+データを開く
-基本情報
登録情報 | データベース: PDB / ID: 8bm0 | ||||||||||||
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タイトル | Structure of GroEL:GroES-ATP complex plunge frozen 200 ms after reaction initiation | ||||||||||||
要素 |
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キーワード | CHAPERONE (シャペロン) / GroEL | ||||||||||||
機能・相同性 | 機能・相同性情報 GroEL-GroES complex / chaperonin ATPase / virion assembly / chaperone cofactor-dependent protein refolding / protein folding chaperone / isomerase activity / ATP-dependent protein folding chaperone / response to radiation / unfolded protein binding / フォールディング ...GroEL-GroES complex / chaperonin ATPase / virion assembly / chaperone cofactor-dependent protein refolding / protein folding chaperone / isomerase activity / ATP-dependent protein folding chaperone / response to radiation / unfolded protein binding / フォールディング / response to heat / protein-folding chaperone binding / protein refolding / magnesium ion binding / ATP hydrolysis activity / ATP binding / 生体膜 / identical protein binding / metal ion binding / 細胞質基質 類似検索 - 分子機能 | ||||||||||||
生物種 | Escherichia coli (大腸菌) | ||||||||||||
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: 3.4 Å | ||||||||||||
データ登録者 | Dhurandhar, M. / Torino, S. / Efremov, R. | ||||||||||||
資金援助 | European Union, ベルギー, 3件
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引用 | ジャーナル: Nat Methods / 年: 2023 タイトル: Time-resolved cryo-EM using a combination of droplet microfluidics with on-demand jetting. 著者: Stefania Torino / Mugdha Dhurandhar / Annelore Stroobants / Raf Claessens / Rouslan G Efremov / 要旨: Single-particle cryogenic electron microscopy (cryo-EM) allows reconstruction of high-resolution structures of proteins in different conformations. Protein function often involves transient ...Single-particle cryogenic electron microscopy (cryo-EM) allows reconstruction of high-resolution structures of proteins in different conformations. Protein function often involves transient functional conformations, which can be resolved using time-resolved cryo-EM (trEM). In trEM, reactions are arrested after a defined delay time by rapid vitrification of protein solution on the EM grid. Despite the increasing interest in trEM among the cryo-EM community, making trEM samples with a time resolution below 100 ms remains challenging. Here we report the design and the realization of a time-resolved cryo-plunger that combines a droplet-based microfluidic mixer with a laser-induced generator of microjets that allows rapid reaction initiation and plunge-freezing of cryo-EM grids. Using this approach, a time resolution of 5 ms was achieved and the protein density map was reconstructed to a resolution of 2.1 Å. trEM experiments on GroEL:GroES chaperonin complex resolved the kinetics of the complex formation and visualized putative short-lived conformations of GroEL-ATP complex. #1: ジャーナル: Nat Commun / 年: 2023 タイトル: BtuB TonB-dependent transporters and BtuG surface lipoproteins form stable complexes for vitamin B uptake in gut Bacteroides. 著者: Javier Abellon-Ruiz / Kalyanashis Jana / Augustinas Silale / Andrew M Frey / Arnaud Baslé / Matthias Trost / Ulrich Kleinekathöfer / Bert van den Berg / 要旨: Vitamin B (cobalamin) is required for most human gut microbes, many of which are dependent on scavenging to obtain this vitamin. Since bacterial densities in the gut are extremely high, competition ...Vitamin B (cobalamin) is required for most human gut microbes, many of which are dependent on scavenging to obtain this vitamin. Since bacterial densities in the gut are extremely high, competition for this keystone micronutrient is severe. Contrasting with Enterobacteria, members of the dominant genus Bacteroides often encode several BtuB vitamin B outer membrane transporters together with a conserved array of surface-exposed B-binding lipoproteins. Here we show that the BtuB transporters from Bacteroides thetaiotaomicron form stable, pedal bin-like complexes with surface-exposed BtuG lipoprotein lids, which bind B with high affinities. Closing of the BtuG lid following B capture causes destabilisation of the bound B by a conserved BtuB extracellular loop, causing translocation of the vitamin to BtuB and subsequent transport. We propose that TonB-dependent, lipoprotein-assisted small molecule uptake is a general feature of Bacteroides spp. that is important for the success of this genus in colonising the human gut. #2: ジャーナル: Nat Methods / 年: 2023 タイトル: Time-resolved cryo-EM using a combination of droplet microfluidics with on-demand jetting. 