+データを開く
-基本情報
登録情報 | データベース: PDB / ID: 5np7 | ||||||
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タイトル | CryoEM structure of Human Rad51 on single-stranded DNA to 4.2A resolution. | ||||||
要素 | DNA repair protein RAD51 homolog 1DNA修復 | ||||||
キーワード | RECOMBINATION (遺伝的組換え) / recombinase / cryoEM (低温電子顕微鏡法) / Human Rad51 (Rad51) / single-stranded DNA (デオキシリボ核酸) | ||||||
機能・相同性 | 機能・相同性情報 presynaptic intermediate filament cytoskeleton / mitotic recombination-dependent replication fork processing / cellular response to camptothecin / chromosome organization involved in meiotic cell cycle / telomere maintenance via telomere lengthening / positive regulation of DNA ligation / double-strand break repair involved in meiotic recombination / nuclear ubiquitin ligase complex / cellular response to hydroxyurea / replication-born double-strand break repair via sister chromatid exchange ...presynaptic intermediate filament cytoskeleton / mitotic recombination-dependent replication fork processing / cellular response to camptothecin / chromosome organization involved in meiotic cell cycle / telomere maintenance via telomere lengthening / positive regulation of DNA ligation / double-strand break repair involved in meiotic recombination / nuclear ubiquitin ligase complex / cellular response to hydroxyurea / replication-born double-strand break repair via sister chromatid exchange / lateral element / telomere maintenance via recombination / DNA recombinase assembly / regulation of DNA damage checkpoint / mitotic recombination / DNA strand invasion / Impaired BRCA2 binding to PALB2 / DNA strand exchange activity / reciprocal meiotic recombination / Defective homologous recombination repair (HRR) due to BRCA1 loss of function / Defective HDR through Homologous Recombination Repair (HRR) due to PALB2 loss of BRCA1 binding function / Defective HDR through Homologous Recombination Repair (HRR) due to PALB2 loss of BRCA2/RAD51/RAD51C binding function / Homologous DNA Pairing and Strand Exchange / Resolution of D-loop Structures through Synthesis-Dependent Strand Annealing (SDSA) / single-stranded DNA helicase activity / Resolution of D-loop Structures through Holliday Junction Intermediates / ATP-dependent DNA damage sensor activity / HDR through Single Strand Annealing (SSA) / Impaired BRCA2 binding to RAD51 / regulation of double-strand break repair via homologous recombination / nuclear chromosome / replication fork processing / DNA unwinding involved in DNA replication / Transcriptional Regulation by E2F6 / Presynaptic phase of homologous DNA pairing and strand exchange / ATP-dependent activity, acting on DNA / interstrand cross-link repair / DNA polymerase binding / condensed chromosome / meiotic cell cycle / condensed nuclear chromosome / male germ cell nucleus / cellular response to ionizing radiation / regulation of protein phosphorylation / double-strand break repair via homologous recombination / HDR through Homologous Recombination (HRR) / PML body / 遺伝的組換え / site of double-strand break / single-stranded DNA binding / double-stranded DNA binding / DNA recombination / chromosome, telomeric region / ミトコンドリアマトリックス / DNA修復 / 中心体 / DNA damage response / chromatin binding / クロマチン / 核小体 / perinuclear region of cytoplasm / enzyme binding / ATP hydrolysis activity / protein-containing complex / ミトコンドリア / 核質 / ATP binding / identical protein binding / 細胞核 / 細胞質基質 / 細胞質 類似検索 - 分子機能 | ||||||
生物種 | Homo sapiens (ヒト) | ||||||
手法 | 電子顕微鏡法 / らせん対称体再構成法 / クライオ電子顕微鏡法 / 解像度: 4.2 Å | ||||||
データ登録者 | Short, J.M. / Venkitaraman, A. | ||||||
引用 | ジャーナル: Nucleic Acids Res / 年: 2016 タイトル: High-resolution structure of the presynaptic RAD51 filament on single-stranded DNA by electron cryo-microscopy. 著者: Judith M Short / Yang Liu / Shaoxia Chen / Neelesh Soni / Mallur S Madhusudhan / Mahmud K K Shivji / Ashok R Venkitaraman / 要旨: Homologous DNA recombination (HR) by the RAD51 recombinase enables error-free DNA break repair. To execute HR, RAD51 first forms a presynaptic filament on single-stranded (ss) DNA, which catalyses ...Homologous DNA recombination (HR) by the RAD51 recombinase enables error-free DNA break repair. To execute HR, RAD51 first forms a presynaptic filament on single-stranded (ss) DNA, which catalyses pairing with homologous double-stranded (ds) DNA. Here, we report a structure for the presynaptic human RAD51 filament at 3.5-5.0Å resolution using electron cryo-microscopy. RAD51 encases ssDNA in a helical filament