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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: PDB / ID: 8fm9 | ||||||
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タイトル | Nodavirus RNA replication proto-crown, detergent-solubliized C12 multimer | ||||||
![]() | RNA-directed RNA polymerase | ||||||
![]() | VIRAL PROTEIN / Nodavirus RNA replication and RNA capping complex / Dodecamer ring / Outer mitochondrial membrane protein complex | ||||||
機能・相同性 | Nodavirus methyltransferase domain / Nodavirus Vmethyltransferase / host cell mitochondrial outer membrane / RNA-directed RNA polymerase / RNA-dependent RNA polymerase activity / DNA/RNA polymerase superfamily / membrane / RNA-directed RNA polymerase![]() | ||||||
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: 3.2 Å | ||||||
![]() | Zhan, H. / Unchwaniwala, N. / Rebolledo Viveros, A. / Pennington, J. / Horswill, M. / Broadberry, R. / Myers, J. / den Boon, J. / Grant, T. / Ahlquist, P. | ||||||
資金援助 | ![]()
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![]() | ![]() タイトル: Nodavirus RNA replication crown architecture reveals proto-crown precursor and viral protein A conformational switching. 著者: Hong Zhan / Nuruddin Unchwaniwala / Andrea Rebolledo-Viveros / Janice Pennington / Mark Horswill / Roma Broadberry / Jonathan Myers / Johan A den Boon / Timothy Grant / Paul Ahlquist / ![]() 要旨: Positive-strand RNA viruses replicate their genomes in virus-induced membrane vesicles, and the resulting RNA replication complexes are a major target for virus control. Nodavirus studies first ...Positive-strand RNA viruses replicate their genomes in virus-induced membrane vesicles, and the resulting RNA replication complexes are a major target for virus control. Nodavirus studies first revealed viral RNA replication proteins forming a 12-fold symmetric "crown" at the vesicle opening to the cytosol, an arrangement recently confirmed to extend to distantly related alphaviruses. Using cryoelectron microscopy (cryo-EM), we show that mature nodavirus crowns comprise two stacked 12-mer rings of multidomain viral RNA replication protein A. Each ring contains an ~19 nm circle of C-proximal polymerase domains, differentiated by strikingly diverged positions of N-proximal RNA capping/membrane binding domains. The lower ring is a "proto-crown" precursor that assembles prior to RNA template recruitment, RNA synthesis, and replication vesicle formation. In this proto-crown, the N-proximal segments interact to form a toroidal central floor, whose 3.1 Å resolution structure reveals many mechanistic details of the RNA capping/membrane binding domains. In the upper ring, cryo-EM fitting indicates that the N-proximal domains extend radially outside the polymerases, forming separated, membrane-binding "legs." The polymerase and N-proximal domains are connected by a long linker accommodating the conformational switch between the two rings and possibly also polymerase movements associated with RNA synthesis and nonsymmetric electron density in the lower center of mature crowns. The results reveal remarkable viral protein multifunctionality, conformational flexibility, and evolutionary plasticity and insights into (+)RNA virus replication and control. | ||||||
履歴 |
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構造の表示
構造ビューア | 分子: ![]() ![]() |
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ダウンロードとリンク
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ダウンロード
PDBx/mmCIF形式 | ![]() | 1.9 MB | 表示 | ![]() |
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PDB形式 | ![]() | 1.4 MB | 表示 | ![]() |
PDBx/mmJSON形式 | ![]() | ツリー表示 | ![]() | |
その他 | ![]() |
-検証レポート
アーカイブディレクトリ | ![]() ![]() | HTTPS FTP |
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-関連構造データ
関連構造データ | ![]() 29289MC ![]() 8fmaC ![]() 8fmbC M: このデータのモデリングに利用したマップデータ C: 同じ文献を引用 ( |
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類似構造データ | 類似検索 - 機能・相同性 ![]() |
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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
#1: タンパク質 | 分子量: 113996.586 Da / 分子数: 24 / 由来タイプ: 組換発現 / 由来: (組換発現) ![]() 発現宿主: ![]() ![]() 参照: UniProt: Q66929, RNA-directed RNA polymerase |
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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: Nodavirus RNA replication proto-crown, detergent-solubliized C12 multimer タイプ: COMPLEX / Entity ID: all / 由来: RECOMBINANT |
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分子量 | 実験値: NO |
由来(天然) | 生物種: ![]() |
由来(組換発現) | 生物種: ![]() ![]() |
緩衝液 | pH: 7.4 |
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: 300 divisions/in. / グリッドのタイプ: Quantifoil R1.2/1.3 |
急速凍結 | 装置: FEI VITROBOT MARK IV / 凍結剤: ETHANE / 湿度: 100 % / 凍結前の試料温度: 277 K |
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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 / 最大 デフォーカス(公称値): 2500 nm / 最小 デフォーカス(公称値): 500 nm / Cs: 2.7 mm / C2レンズ絞り径: 100 µm |
撮影 | 電子線照射量: 100 e/Å2 / 検出モード: INTEGRATING フィルム・検出器のモデル: FEI FALCON III (4k x 4k) |
画像スキャン | 横: 4096 / 縦: 4096 |
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解析
ソフトウェア | 名称: PHENIX / バージョン: 1.20.1_4487: / 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 | ||||||||||||||||||||||||||||||||||||||||||||||||
対称性 | 点対称性: C12 (12回回転対称) | ||||||||||||||||||||||||||||||||||||||||||||||||
3次元再構成 | 解像度: 3.2 Å / 解像度の算出法: FSC 0.143 CUT-OFF / 粒子像の数: 11093 / 対称性のタイプ: POINT | ||||||||||||||||||||||||||||||||||||||||||||||||
原子モデル構築 | プロトコル: FLEXIBLE FIT 詳細: The final model includes 24 chains representing 12 protein A polypeptides. Chains A-L represent the N-terminal floor segment built into the high-resolution EM density. Chains M-X represent a ...詳細: The final model includes 24 chains representing 12 protein A polypeptides. Chains A-L represent the N-terminal floor segment built into the high-resolution EM density. Chains M-X represent a mainchain trace of the polymerase segment rigid body fit into the map and are included to give a more complete picture of the molecule. The polymerase mainchain trace was refined from an Alphafold prediction into a map obtained by symmetry expansion with subtraction. The polymerase map is uploaded as a separate entry. The two segments have separate chain IDs as it is unclear from the density which polymerase connects to which floor segment. | ||||||||||||||||||||||||||||||||||||||||||||||||
拘束条件 |
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