著者: Stefania Torino / Mugdha Dhurandhar / Annelore Stroobants / Raf Claessens / Rouslan G Efremov / 要旨: Single-particle cryogenic electron microscopy (cryo-EM) allows reconstruction of high-resolution structures of proteins in different conformations. Protein function often involves transient ...Single-particle cryogenic electron microscopy (cryo-EM) allows reconstruction of high-resolution structures of proteins in different conformations. Protein function often involves transient functional conformations, which can be resolved using time-resolved cryo-EM (trEM). In trEM, reactions are arrested after a defined delay time by rapid vitrification of protein solution on the EM grid. Despite the increasing interest in trEM among the cryo-EM community, making trEM samples with a time resolution below 100 ms remains challenging. Here we report the design and the realization of a time-resolved cryo-plunger that combines a droplet-based microfluidic mixer with a laser-induced generator of microjets that allows rapid reaction initiation and plunge-freezing of cryo-EM grids. Using this approach, a time resolution of 5 ms was achieved and the protein density map was reconstructed to a resolution of 2.1 Å. trEM experiments on GroEL:GroES chaperonin complex resolved the kinetics of the complex formation and visualized putative short-lived conformations of GroEL-ATP complex. | ||||||||||||
履歴 |
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-構造の表示
構造ビューア | 分子: MolmilJmol/JSmol |
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-ダウンロードとリンク
-ダウンロード
PDBx/mmCIF形式 | 8bm0.cif.gz | 1.4 MB | 表示 | PDBx/mmCIF形式 |
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PDB形式 | pdb8bm0.ent.gz | 1.2 MB | 表示 | PDB形式 |
PDBx/mmJSON形式 | 8bm0.json.gz | ツリー表示 | PDBx/mmJSON形式 | |
その他 | その他のダウンロード |
-検証レポート
アーカイブディレクトリ | https://data.pdbj.org/pub/pdb/validation_reports/bm/8bm0 ftp://data.pdbj.org/pub/pdb/validation_reports/bm/8bm0 | HTTPS FTP |
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-関連構造データ
関連構造データ | 16116MC 8bk7C 8bk8C 8bk9C 8bkaC 8bkbC 8bkgC 8bkzC 8bl2C 8bl7C 8blcC 8bldC 8bleC 8blfC 8blyC 8bm1C 8bmdC 8bmoC 8bmtC C: 同じ文献を引用 (文献) M: このデータのモデリングに利用したマップデータ |
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類似構造データ | 類似検索 - 機能・相同性F&H 検索 |
-リンク
-集合体
登録構造単位 |
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-要素
#1: タンパク質 | 分子量: 57391.711 Da / 分子数: 14 / 由来タイプ: 組換発現 / 由来: (組換発現) Escherichia coli (大腸菌) / 遺伝子: groEL, groL, mopA, b4143, JW4103 / 発現宿主: Escherichia coli (大腸菌) / 参照: UniProt: P0A6F5, chaperonin ATPase #2: タンパク質 | 分子量: 10472.016 Da / 分子数: 7 / 由来タイプ: 組換発現 / 由来: (組換発現) Escherichia coli (大腸菌) / 遺伝子: groES, groS, mopB, b4142, JW4102 / 発現宿主: Escherichia coli (大腸菌) / 参照: UniProt: P0A6F9 #3: 化合物 | ChemComp-MG / #4: 化合物 | ChemComp-K / #5: 化合物 | ChemComp-ATP / 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 |
-試料調製
構成要素 | 名称: GroEL:GroES-ATP complex / タイプ: COMPLEX / Entity ID: #1-#2 / 由来: RECOMBINANT | |||||||||||||||||||||||||||||||||||
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分子量 | 値: 0.95 MDa / 実験値: NO | |||||||||||||||||||||||||||||||||||
由来(天然) | 生物種: Escherichia coli (大腸菌) / 細胞内の位置: cytoplasm | |||||||||||||||||||||||||||||||||||
由来(組換発現) | 生物種: Escherichia coli (大腸菌) | |||||||||||||||||||||||||||||||||||
緩衝液 | pH: 7.5 | |||||||||||||||||||||||||||||||||||
緩衝液成分 |
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試料 | 濃度: 8 mg/ml / 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 R2/1 | |||||||||||||||||||||||||||||||||||
急速凍結 | 凍結剤: ETHANE |
-電子顕微鏡撮影
顕微鏡 | モデル: JEOL CRYO ARM 300 |
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電子銃 | 電子線源: FIELD EMISSION GUN / 加速電圧: 300 kV / 照射モード: FLOOD BEAM |
電子レンズ | モード: BRIGHT FIELDBright-field microscopy / 倍率(公称値): 60000 X / 最大 デフォーカス(公称値): 3500 nm / 最小 デフォーカス(公称値): 500 nm / Cs: 2.55 mm / C2レンズ絞り径: 100 µm / アライメント法: COMA FREE |
試料ホルダ | 凍結剤: NITROGEN / 試料ホルダーモデル: JEOL CRYOSPECPORTER / 最低温度: 80 K |
撮影 | 電子線照射量: 63.6 e/Å2 / フィルム・検出器のモデル: GATAN K3 (6k x 4k) |
電子光学装置 | エネルギーフィルター名称: In-column Omega Filter |
-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: C7 (7回回転対称) | ||||||||||||||||||||||||||||||||||||
3次元再構成 | 解像度: 3.4 Å / 解像度の算出法: FSC 0.143 CUT-OFF / 粒子像の数: 17592 / 対称性のタイプ: POINT | ||||||||||||||||||||||||||||||||||||
原子モデル構築 | プロトコル: FLEXIBLE FIT / 空間: REAL | ||||||||||||||||||||||||||||||||||||
拘束条件 |
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