+データを開く
-基本情報
登録情報 | データベース: PDB / ID: 8ouz | ||||||||||||||||||
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タイトル | Human RAD51B-RAD51C-RAD51D-XRCC2 (BCDX2) complex, 2.2 A resolution | ||||||||||||||||||
要素 |
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キーワード | DNA BINDING PROTEIN / Complex / ssDNA-binding / ATPase / RAD51 | ||||||||||||||||||
機能・相同性 | 機能・相同性情報 meiotic DNA recombinase assembly / Rad51C-XRCC3 complex / Rad51B-Rad51C-Rad51D-XRCC2 complex / female meiosis sister chromatid cohesion / blastocyst growth / crossover junction DNA endonuclease activity / somite development / telomere maintenance via recombination / Impaired BRCA2 binding to PALB2 / DNA strand invasion ...meiotic DNA recombinase assembly / Rad51C-XRCC3 complex / Rad51B-Rad51C-Rad51D-XRCC2 complex / female meiosis sister chromatid cohesion / blastocyst growth / crossover junction DNA endonuclease activity / somite development / telomere maintenance via recombination / Impaired BRCA2 binding to PALB2 / DNA strand invasion / gamma-tubulin binding / reciprocal meiotic recombination / sister chromatid cohesion / regulation of fibroblast apoptotic process / centrosome cycle / 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) / Resolution of D-loop Structures through Holliday Junction Intermediates / positive regulation of neurogenesis / ATP-dependent DNA damage sensor activity / male meiosis I / Presynaptic phase of homologous DNA pairing and strand exchange / response to X-ray / ATP-dependent activity, acting on DNA / somitogenesis / interstrand cross-link repair / four-way junction DNA binding / positive regulation of G2/M transition of mitotic cell cycle / telomere maintenance / neurogenesis / meiotic cell cycle / replication fork / response to gamma radiation / TP53 Regulates Transcription of DNA Repair Genes / double-strand break repair via homologous recombination / HDR through Homologous Recombination (HRR) / multicellular organism growth / Meiotic recombination / cell junction / mitotic cell cycle / single-stranded DNA binding / Factors involved in megakaryocyte development and platelet production / spermatogenesis / double-stranded DNA binding / DNA recombination / in utero embryonic development / negative regulation of neuron apoptotic process / chromosome, telomeric region / regulation of cell cycle / intracellular membrane-bounded organelle / DNA repair / centrosome / positive regulation of cell population proliferation / perinuclear region of cytoplasm / ATP hydrolysis activity / mitochondrion / DNA binding / nucleoplasm / ATP binding / nucleus / 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 / クライオ電子顕微鏡法 / 解像度: 2.2 Å | ||||||||||||||||||
データ登録者 | Greenhough, L.A. / Liang, C.C. / West, S.C. | ||||||||||||||||||
資金援助 | 英国, European Union, 5件
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引用 | ジャーナル: Nature / 年: 2023 タイトル: Structure and function of the RAD51B-RAD51C-RAD51D-XRCC2 tumour suppressor. 