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基本情報
登録情報 | データベース: PDB / ID: 7opl | ||||||
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タイトル | CryoEM structure of DNA Polymerase alpha - primase bound to SARS CoV nsp1 | ||||||
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![]() | DNA BINDING PROTEIN / DNA polymerase / Primase / viral protein | ||||||
機能・相同性 | ![]() positive regulation of DNA primase activity / DNA primase AEP / ribonucleotide binding / DNA replication initiation / DNA/RNA hybrid binding / Telomere C-strand synthesis initiation / Inhibition of replication initiation of damaged DNA by RB1/E2F1 / alpha DNA polymerase:primase complex / Polymerase switching / regulation of type I interferon production ...positive regulation of DNA primase activity / DNA primase AEP / ribonucleotide binding / DNA replication initiation / DNA/RNA hybrid binding / Telomere C-strand synthesis initiation / Inhibition of replication initiation of damaged DNA by RB1/E2F1 / alpha DNA polymerase:primase complex / Polymerase switching / regulation of type I interferon production / Processive synthesis on the lagging strand / Assembly of the SARS-CoV-1 Replication-Transcription Complex (RTC) / Maturation of replicase proteins / Transcription of SARS-CoV-1 sgRNAs / DNA primase activity / Removal of the Flap Intermediate / Polymerase switching on the C-strand of the telomere / lagging strand elongation / DNA replication, synthesis of primer / mitotic DNA replication initiation / Translation of Replicase and Assembly of the Replication Transcription Complex / K48-linked deubiquitinase activity / Replication of the SARS-CoV-1 genome / host cell endoplasmic reticulum / K63-linked deubiquitinase activity / DNA strand elongation involved in DNA replication / DNA synthesis involved in DNA repair / G1/S-Specific Transcription / leading strand elongation / SARS-CoV-1 modulates host translation machinery / DNA replication origin binding / Activation of the pre-replicative complex / DNA replication initiation / viral genome replication / methyltransferase activity / Defective pyroptosis / nuclear matrix / double-strand break repair via nonhomologous end joining / protein import into nucleus / SARS-CoV-1 activates/modulates innate immune responses / nuclear envelope / single-stranded DNA binding / 4 iron, 4 sulfur cluster binding / double membrane vesicle viral factory outer membrane / SARS coronavirus main proteinase / host cell endosome / symbiont-mediated degradation of host mRNA / mRNA guanylyltransferase / symbiont-mediated suppression of host ISG15-protein conjugation / G-quadruplex RNA binding / omega peptidase activity / symbiont-mediated suppression of host cytoplasmic pattern recognition receptor signaling pathway via inhibition of IRF3 activity / methylation / host cell Golgi apparatus / symbiont-mediated perturbation of host ubiquitin-like protein modification / endonuclease activity / DNA replication / ubiquitinyl hydrolase 1 / cysteine-type deubiquitinase activity / 加水分解酵素; プロテアーゼ; ペプチド結合加水分解酵素; システインプロテアーゼ / DNA-directed DNA polymerase / single-stranded RNA binding / DNA-directed DNA polymerase activity / host cell perinuclear region of cytoplasm / viral protein processing / lyase activity / symbiont-mediated suppression of host type I interferon-mediated signaling pathway / induction by virus of host autophagy / cysteine-type endopeptidase activity / RNA-dependent RNA polymerase activity / DNA repair / nucleotide binding / virus-mediated perturbation of host defense response / chromatin binding / chromatin / nucleolus / protein kinase binding / magnesium ion binding / proteolysis / DNA binding / zinc ion binding / nucleoplasm / identical protein binding / membrane / nucleus / metal ion binding / cytosol 類似検索 - 分子機能 | ||||||
生物種 | ![]() ![]() | ||||||
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: 4.12 Å | ||||||
![]() | Kilkenny, M.L. / Pellegrini, L. | ||||||
資金援助 | ![]()
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![]() | ![]() タイトル: Structural basis for the interaction of SARS-CoV-2 virulence factor nsp1 with DNA polymerase α-primase. 