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基本情報
登録情報 | データベース: PDB / ID: 6mw3 | |||||||||
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タイトル | EM structure of Bacillus subtilis ribonucleotide reductase inhibited filament composed of NrdE alpha subunit and NrdF beta subunit with dATP | |||||||||
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![]() | OXIDOREDUCTASE / ribonucleotide reductase / allostery / nucleotide metabolism / filament / dATP / ATP | |||||||||
機能・相同性 | ![]() ribonucleoside-diphosphate reductase complex / ribonucleoside-diphosphate reductase / ribonucleoside-diphosphate reductase activity, thioredoxin disulfide as acceptor / deoxyribonucleotide biosynthetic process / DNA replication / ATP binding 類似検索 - 分子機能 | |||||||||
生物種 | ![]() ![]() | |||||||||
手法 | 電子顕微鏡法 / らせん対称体再構成法 / クライオ電子顕微鏡法 / 解像度: 4.65 Å | |||||||||
![]() | Thomas, W.C. / Bacik, J.P. / Kaelber, J.T. / Ando, N. | |||||||||
資金援助 | ![]()
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![]() | ![]() タイトル: Convergent allostery in ribonucleotide reductase. 著者: William C Thomas / F Phil Brooks / Audrey A Burnim / John-Paul Bacik / JoAnne Stubbe / Jason T Kaelber / James Z Chen / Nozomi Ando / ![]() 要旨: Ribonucleotide reductases (RNRs) use a conserved radical-based mechanism to catalyze the conversion of ribonucleotides to deoxyribonucleotides. Within the RNR family, class Ib RNRs are notable for ...Ribonucleotide reductases (RNRs) use a conserved radical-based mechanism to catalyze the conversion of ribonucleotides to deoxyribonucleotides. Within the RNR family, class Ib RNRs are notable for being largely restricted to bacteria, including many pathogens, and for lacking an evolutionarily mobile ATP-cone domain that allosterically controls overall activity. In this study, we report the emergence of a distinct and unexpected mechanism of activity regulation in the sole RNR of the model organism Bacillus subtilis. Using a hypothesis-driven structural approach that combines the strengths of small-angle X-ray scattering (SAXS), crystallography, and cryo-electron microscopy (cryo-EM), we describe the reversible interconversion of six unique structures, including a flexible active tetramer and two inhibited helical filaments. These structures reveal the conformational gymnastics necessary for RNR activity and the molecular basis for its control via an evolutionarily convergent form of allostery. | |||||||||
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 | 分子: ![]() ![]() |
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-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
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リンク
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集合体
登録構造単位 | ![]()
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1 | ![]()
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対称性 | らせん対称: (回転対称性: 1 / Dyad axis: yes / N subunits divisor: 1 / Num. of operations: 6 / Rise per n subunits: 73.8 Å / Rotation per n subunits: 88.6 °) |
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要素
#1: タンパク質 | 分子量: 80791.469 Da / 分子数: 2 / 由来タイプ: 組換発現 / 由来: (組換発現) ![]() ![]() ![]() ![]() 参照: UniProt: A0A162Q3J9, UniProt: P50620*PLUS, ribonucleoside-diphosphate reductase #2: タンパク質・ペプチド | 分子量: 698.854 Da / 分子数: 2 / 由来タイプ: 組換発現 / 詳細: C-terminus modeled as a polyalanine chain / 由来: (組換発現) ![]() ![]() ![]() ![]() #3: 化合物 | ChemComp-DTP / 配列の詳細 | B. subtilis Ribonucleoside-diphosphate reductase beta subunit (NrdF) is modeled as an 8-residue ...B. subtilis Ribonucleoside-diphosphate reductase beta subunit (NrdF) is modeled as an 8-residue polyA chain. The complete sequence is provided below: MGSSHHHHHH | |
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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: FILAMENT / 3次元再構成法: らせん対称体再構成法 |
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試料調製
構成要素 | 名称: Inhibited filament of ribonucleoside-diphosphate reductase composed of NrdE alpha subunits and NrdF beta subunit tails タイプ: COMPLEX 詳細: Beta subunit core density only visible at low threshold. Beta subunit tail is bound with strong density to alpha subunit and modeled as a poly-A peptide in the model. Entity ID: #1-#2 / 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: 7.6 詳細: Glycerol in original storage buffer was diluted to < 0.25% w/v. | |||||||||||||||||||||||||
緩衝液成分 |
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試料 | 濃度: 0.4 mg/ml / 包埋: NO / シャドウイング: NO / 染色: NO / 凍結: YES 詳細: Cryo-EM samples of the NrdEF filament were prepared by mixing 20 uM C382S holo-NrdE with 20 or 40 uM Mn-reconstituted NrdF in assay buffer with 100 uM dATP and 1 mM CDP, prior to dilution ...詳細: Cryo-EM samples of the NrdEF filament were prepared by mixing 20 uM C382S holo-NrdE with 20 or 40 uM Mn-reconstituted NrdF in assay buffer with 100 uM dATP and 1 mM CDP, prior to dilution with nucleotide-containing buffer to a concentration of 5 uM protein. A subset of the grids were pre-coated with a support film of continuous, amorphous carbon by flotation of cleaved mica. For these grids, the sample was diluted to a final protein concentration of 2 uM. | |||||||||||||||||||||||||
試料支持 | 詳細: unspecified | |||||||||||||||||||||||||
急速凍結 | 装置: LEICA EM GP / 凍結剤: ETHANE / 湿度: 95 % |
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電子顕微鏡撮影
実験機器 | ![]() モデル: Talos Arctica / 画像提供: FEI Company |
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顕微鏡 | モデル: FEI TALOS ARCTICA |
電子銃 | 電子線源: ![]() |
電子レンズ | モード: BRIGHT FIELD / 倍率(公称値): 130000 X / Cs: 2.7 mm / C2レンズ絞り径: 50 µm / アライメント法: ZEMLIN TABLEAU |
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 | 電子線照射量: 8 e/Å2 / 検出モード: COUNTING フィルム・検出器のモデル: GATAN K2 SUMMIT (4k x 4k) 実像数: 2843 |
電子光学装置 | エネルギーフィルタースリット幅: 20 eV |
画像スキャン | 横: 3838 / 縦: 3710 |
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解析
EMソフトウェア |
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CTF補正 | タイプ: PHASE FLIPPING AND AMPLITUDE CORRECTION | ||||||||||||||||||||||||||||||||||||
らせん対称 | 回転角度/サブユニット: 88.6 ° / 軸方向距離/サブユニット: 73.8 Å / らせん対称軸の対称性: C1 | ||||||||||||||||||||||||||||||||||||
粒子像の選択 | 選択した粒子像数: 281591 | ||||||||||||||||||||||||||||||||||||
3次元再構成 | 解像度: 4.65 Å / 解像度の算出法: FSC 0.143 CUT-OFF / 粒子像の数: 126224 / アルゴリズム: FOURIER SPACE / 対称性のタイプ: HELICAL | ||||||||||||||||||||||||||||||||||||
原子モデル構築 | プロトコル: OTHER / 空間: REAL / Target criteria: Correlation coefficient | ||||||||||||||||||||||||||||||||||||
原子モデル構築 | PDB-ID: 6CGN Accession code: 6CGN / Source name: PDB / タイプ: experimental model |