著者: Luke A Greenhough / Chih-Chao Liang / Ondrej Belan / Simone Kunzelmann / Sarah Maslen / Monica C Rodrigo-Brenni / Roopesh Anand / Mark Skehel / Simon J Boulton / Stephen C West / 要旨: Homologous recombination is a fundamental process of life. It is required for the protection and restart of broken replication forks, the repair of chromosome breaks and the exchange of genetic ...Homologous recombination is a fundamental process of life. It is required for the protection and restart of broken replication forks, the repair of chromosome breaks and the exchange of genetic material during meiosis. Individuals with mutations in key recombination genes, such as BRCA2 (also known as FANCD1), or the RAD51 paralogues RAD51B, RAD51C (also known as FANCO), RAD51D, XRCC2 (also known as FANCU) and XRCC3, are predisposed to breast, ovarian and prostate cancers and the cancer-prone syndrome Fanconi anaemia. The BRCA2 tumour suppressor protein-the product of BRCA2-is well characterized, but the cellular functions of the RAD51 paralogues remain unclear. Genetic knockouts display growth defects, reduced RAD51 focus formation, spontaneous chromosome abnormalities, sensitivity to PARP inhibitors and replication fork defects, but the precise molecular roles of RAD51 paralogues in fork stability, DNA repair and cancer avoidance remain unknown. Here we used cryo-electron microscopy, AlphaFold2 modelling and structural proteomics to determine the structure of the RAD51B-RAD51C-RAD51D-XRCC2 complex (BCDX2), revealing that RAD51C-RAD51D-XRCC2 mimics three RAD51 protomers aligned within a nucleoprotein filament, whereas RAD51B is highly dynamic. Biochemical and single-molecule analyses showed that BCDX2 stimulates the nucleation and extension of RAD51 filaments-which are essential for recombinational DNA repair-in reactions that depend on the coupled ATPase activities of RAD51B and RAD51C. Our studies demonstrate that BCDX2 orchestrates RAD51 assembly on single stranded DNA for replication fork protection and double strand break repair, in reactions that are critical for tumour avoidance. | ||||||||||||||||||
履歴 |
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-構造の表示
構造ビューア | 分子: MolmilJmol/JSmol |
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-ダウンロードとリンク
-ダウンロード
PDBx/mmCIF形式 | 8ouz.cif.gz | 364.7 KB | 表示 | PDBx/mmCIF形式 |
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PDB形式 | pdb8ouz.ent.gz | 294.7 KB | 表示 | PDB形式 |
PDBx/mmJSON形式 | 8ouz.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 | 8ouz_validation.pdf.gz | 1.2 MB | 表示 | wwPDB検証レポート |
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文書・詳細版 | 8ouz_full_validation.pdf.gz | 1.2 MB | 表示 | |
XML形式データ | 8ouz_validation.xml.gz | 41.4 KB | 表示 | |
CIF形式データ | 8ouz_validation.cif.gz | 62.5 KB | 表示 | |
アーカイブディレクトリ | https://data.pdbj.org/pub/pdb/validation_reports/ou/8ouz ftp://data.pdbj.org/pub/pdb/validation_reports/ou/8ouz | 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
-DNA repair protein RAD51 homolog ... , 3種, 3分子 ABC
#1: タンパク質 | 分子量: 38296.984 Da / 分子数: 1 / 由来タイプ: 組換発現 / 由来: (組換発現) Homo sapiens (ヒト) / 遺伝子: RAD51B, RAD51L1, REC2 / プラスミド: pBIG-BCDX2 / 細胞株 (発現宿主): Sf9 発現宿主: Spodoptera frugiperda (ツマジロクサヨトウ) 参照: UniProt: O15315 |
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#2: タンパク質 | 分子量: 42244.609 Da / 分子数: 1 / 由来タイプ: 組換発現 / 由来: (組換発現) Homo sapiens (ヒト) / 遺伝子: RAD51C, RAD51L2 / プラスミド: pBIG-BCDX2 / 細胞株 (発現宿主): Sf9 発現宿主: Spodoptera frugiperda (ツマジロクサヨトウ) 参照: UniProt: O43502 |
#3: タンパク質 | 分子量: 35084.254 Da / 分子数: 1 / 由来タイプ: 組換発現 / 由来: (組換発現) Homo sapiens (ヒト) / 遺伝子: RAD51D, RAD51L3 / プラスミド: pBIG-BCDX2 / 細胞株 (発現宿主): Sf9 発現宿主: Spodoptera frugiperda (ツマジロクサヨトウ) 参照: UniProt: O75771 |
-タンパク質 , 1種, 1分子 D
