+データを開く
-基本情報
登録情報 | データベース: PDB / ID: 5kz5 | ||||||
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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 | ||||||
要素 |
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キーワード | TRANSFERASE/OXIDOREDUCTASE / frataxin / iron-sulfur protein / mitochondria / protein complex / TRANSFERASE-OXIDOREDUCTASE complex | ||||||
機能・相同性 | 機能・相同性情報 regulation of ferrochelatase activity / negative regulation of iron ion import across plasma membrane / molybdopterin cofactor metabolic process / Molybdenum cofactor biosynthesis / proprioception / [4Fe-4S] cluster assembly / iron incorporation into metallo-sulfur cluster / positive regulation of lyase activity / positive regulation of succinate dehydrogenase activity / L-cysteine desulfurase complex ...regulation of ferrochelatase activity / negative regulation of iron ion import across plasma membrane / molybdopterin cofactor metabolic process / Molybdenum cofactor biosynthesis / proprioception / [4Fe-4S] cluster assembly / iron incorporation into metallo-sulfur cluster / positive regulation of lyase activity / positive regulation of succinate dehydrogenase activity / L-cysteine desulfurase complex / Mitochondrial iron-sulfur cluster biogenesis / mitochondrial [2Fe-2S] assembly complex / positive regulation of aconitate hydratase activity / positive regulation of mitochondrial electron transport, NADH to ubiquinone / iron chaperone activity / Maturation of TCA enzymes and regulation of TCA cycle / cysteine desulfurase / cysteine desulfurase activity / negative regulation of organ growth / Mo-molybdopterin cofactor biosynthetic process / iron-sulfur cluster assembly complex / Mitochondrial protein import / [2Fe-2S] cluster assembly / oxidative phosphorylation / adult walking behavior / response to iron ion / embryo development ending in birth or egg hatching / iron-sulfur cluster assembly / heme biosynthetic process / negative regulation of multicellular organism growth / organ growth / positive regulation of catalytic activity / muscle cell cellular homeostasis / ferroxidase / negative regulation of release of cytochrome c from mitochondria / iron-sulfur cluster binding / protein autoprocessing / ferroxidase activity / ferric iron binding / mitochondrion organization / ferrous iron binding / 2 iron, 2 sulfur cluster binding / cellular response to hydrogen peroxide / pyridoxal phosphate binding / Maturation of replicase proteins / positive regulation of cell growth / iron ion transport / intracellular iron ion homeostasis / molecular adaptor activity / mitochondrial matrix / iron ion binding / centrosome / positive regulation of cell population proliferation / negative regulation of apoptotic process / protein homodimerization activity / mitochondrion / zinc ion binding / nucleoplasm / nucleus / metal ion binding / cytoplasm / cytosol 類似検索 - 分子機能 | ||||||
生物種 | Homo sapiens (ヒト) | ||||||
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: 14.3 Å | ||||||
データ登録者 | Gakh, O. / Ranatunga, W. / Smith, D.Y. / Ahlgren, E.C. / Al-Karadaghi, S. / Thompson, J.R. / Isaya, G. | ||||||
資金援助 | 米国, 1件
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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. | ||||||
履歴 |
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-構造の表示
ムービー |
ムービービューア |
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構造ビューア | 分子: MolmilJmol/JSmol |
-ダウンロードとリンク
-ダウンロード
PDBx/mmCIF形式 | 5kz5.cif.gz | 1.3 MB | 表示 | PDBx/mmCIF形式 |
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PDB形式 | pdb5kz5.ent.gz | 1.1 MB | 表示 | PDB形式 |
PDBx/mmJSON形式 | 5kz5.json.gz | ツリー表示 | PDBx/mmJSON形式 | |
その他 | その他のダウンロード |
-検証レポート
文書・要旨 | 5kz5_validation.pdf.gz | 1.1 MB | 表示 | wwPDB検証レポート |
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文書・詳細版 | 5kz5_full_validation.pdf.gz | 1.2 MB | 表示 | |
XML形式データ | 5kz5_validation.xml.gz | 194.5 KB | 表示 | |
CIF形式データ | 5kz5_validation.cif.gz | 304.5 KB | 表示 | |
アーカイブディレクトリ | https://data.pdbj.org/pub/pdb/validation_reports/kz/5kz5 ftp://data.pdbj.org/pub/pdb/validation_reports/kz/5kz5 | HTTPS FTP |
-関連構造データ
-リンク
-集合体
登録構造単位 |
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1 |
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-要素
#1: タンパク質 | 分子量: 43429.648 Da / 分子数: 12 / 由来タイプ: 組換発現 / 由来: (組換発現) Homo sapiens (ヒト) / 遺伝子: NFS1, NIFS, HUSSY-08 / 発現宿主: Escherichia coli #1/H766 (大腸菌) / 参照: UniProt: Q9Y697, cysteine desulfurase #2: タンパク質 | 分子量: 18849.025 Da / 分子数: 12 / 由来タイプ: 組換発現 / 由来: (組換発現) Homo sapiens (ヒト) / 遺伝子: FXN, FRDA, X25 / 発現宿主: Escherichia coli #1/H766 (大腸菌) / 参照: UniProt: Q16595, ferroxidase #3: タンパク質 | 分子量: 12525.580 Da / 分子数: 12 / 由来タイプ: 組換発現 / 由来: (組換発現) Homo sapiens (ヒト) / 遺伝子: ISCU, NIFUN / 発現宿主: Escherichia coli #1/H766 (大腸菌) / 参照: UniProt: Q9H1K1 |
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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 |
-試料調製
構成要素 | 名称: NFS1-ISD11-ISCU-FXN / タイプ: COMPLEX / Entity ID: all / 由来: RECOMBINANT | |||||||||||||||
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由来(天然) | 生物種: Homo sapiens (ヒト) | |||||||||||||||
由来(組換発現) | 生物種: Escherichia coli (大腸菌) / プラスミド: pCDF, pET | |||||||||||||||
緩衝液 | pH: 8 | |||||||||||||||
緩衝液成分 |
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試料 | 濃度: 0.3 mg/ml / 包埋: NO / シャドウイング: NO / 染色: YES / 凍結: NO / 詳細: 50 mM Tris-HCl, pH 8.0, 150 mM NaCl | |||||||||||||||
染色 | タイプ: NEGATIVE / 詳細: 5 and 30 seconds / 染色剤: 1% uranyl acetate | |||||||||||||||
試料支持 | 詳細: DV-502A vacuum evaporator (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 copper grids, EMS |
-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: FIELD EMISSION GUN / 加速電圧: 300 kV / 照射モード: OTHER |
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): 3000 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): 3000 nm / Cs: 2 mm |
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: 466 |
画像スキャン | 横: 4096 / 縦: 4096 |
-解析
EMソフトウェア |
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画像処理 | 詳細: 432 symmetry | ||||||||||||||||||||||||||||||||
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: 4124 | ||||||||||||||||||||||||||||||||
対称性 | 点対称性: O (正8面体型対称) | ||||||||||||||||||||||||||||||||
3次元再構成 | 解像度: 14.3 Å / 解像度の算出法: FSC 0.143 CUT-OFF / 粒子像の数: 4124 / 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 |