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基本情報
登録情報 | データベース: EMDB / ID: EMD-20440 | |||||||||
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タイトル | Cryo-EM structure of AdnA(D934A)-AdnB(D1014A) in complex with AMPPNP | |||||||||
![]() | AdnA(D934A)-AdnB(D1014A) in complex with AMPPNP | |||||||||
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![]() | DNA BINDING PROTEIN | |||||||||
機能・相同性 | ![]() DNA helicase complex / DNA 3'-5' helicase / recombinational repair / exonuclease activity / 3'-5' DNA helicase activity / DNA helicase activity / isomerase activity / DNA helicase / hydrolase activity / DNA repair ...DNA helicase complex / DNA 3'-5' helicase / recombinational repair / exonuclease activity / 3'-5' DNA helicase activity / DNA helicase activity / isomerase activity / DNA helicase / hydrolase activity / DNA repair / DNA binding / ATP binding / cytosol 類似検索 - 分子機能 | |||||||||
生物種 | ![]() ![]() | |||||||||
手法 | 単粒子再構成法 / クライオ電子顕微鏡法 / 解像度: 3.5 Å | |||||||||
![]() | Jia N / Unciuleac M / Shuman S / Patel DJ | |||||||||
![]() | ![]() タイトル: Structures and single-molecule analysis of bacterial motor nuclease AdnAB illuminate the mechanism of DNA double-strand break resection. 著者: Ning Jia / Mihaela C Unciuleac / Chaoyou Xue / Eric C Greene / Dinshaw J Patel / Stewart Shuman / ![]() 要旨: Mycobacterial AdnAB is a heterodimeric helicase-nuclease that initiates homologous recombination by resecting DNA double-strand breaks (DSBs). The AdnA and AdnB subunits are each composed of an N- ...Mycobacterial AdnAB is a heterodimeric helicase-nuclease that initiates homologous recombination by resecting DNA double-strand breaks (DSBs). The AdnA and AdnB subunits are each composed of an N-terminal motor domain and a C-terminal nuclease domain. Here we report cryoelectron microscopy (cryo-EM) structures of AdnAB in three functional states: in the absence of DNA and in complex with forked duplex DNAs before and after cleavage of the 5' single-strand DNA (ssDNA) tail by the AdnA nuclease. The structures reveal the path of the 5' ssDNA through the AdnA nuclease domain and the mechanism of 5' strand cleavage; the path of the 3' tracking strand through the AdnB motor and the DNA contacts that couple ATP hydrolysis to mechanical work; the position of the AdnA iron-sulfur cluster subdomain at the Y junction and its likely role in maintaining the split trajectories of the unwound 5' and 3' strands. Single-molecule DNA curtain analysis of DSB resection reveals that AdnAB is highly processive but prone to spontaneous pausing at random sites on duplex DNA. A striking property of AdnAB is that the velocity of DSB resection slows after the enzyme experiences a spontaneous pause. Our results highlight shared as well as distinctive properties of AdnAB vis-à-vis the RecBCD and AddAB clades of bacterial DSB-resecting motor nucleases. | |||||||||
履歴 |
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構造の表示
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構造ビューア | EMマップ: ![]() ![]() ![]() |
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画像 | ![]() | 75.4 KB | ||