#4: タンパク質 | 分子量: 31921.381 Da / 分子数: 1 / 由来タイプ: 組換発現 / 由来: (組換発現) Homo sapiens (ヒト) / 遺伝子: XRCC2 / プラスミド: pBIG-BCDX2 / 細胞株 (発現宿主): Sf9 発現宿主: Spodoptera frugiperda (ツマジロクサヨトウ) 参照: UniProt: O43543 |
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-非ポリマー , 4種, 148分子
#5: 化合物 | ChemComp-ADP / | ||||
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#6: 化合物 | #7: 化合物 | #8: 水 | ChemComp-HOH / | |
-詳細
研究の焦点であるリガンドがあるか | Y |
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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: RAD51B-RAD51C-RAD51D-XRCC2 (BCDX2) / タイプ: COMPLEX 詳細: Recombinant BCDX2 complexes purified from Sf9 insect cells, crosslinked with 0.005% glutaraldehyde (RT, 10 minutes) in the presence of 30 nt ssDNA, and vitrified in the presence of ADP.AlFx. ...詳細: Recombinant BCDX2 complexes purified from Sf9 insect cells, crosslinked with 0.005% glutaraldehyde (RT, 10 minutes) in the presence of 30 nt ssDNA, and vitrified in the presence of ADP.AlFx. THERE IS NO ssDNA BOUND TO BCDX2 IN THIS STRUCTURE Entity ID: #1-#4 / 由来: RECOMBINANT | ||||||||||||||||||||||||||||||||||||||||||||||||||
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分子量 | 実験値: NO | ||||||||||||||||||||||||||||||||||||||||||||||||||
由来(天然) | 生物種: Homo sapiens (ヒト) | ||||||||||||||||||||||||||||||||||||||||||||||||||
由来(組換発現) | 生物種: Spodoptera frugiperda (ツマジロクサヨトウ) 株: Sf9 / プラスミド: pBIG-BCDX2 | ||||||||||||||||||||||||||||||||||||||||||||||||||
緩衝液 | pH: 7.5 詳細: 25 mM HEPES pH 7.5, 100 mM NaCl, 2.5 mM MgCl2, 0.5 mM ADP.AlFx (0.5 mM ADP, 0.5 mM AlCl3, 10 mM NaF), 0.25 mM TCEP, 0.00075% Tween20, 0.075 mM CHAPSO | ||||||||||||||||||||||||||||||||||||||||||||||||||
緩衝液成分 |
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試料 | 濃度: 0.25 mg/ml / 包埋: NO / シャドウイング: NO / 染色: NO / 凍結: YES | ||||||||||||||||||||||||||||||||||||||||||||||||||
試料支持 | 詳細: 45 mA / グリッドの材料: GOLD / グリッドのサイズ: 200 divisions/in. / グリッドのタイプ: UltrAuFoil R2/2 | ||||||||||||||||||||||||||||||||||||||||||||||||||
急速凍結 | 装置: FEI VITROBOT MARK IV / 凍結剤: ETHANE / 湿度: 95 % / 凍結前の試料温度: 277.15 K |
-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: SPOT SCAN |
電子レンズ | モード: BRIGHT FIELD / 倍率(公称値): 105000 X / 最大 デフォーカス(公称値): 2250 nm / 最小 デフォーカス(公称値): 750 nm / Cs: 2.7 mm / C2レンズ絞り径: 50 µm / 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 | 平均露光時間: 2.78 sec. / 電子線照射量: 53.2 e/Å2 フィルム・検出器のモデル: GATAN K3 BIOQUANTUM (6k x 4k) 撮影したグリッド数: 1 / 実像数: 35305 |
-解析
ソフトウェア |
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EMソフトウェア |
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CTF補正 | タイプ: PHASE FLIPPING AND AMPLITUDE CORRECTION | ||||||||||||||||||||||||||||||||||||||||
粒子像の選択 | 選択した粒子像数: 25886382 | ||||||||||||||||||||||||||||||||||||||||
対称性 | 点対称性: C1 (非対称) | ||||||||||||||||||||||||||||||||||||||||
3次元再構成 | 解像度: 2.2 Å / 解像度の算出法: FSC 0.143 CUT-OFF / 粒子像の数: 1371033 / アルゴリズム: FOURIER SPACE / クラス平均像の数: 1 / 対称性のタイプ: POINT | ||||||||||||||||||||||||||||||||||||||||
原子モデル構築 | B value: 50.38 / プロトコル: RIGID BODY FIT / 空間: REAL 詳細: PDB-8OUY was initially docked into the postprocessed map using ChimeraX 1.4, and iteratively real space refined with Phenix and manually modified using COOT | ||||||||||||||||||||||||||||||||||||||||
原子モデル構築 | PDB-ID: 8OUY Accession code: 8OUY / Source name: PDB / タイプ: experimental model | ||||||||||||||||||||||||||||||||||||||||
精密化 | 交差検証法: NONE | ||||||||||||||||||||||||||||||||||||||||
拘束条件 |
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