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データを開く
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基本情報
登録情報 | データベース: EMDB / ID: EMD-30310 | ||||||||||||
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タイトル | Human RAD51 post-synaptic complexes mutant (V273P, D274G) | ||||||||||||
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![]() | meitotic homologous recombination / DNA repair / ATPase / RECOMBINATION | ||||||||||||
機能・相同性 | ![]() 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 / nuclear ubiquitin ligase complex / double-strand break repair involved in meiotic recombination / 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 / nuclear ubiquitin ligase complex / double-strand break repair involved in meiotic recombination / 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 / Impaired BRCA2 binding to PALB2 / DNA strand invasion / 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 / condensed nuclear chromosome / meiotic cell cycle / 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 / Meiotic recombination / site of double-strand break / single-stranded DNA binding / double-stranded DNA binding / DNA recombination / chromosome, telomeric region / mitochondrial matrix / DNA repair / centrosome / DNA damage response / chromatin binding / chromatin / nucleolus / perinuclear region of cytoplasm / enzyme binding / ATP hydrolysis activity / protein-containing complex / mitochondrion / nucleoplasm / ATP binding / identical protein binding / nucleus / cytosol / cytoplasm 類似検索 - 分子機能 | ||||||||||||
生物種 | ![]() | ||||||||||||
手法 | らせん対称体再構成法 / クライオ電子顕微鏡法 / 解像度: 3.43 Å | ||||||||||||
![]() | Chi HY / Ho MC / Tsai MD / Luo SC / Yeh HY | ||||||||||||
資金援助 | ![]()
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![]() | ![]() タイトル: Identification of fidelity-governing factors in human recombinases DMC1 and RAD51 from cryo-EM structures. 著者: Shih-Chi Luo / Hsin-Yi Yeh / Wei-Hsuan Lan / Yi-Min Wu / Cheng-Han Yang / Hao-Yen Chang / Guan-Chin Su / Chia-Yi Lee / Wen-Jin Wu / Hung-Wen Li / Meng-Chiao Ho / Peter Chi / Ming-Daw Tsai / ![]() 要旨: Both high-fidelity and mismatch-tolerant recombination, catalyzed by RAD51 and DMC1 recombinases, respectively, are indispensable for genomic integrity. Here, we use cryo-EM, MD simulation and ...Both high-fidelity and mismatch-tolerant recombination, catalyzed by RAD51 and DMC1 recombinases, respectively, are indispensable for genomic integrity. Here, we use cryo-EM, MD simulation and functional analysis to elucidate the structural basis for the mismatch tolerance of DMC1. Structural analysis of DMC1 presynaptic and postsynaptic complexes suggested that the lineage-specific Loop 1 Gln244 (Met243 in RAD51) may help stabilize DNA backbone, whereas Loop 2 Pro274 and Gly275 (Val273/Asp274 in RAD51) may provide an open "triplet gate" for mismatch tolerance. In support, DMC1-Q244M displayed marked increase in DNA dynamics, leading to unobservable DNA map. MD simulation showed highly dispersive mismatched DNA ensemble in RAD51 but well-converged DNA in DMC1 and RAD51-V273P/D274G. Replacing Loop 1 or Loop 2 residues in DMC1 with RAD51 counterparts enhanced DMC1 fidelity, while reciprocal mutations in RAD51 attenuated its fidelity. Our results show that three Loop 1/Loop 2 residues jointly enact contrasting fidelities of DNA recombinases. | ||||||||||||