of 103Å pitch, comprising 6.4 protomers per turn, with a rise of 16.1Å and a twist of 56.2°. Inter-protomer distance correlates with rotation of an α-helical region in the core catalytic domain that is juxtaposed to ssDNA, suggesting how the RAD51-DNA interaction modulates protomer spacing and filament pitch. We map Fanconi anaemia-like disease-associated RAD51 mutations, clarifying potential phenotypes. We predict binding sites on the presynaptic filament for two modules present in each BRC repeat of the BRCA2 tumour suppressor, a critical HR mediator. Structural modelling suggests that changes in filament pitch mask or expose one binding site with filament-inhibitory potential, rationalizing the paradoxical ability of the BRC repeats to either stabilize or inhibit filament formation at different steps during HR. Collectively, our findings provide fresh insight into the structural mechanism of HR and its dysregulation in human disease. | ||||||
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 | 5np7.cif.gz | 420.6 KB | 表示 | PDBx/mmCIF形式 |
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PDB形式 | pdb5np7.ent.gz | 341.2 KB | 表示 | PDB形式 |
PDBx/mmJSON形式 | 5np7.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
アーカイブディレクトリ | https://data.pdbj.org/pub/pdb/validation_reports/np/5np7 ftp://data.pdbj.org/pub/pdb/validation_reports/np/5np7 | HTTPS FTP |
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-関連構造データ
-リンク
-集合体
登録構造単位 |
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-要素
#1: タンパク質 | 分子量: 37009.125 Da / 分子数: 7 / 由来タイプ: 組換発現 / 由来: (組換発現) Homo sapiens (ヒト) / 遺伝子: RAD51, RAD51A, RECA / 発現宿主: Escherichia coli (大腸菌) / 参照: UniProt: Q06609 #2: 化合物 | ChemComp-ANP / |
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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: HELICAL ARRAY / 3次元再構成法: らせん対称体再構成法 |
-試料調製
構成要素 | 名称: Helical filament of HRAD51 on single-stranded DNA with AMPPNP タイプ: COMPLEX / Entity ID: #1 / 由来: RECOMBINANT |
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分子量 | 値: 259 kDa/nm / 実験値: NO |
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) |
緩衝液 | pH: 7 |
試料 | 包埋: NO / シャドウイング: NO / 染色: NO / 凍結: YES |
試料支持 | 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: Quantifoil |
急速凍結 | 凍結剤: NITROGEN |
-電子顕微鏡撮影
実験機器 | モデル: Titan Krios / 画像提供: FEI Company |
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顕微鏡 | モデル: FEI TITAN KRIOS |
電子銃 | 電子線源: FIELD EMISSION GUN / 加速電圧: 300 kV / 照射モード: FLOOD BEAM |
電子レンズ | モード: BRIGHT FIELDBright-field microscopy / 倍率(公称値): 59000 X / 倍率(補正後): 104477 X / 最小 デフォーカス(公称値): 2000 nm / Cs: 2.7 mm / アライメント法: COMA FREE |
試料ホルダ | 凍結剤: NITROGEN 試料ホルダーモデル: FEI TITAN KRIOS AUTOGRID HOLDER |
撮影 | 電子線照射量: 20 e/Å2 / 検出モード: INTEGRATING フィルム・検出器のモデル: FEI FALCON II (4k x 4k) |
画像スキャン | サンプリングサイズ: 14 µm / 横: 4096 / 縦: 4096 / 動画フレーム数/画像: 34 |
-解析
ソフトウェア | 名称: REFMAC / バージョン: 5.8.0166 / 分類: 精密化 | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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EMソフトウェア |
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CTF補正 | 詳細: per segment / タイプ: NONE | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
らせん対称 | 回転角度/サブユニット: 56.2 ° / 軸方向距離/サブユニット: 16 Å / らせん対称軸の対称性: C1 | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
粒子像の選択 | 選択した粒子像数: 70000 | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
3次元再構成 | 解像度: 4.2 Å / 解像度の算出法: FSC 0.143 CUT-OFF / 粒子像の数: 60000 詳細: Helical segments were divided in half, not drawn randomly or odd/even as this would result in some segments from the same filament in both half models. Thus, it is strictly not gold-standard. 対称性のタイプ: HELICAL | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
原子モデル構築 | B value: 94.574 / プロトコル: FLEXIBLE FIT / 空間: RECIPROCAL / Target criteria: Maximum likelihood 詳細: Backbone fitting is good but many of the sidechains cannot be relied on as the map resolution was insufficient. | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
精密化 | 解像度: 4.2→180 Å / Cor.coef. Fo:Fc: 0.937 / SU B: 46.567 / SU ML: 0.572 立体化学のターゲット値: MAXIMUM LIKELIHOOD WITH PHASES
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溶媒の処理 | 溶媒モデル: PARAMETERS FOR MASK CACLULATION | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
原子変位パラメータ | Biso mean: 131.509 Å2
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精密化ステップ | サイクル: 1 / 合計: 16754 | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
拘束条件 |
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