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-関連構造データ
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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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ファイル | ![]() | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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注釈 | AdnA(D934A)-AdnB(D1014A) in complex with AMPPNP | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
ボクセルのサイズ | X=Y=Z: 0.8613 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 : AdnAB-D934A-D1014A mutant in complex with AMPPNP
全体 | 名称: AdnAB-D934A-D1014A mutant in complex with AMPPNP |
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要素 |
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-超分子 #1: AdnAB-D934A-D1014A mutant in complex with AMPPNP
超分子 | 名称: AdnAB-D934A-D1014A mutant in complex with AMPPNP / タイプ: complex / ID: 1 / 親要素: 0 / 含まれる分子: #1-#2 |
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由来(天然) | 生物種: ![]() |
分子量 | 理論値: 200 KDa |
-分子 #1: UvrD/REP helicase
分子 | 名称: UvrD/REP helicase / タイプ: protein_or_peptide / ID: 1 / コピー数: 1 / 光学異性体: LEVO / EC番号: DNA helicase |
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由来(天然) | 生物種: ![]() |
分子量 | 理論値: 114.735938 KDa |
組換発現 | 生物種: ![]() ![]() |
配列 | 文字列: MTQVASPVVQ ARYSPVELSA ALGLFPPTDE QAAVIAAPPG PLVVIAGAGA GKTETMAARV VWLVANGFAT PSQVLGLTFT RKAAGQLLR RVRTRLARLA GAGLAPGSGA SDESATVSTY HAFAGTLLRE HGLLLPVEPD TRLLSETELW QLAYDVVCAH P GHLDTEKT ...文字列: MTQVASPVVQ ARYSPVELSA ALGLFPPTDE QAAVIAAPPG PLVVIAGAGA GKTETMAARV VWLVANGFAT PSQVLGLTFT RKAAGQLLR RVRTRLARLA GAGLAPGSGA SDESATVSTY HAFAGTLLRE HGLLLPVEPD TRLLSETELW QLAYDVVCAH P GHLDTEKT PAAVTAMVLR LSGALAEHLV DTDQLR(UNK)(UNK)(UNK)(UNK) (UNK)(UNK)(UNK)(UNK)(UNK) (UNK)(UNK)(UNK)(UNK) (UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK) (UNK)(UNK) (UNK)(UNK)LLRML ATQTERTELV PLIDALHQRM RAEKVMDFGM QMAAAARLAA RFPQVGEQLR QRFRVVLLDE YQDTG HAQR IALSSLFGGG ADDGLALTAV GDPIQSIYGW RGASATNLPR FTTDFPYSDG TPAPTLELRT (UNK)(UNK)(UNK) (UNK)(UNK)(UNK)(UNK)(UNK)(UNK) (UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK) (UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK) (UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK) (UNK)(UNK) (UNK)TIRCALLNN VAAERDWVAD HLARAYHGAI GRGEAAPTAA VLVRRNADAA PMAEALTARG VPVEVV GVA GLLAVPEVAD LVAMLRLIAD PTAGSAVMRI LTGPRWRFGA RDIAALWRRA VELDDRPKGE LGTADIVAQA APDADTA CV ADAICDPGDA ERYSPAGYER IVALGRELTM LRAHLGHPLP ELVAEVRRVL GLDAEARAAR PVAAGWAGTE NLDRFSDL V SDFAGHAGAS VSALLAYLDA AVEVENGLAP AELTVSHDRV QILTVHAAKG LEWQVVAVPH LSARVFPSTT QARTWLTDA SDLPPLLRGD RATESEIGVP VLDTSDIYDR KILSDKISDH KKSLDQRRVD EERRLLYVAI TRAEDTLLLS GHHWGATESK PRGPSEFLC ELKTILEEAT AAGTPCGEIE HWAPDPAPGE TNPLRDQVVE ALWPPVASAD DHVHRGAQLV AAAMAGEVSA E ADQEGWAA DVDALLAERE RP(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK) (UNK)(UNK)(UNK)(UNK) (UNK)(UNK)(UNK)(UNK)(UNK) (UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK) (UNK) (UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK) (UNK)(UNK)(UNK)(UNK)(UNK)HALLG TTFHEWVQRY FHAERLFDL DDLPGAVDSD SGRAVEESLA ELQDAFVKSP WAARTPVEVE VPFDMVLGET VVRGRIDAVF AEPDGTTMVL A WKTGDPPE TPEAKEHAAV QLAVYRLAWA AMRGCPPESV RAAFHYVRSG (UNK)(UNK)(UNK)(UNK)(UNK)(UNK) (UNK)(UNK)(UNK) (UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)LA AAPTETAEEA DRIT UniProtKB: DNA helicase, DNA helicase, DNA helicase, DNA helicase |
