- EMDB-8301: Architecture of the Human Mitochondrial Iron-Sulfur Cluster Assem... -
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基本情報
登録情報
データベース: EMDB / ID: EMD-8301
タイトル
Architecture of the Human Mitochondrial Iron-Sulfur Cluster Assembly Machinery: the Complex Formed by the Iron Donor, the Sulfur Donor, and the Scaffold
マップデータ
Human mitochondrial iron-sulfur cluster assembly machinery: grouping of segments corresponding to one half of the full map (EMD-8293)
National Institutes of Health/National Institute on Aging (NIH/NIA)
AG15709-19
米国
引用
ジャーナル: J Biol Chem / 年: 2016 タイトル: Architecture of the Human Mitochondrial Iron-Sulfur Cluster Assembly Machinery. 著者: Oleksandr Gakh / Wasantha Ranatunga / Douglas Y Smith / Eva-Christina Ahlgren / Salam Al-Karadaghi / James R Thompson / Grazia Isaya / 要旨: Fe-S clusters, essential cofactors needed for the activity of many different enzymes, are assembled by conserved protein machineries inside bacteria and mitochondria. As the architecture of the human ...Fe-S clusters, essential cofactors needed for the activity of many different enzymes, are assembled by conserved protein machineries inside bacteria and mitochondria. As the architecture of the human machinery remains undefined, we co-expressed in Escherichia coli the following four proteins involved in the initial step of Fe-S cluster synthesis: FXN (iron donor); [NFS1]·[ISD11] (sulfur donor); and ISCU (scaffold upon which new clusters are assembled). We purified a stable, active complex consisting of all four proteins with 1:1:1:1 stoichiometry. Using negative staining transmission EM and single particle analysis, we obtained a three-dimensional model of the complex with ∼14 Å resolution. Molecular dynamics flexible fitting of protein structures docked into the EM map of the model revealed a [FXN]·[NFS1]·[ISD11]·[ISCU] complex, consistent with the measured 1:1:1:1 stoichiometry of its four components. The complex structure fulfills distance constraints obtained from chemical cross-linking of the complex at multiple recurring interfaces, involving hydrogen bonds, salt bridges, or hydrophobic interactions between conserved residues. The complex consists of a central roughly cubic [FXN]·[ISCU] sub-complex with one symmetric ISCU trimer bound on top of one symmetric FXN trimer at each of its eight vertices. Binding of 12 [NFS1]·[ISD11] sub-complexes to the surface results in a globular macromolecule with a diameter of ∼15 nm and creates 24 Fe-S cluster assembly centers. The organization of each center recapitulates a previously proposed conserved mechanism for sulfur donation from NFS1 to ISCU and reveals, for the first time, a path for iron donation from FXN to ISCU.
試料ホルダーモデル: SIDE ENTRY, EUCENTRIC / ホルダー冷却材: NITROGEN
実験機器
モデル: Tecnai F30 / 画像提供: FEI Company
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画像解析
詳細
432 symmetry
粒子像選択
選択した数: 4124
CTF補正
ソフトウェア - 名称: EMAN2 (ver. 2.06) / 詳細: The ctf.auto function from EMAN2 was applied.
最終 再構成
想定した対称性 - 点群: O (正8面体型対称) / アルゴリズム: FOURIER SPACE / 解像度のタイプ: BY AUTHOR / 解像度: 14.3 Å / 解像度の算出法: FSC 0.143 CUT-OFF / ソフトウェア - 名称: EMAN2 (ver. 2.06) / 使用した粒子像数: 4124
初期 角度割当
タイプ: NOT APPLICABLE
最終 角度割当
タイプ: NOT APPLICABLE
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: RIGID BODY FIT
得られたモデル
PDB-5kz5: Architecture of the Human Mitochondrial Iron-Sulfur Cluster Assembly Machinery: the Complex Formed by the Iron Donor, the Sulfur Donor, and the Scaffold