対称性 | 点対称性: O (正8面体型対称) | ||||||||||||||||||||||||||||||||||||
3次元再構成 | 解像度: 15.6 Å / 解像度の算出法: FSC 0.143 CUT-OFF / 粒子像の数: 4218 / アルゴリズム: FOURIER SPACE / 対称性のタイプ: POINT | ||||||||||||||||||||||||||||||||||||
原子モデル構築 | プロトコル: RIGID BODY FIT / 空間: REAL |