履歴 |
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構造の表示
ムービー |
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構造ビューア | EMマップ: ![]() ![]() ![]() |
添付画像 |
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マップデータ | ![]() | 202.5 MB | ![]() | |
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ヘッダ (付随情報) | ![]() ![]() | 16.8 KB 16.8 KB | 表示 表示 | ![]() |
FSC (解像度算出) | ![]() | 13.7 KB | 表示 | ![]() |
画像 | ![]() | 106 KB | ||
Filedesc metadata | ![]() | 6.8 KB | ||
アーカイブディレクトリ | ![]() ![]() | HTTPS FTP |
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リンク
EMDBのページ | ![]() ![]() |
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「今月の分子」の関連する項目 |
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マップ
ファイル | ![]() | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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ボクセルのサイズ | X=Y=Z: 0.84 Å | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
密度 |
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対称性 | 空間群: 1 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
詳細 | EMDB XML:
CCP4マップ ヘッダ情報:
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-添付データ
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試料の構成要素
-全体 : RAD51-dsDNA filament (V273P D274G mutant)
全体 | 名称: RAD51-dsDNA filament (V273P D274G mutant) |
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要素 |
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-超分子 #1: RAD51-dsDNA filament (V273P D274G mutant)
超分子 | 名称: RAD51-dsDNA filament (V273P D274G mutant) / タイプ: complex / ID: 1 / 親要素: 0 / 含まれる分子: #1-#3 |
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由来(天然) | 生物種: ![]() |
-分子 #1: DNA repair protein RAD51 homolog 1
分子 | 名称: DNA repair protein RAD51 homolog 1 / タイプ: protein_or_peptide / ID: 1 / コピー数: 3 / 光学異性体: LEVO |
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由来(天然) | 生物種: ![]() |
分子量 | 理論値: 36.949074 KDa |
組換発現 | 生物種: ![]() ![]() |
配列 | 文字列: MAMQMQLEAN ADTSVEEESF GPQPISRLEQ CGINANDVKK LEEAGFHTVE AVAYAPKKEL INIKGISEAK ADKILAEAAK LVPMGFTTA TEFHQRRSEI IQITTGSKEL DKLLQGGIET GSITEMFGEF RTGKTQICHT LAVTCQLPID RGGGEGKAMY I DTEGTFRP ...文字列: MAMQMQLEAN ADTSVEEESF GPQPISRLEQ CGINANDVKK LEEAGFHTVE AVAYAPKKEL INIKGISEAK ADKILAEAAK LVPMGFTTA TEFHQRRSEI IQITTGSKEL DKLLQGGIET GSITEMFGEF RTGKTQICHT LAVTCQLPID RGGGEGKAMY I DTEGTFRP ERLLAVAERY GLSGSDVLDN VAYARAFNTD HQTQLLYQAS AMMVESRYAL LIVDSATALY RTDYSGRGEL SA RQMHLAR FLRMLLRLAD EFGVAVVITN QVVAQPGGAA MFAADPKKPI GGNIIAHAST TRLYLRKGRG ETRICKIYDS PCL PEAEAM FAINADGVGD AKD UniProtKB: DNA repair protein RAD51 homolog 1 |
-分子 #2: DNA (5'-D(P*TP*TP*TP*TP*TP*TP*TP*TP*T)-3')
分子 | 名称: DNA (5'-D(P*TP*TP*TP*TP*TP*TP*TP*TP*T)-3') / タイプ: dna / ID: 2 詳細: Authors know the sequence of chains D/E. Chain D: TCA GCT GTT GCC CGT. Chain E: ACG GGC AAC AGC TGA. Since the DNA sequence could not be revealed by the helical reconstruction method, authors ...詳細: Authors know the sequence of chains D/E. Chain D: TCA GCT GTT GCC CGT. Chain E: ACG GGC AAC AGC TGA. Since the DNA sequence could not be revealed by the helical reconstruction method, authors have used poly-T for chain D, poly-A for chain E respectively for model building. コピー数: 1 / 分類: DNA |