著者: Mairi L Kilkenny / Charlotte E Veale / Amir Guppy / Steven W Hardwick / Dimitri Y Chirgadze / Neil J Rzechorzek / Joseph D Maman / Luca Pellegrini / ![]() 要旨: The molecular mechanisms that drive the infection by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-the causative agent of coronavirus disease 2019 (COVID-19)-are under intense ...The molecular mechanisms that drive the infection by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-the causative agent of coronavirus disease 2019 (COVID-19)-are under intense current scrutiny to understand how the virus operates and to uncover ways in which the disease can be prevented or alleviated. Recent proteomic screens of the interactions between viral and host proteins have identified the human proteins targeted by SARS-CoV-2. The DNA polymerase α (Pol α)-primase complex or primosome-responsible for initiating DNA synthesis during genomic duplication-was identified as a target of nonstructural protein 1 (nsp1), a major virulence factor in the SARS-CoV-2 infection. Here, we validate the published reports of the interaction of nsp1 with the primosome by demonstrating direct binding with purified recombinant components and providing a biochemical characterization of their interaction. Furthermore, we provide a structural basis for the interaction by elucidating the cryo-electron microscopy structure of nsp1 bound to the primosome. Our findings provide biochemical evidence for the reported targeting of Pol α by the virulence factor nsp1 and suggest that SARS-CoV-2 interferes with Pol α's putative role in the immune response during the viral infection. | ||||||
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構造の表示
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構造ビューア | 分子: ![]() ![]() |
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PDBx/mmCIF形式 | ![]() | 458.9 KB | 表示 | ![]() |
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PDB形式 | ![]() | 359 KB | 表示 | ![]() |
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その他 | ![]() |
-検証レポート
文書・要旨 | ![]() | 1.1 MB | 表示 | ![]() |
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文書・詳細版 | ![]() | 1.2 MB | 表示 | |
XML形式データ | ![]() | 72.3 KB | 表示 | |
CIF形式データ | ![]() | 110.2 KB | 表示 | |
アーカイブディレクトリ | ![]() ![]() | HTTPS FTP |
-関連構造データ
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リンク
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集合体
登録構造単位 | ![]()
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要素
-DNA polymerase alpha ... , 2種, 2分子 AB
#1: タンパク質 | 分子量: 133702.562 Da / 分子数: 1 / 由来タイプ: 組換発現 / 由来: (組換発現) ![]() 発現宿主: ![]() ![]() 参照: UniProt: P09884, DNA-directed DNA polymerase |
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#2: タンパク質 | 分子量: 49855.434 Da / 分子数: 1 / 由来タイプ: 組換発現 / 由来: (組換発現) ![]() 発現宿主: ![]() ![]() 参照: UniProt: Q14181 |
-タンパク質 , 3種, 3分子 CDE
#3: タンパク質 | 分子量: 52590.801 Da / 分子数: 1 / 由来タイプ: 組換発現 / 由来: (組換発現) ![]() 発現宿主: ![]() ![]() 参照: UniProt: P49642, 転移酵素; リンを含む基を移すもの; 核酸を移すもの |
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#4: タンパク質 | 分子量: 58890.918 Da / 分子数: 1 / 由来タイプ: 組換発現 / 由来: (組換発現) ![]() 発現宿主: ![]() ![]() 参照: UniProt: P49643 |
#5: タンパク質 | 分子量: 12955.852 Da / 分子数: 1 / 由来タイプ: 組換発現 由来: (組換発現) ![]() 遺伝子: 1a / 発現宿主: ![]() ![]() |
-非ポリマー , 2種, 4分子 ![](data/chem/img/ZN.gif)
![](data/chem/img/SF4.gif)
![](data/chem/img/SF4.gif)
#6: 化合物 | #7: 化合物 | ChemComp-SF4 / | |
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-詳細
研究の焦点であるリガンドがあるか | N |
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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 |
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試料調製
構成要素 | 名称: Complex of DNA polymerase alpha - primase bound to SARS COV-2 nsp1 タイプ: COMPLEX / Entity ID: #1-#5 / 由来: RECOMBINANT | ||||||||||||||||
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分子量 | 値: 0.31 MDa / 実験値: NO | ||||||||||||||||
由来(天然) | 生物種: ![]() | ||||||||||||||||
由来(組換発現) | 生物種: ![]() ![]() | ||||||||||||||||
緩衝液 | pH: 7.2 | ||||||||||||||||
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: NO / 凍結: YES 詳細: The nsp1 protein was added in 10-fold stoichiometric excess | ||||||||||||||||
試料支持 | グリッドの材料: GOLD / グリッドのサイズ: 300 divisions/in. / グリッドのタイプ: UltrAuFoil R1.2/1.3 | ||||||||||||||||
急速凍結 | 装置: FEI VITROBOT MARK IV / 凍結剤: ETHANE |
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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: ![]() |
電子レンズ | モード: BRIGHT FIELD / 倍率(公称値): 130000 X / 最大 デフォーカス(公称値): -0.7 nm / 最小 デフォーカス(公称値): -2.5 nm / C2レンズ絞り径: 50 µm |
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: 1.31 sec. / 電子線照射量: 46.91 e/Å2 フィルム・検出器のモデル: GATAN K3 BIOQUANTUM (6k x 4k) 実像数: 2919 |
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解析
ソフトウェア | 名称: PHENIX / バージョン: 1.19.1_4122: / 分類: 精密化 | ||||||||||||||||||||||||||||||||||||||||
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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: 709068 | ||||||||||||||||||||||||||||||||||||||||
対称性 | 点対称性: C1 (非対称) | ||||||||||||||||||||||||||||||||||||||||
3次元再構成 | 解像度: 4.12 Å / 解像度の算出法: FSC 0.143 CUT-OFF / 粒子像の数: 233476 / 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 | ||||||||||||||||||||||||||||||||||||||||
原子モデル構築 | 3D fitting-ID: 1 / Source name: PDB / タイプ: experimental model
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拘束条件 |
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