-分子 #2: ATP-dependent DNA helicase (UvrD/REP)
分子 | 名称: ATP-dependent DNA helicase (UvrD/REP) / タイプ: protein_or_peptide / ID: 2 / コピー数: 1 / 光学異性体: LEVO / EC番号: DNA helicase |
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由来(天然) | 生物種: ![]() |
分子量 | 理論値: 110.899562 KDa |
組換発現 | 生物種: ![]() ![]() |
配列 | 文字列: MTTRPAESAP QTASTLLEPG SNGVVRLLGG PGTGKSSLLV DTAVQHILAG ADPESVLLLT GSARLRTAAR AAITARLLGA GTVGVVREP LVRTVHSYAF AVLRLAAQRN GDPPPRLITS AEQDGIIREL LAGDLEDGHR SPVGWPEQLW PALTTAGFAT E LRDLMARC ...文字列: MTTRPAESAP QTASTLLEPG SNGVVRLLGG PGTGKSSLLV DTAVQHILAG ADPESVLLLT GSARLRTAAR AAITARLLGA GTVGVVREP LVRTVHSYAF AVLRLAAQRN GDPPPRLITS AEQDGIIREL LAGDLEDGHR SPVGWPEQLW PALTTAGFAT E LRDLMARC TERGVDPIAL QRLGRTAKRP EWLAAGRFAQ AYEQIMLLRS AVGMAAPQAT VPALGAAELV GAALEALGAD DE LLDTERN RIKLLLVDDA QHLDPQAARL VRALAAGTGL TVIAGDPDQS VFGYRGADPV LLRDDTHPAI TLTQSYRCAP EIA SAITGL GQRLPGVSDT RHWTGNPQRE GTVTVRLAAS THAEGTMIAD ALRRAHLVDG IPWSQMAVIV RSVPRVGTAL ARAL TAAGV PVQDNGTDVP VGRQPAAAAL LTVLDVTATG HLDADSAVAL LTGPIGRVDP VTLRQLRRAL RRADGSQPPR DFGDL LVDA IEREPKGLSA EHARTLRRLR AVLTAARRSD ASGADPRYTL WQAWHASGLQ RRWLAASERG GSVGAQADRD LDAVTT LFD VADQYVNRTA GASLRGLVDH VTRLGAAVAR TEPETAAEAV AVLSVHGALA GEWDFVVIAG VQEGLWPNMI PRGGVLG TQ HLVDVLDGVA DMTDRTVSTR APLVAEERRL LMAAMGRART RVMITAVDSD TGDESLLPSP FCAEISAWAT EPVAEPPL V APRVLAPSAL VGRLRAVVCA PDGAVDDDAR ACAAAQLARL AAAGVPGADP SQWHAMTSLT TEEPLWSEPG HVVTLSPST LQMLTDCPLR WLLERHGGDD GRDVRSTVGS LVHALVSEPG KTESQLVNEL EKVWDDLPYD AKWYSDNELA RHRAMLETFT RWREDTRRQ LTEVATEIPV EGIVVEPGEN TPGVRVRGRL DRLERDEAGR LVVVALKTGK SPVTKDDAQN HAQLAMYQLA V AAGLLDDG DEPGGGKLVY LGKAGAAGAT EREQDPLTPD KRAEWLETVG EAAAATAGPR FVARVNNGCA NCPVRSSCPA QA NGDRP UniProtKB: DNA helicase |
-分子 #3: IRON/SULFUR CLUSTER
分子 | 名称: IRON/SULFUR CLUSTER / タイプ: ligand / ID: 3 / コピー数: 1 / 式: SF4 |
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分子量 | 理論値: 351.64 Da |
Chemical component information | ![]() ChemComp-FS1: |
-分子 #4: PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER
分子 | 名称: PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER / タイプ: ligand / ID: 4 / コピー数: 1 / 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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![]() | 単粒子再構成法 |
試料の集合状態 | particle |
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試料調製
濃度 | 1.5 mg/mL |
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緩衝液 | pH: 7.5 / 構成要素 - 式: Tris / 詳細: 20 mM Tris-HCl, pH 7.5, 150 mM NaCl |
凍結 | 凍結剤: ETHANE / チャンバー内湿度: 100 % / 装置: FEI VITROBOT MARK IV |
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電子顕微鏡法
顕微鏡 | FEI TITAN KRIOS |
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撮影 | フィルム・検出器のモデル: GATAN K2 SUMMIT (4k x 4k) 平均電子線量: 2.16 e/Å2 |
電子線 | 加速電圧: 300 kV / 電子線源: ![]() |
電子光学系 | 照射モード: FLOOD BEAM / 撮影モード: BRIGHT FIELD |
実験機器 | ![]() モデル: Titan Krios / 画像提供: FEI Company |