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由来(天然) | 生物種: ![]() |
分子量 | 理論値: 2.692778 KDa |
配列 | 文字列: (DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT) |
-分子 #3: DNA (5'-D(P*AP*AP*AP*AP*AP*AP*AP*AP*A)-3')
分子 | 名称: DNA (5'-D(P*AP*AP*AP*AP*AP*AP*AP*AP*A)-3') / タイプ: dna / ID: 3 詳細: Authors know the sequence of chains D/E. Chain D: TCA GCT GTT GCC CGT. Chain E: ACG GGC AAC AGC TGA. Since the DNA sequence could not be revealed by the helical reconstruction method, authors ...詳細: Authors know the sequence of chains D/E. Chain D: TCA GCT GTT GCC CGT. Chain E: ACG GGC AAC AGC TGA. Since the DNA sequence could not be revealed by the helical reconstruction method, authors have used poly-T for chain D, poly-A for chain E respectively for model building. コピー数: 1 / 分類: DNA |
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由来(天然) | 生物種: ![]() |
分子量 | 理論値: 2.773904 KDa |
配列 | 文字列: (DA)(DA)(DA)(DA)(DA)(DA)(DA)(DA)(DA) |
-分子 #4: CALCIUM ION
分子 | 名称: CALCIUM ION / タイプ: ligand / ID: 4 / コピー数: 3 / 式: CA |
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分子量 | 理論値: 40.078 Da |
-分子 #5: PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER
分子 | 名称: PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER / タイプ: ligand / ID: 5 / コピー数: 3 / 式: ANP |
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分子量 | 理論値: 506.196 Da |
Chemical component information | ![]() ChemComp-ANP: |
-実験情報
-構造解析
手法 | クライオ電子顕微鏡法 |
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![]() | らせん対称体再構成法 |
試料の集合状態 | filament |
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試料調製
緩衝液 | pH: 7.5 / 構成要素 - 濃度: 25.0 mM / 構成要素 - 式: Tris-HCl 詳細: 25 mM Tris-HCl, pH 7.5, 50 mM KCl and 1 mM dithiothreitol) containing 2 mM AMP-PNP and 5 mM CaCl2 |
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グリッド | モデル: Quantifoil R1.2/1.3 / 材質: COPPER / メッシュ: 200 / 支持フィルム - 材質: GRAPHENE OXIDE / 支持フィルム - トポロジー: CONTINUOUS / 前処理 - タイプ: GLOW DISCHARGE / 前処理 - 時間: 15 sec. |
凍結 | 凍結剤: ETHANE / チャンバー内湿度: 100 % / チャンバー内温度: 295 K / 装置: FEI VITROBOT MARK IV 詳細: The grids were blotted for 1 sec at 22 degree C with 100% relative humidity and plunge-frozen in liquid ethane cooled by liquid nitrogen using a Vitrobot Mark IV (Thermo Fisher).. |
詳細 | protein sample were applied onto a pre-glow-discharged graphene-oxide coated Quantifoil holey carbon grid (1.2/1.3, 200 mesh) using published protocol |
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電子顕微鏡法
顕微鏡 | FEI TITAN KRIOS |
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アライメント法 | Coma free - Residual tilt: 10.0 mrad |
特殊光学系 | エネルギーフィルター - 名称: GIF Quantum LS / エネルギーフィルター - スリット幅: 30 eV |
撮影 | フィルム・検出器のモデル: GATAN K2 QUANTUM (4k x 4k) 検出モード: COUNTING / デジタル化 - サイズ - 横: 3838 pixel / デジタル化 - サイズ - 縦: 3710 pixel / デジタル化 - 画像ごとのフレーム数: 1-50 / 撮影したグリッド数: 1 / 平均露光時間: 5.0 sec. / 平均電子線量: 50.0 e/Å2 |
電子線 | 加速電圧: 300 kV / 電子線源: ![]() |
電子光学系 | C2レンズ絞り径: 70.0 µm / 照射モード: FLOOD BEAM / 撮影モード: BRIGHT FIELD / Cs: 2.7 mm / 倍率(公称値): 165000 |
試料ステージ | 試料ホルダーモデル: FEI TITAN KRIOS AUTOGRID HOLDER ホルダー冷却材: NITROGEN |
実験機器 | ![]() モデル: Titan Krios / 画像提供: FEI Company |