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基本情報
登録情報 | データベース: PDB / ID: 7szi | |||||||||
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タイトル | Cryo-EM structure of OmpK36-TraN mating pair stabilization proteins from carbapenem-resistant Klebsiella pneumoniae | |||||||||
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![]() | MEMBRANE PROTEIN / Bacterial Conjugation / Mating pair stabilization / Single Particle Analysis / Outer Membrane Protein Complex | |||||||||
機能・相同性 | ![]() porin activity / pore complex / monoatomic ion transmembrane transport / cell outer membrane / metal ion binding 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: 2.7 Å | |||||||||
![]() | Beltran, L.C. / Seddon, C. / Beis, K. / Frankel, G. / Egelman, E.H. | |||||||||
資金援助 | ![]()
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![]() | ![]() タイトル: Mating pair stabilization mediates bacterial conjugation species specificity. 著者: Wen Wen Low / Joshua L C Wong / Leticia C Beltran / Chloe Seddon / Sophia David / Hok-Sau Kwong / Tatiana Bizeau / Fengbin Wang / Alejandro Peña / Tiago R D Costa / Bach Pham / Min Chen / ...著者: Wen Wen Low / Joshua L C Wong / Leticia C Beltran / Chloe Seddon / Sophia David / Hok-Sau Kwong / Tatiana Bizeau / Fengbin Wang / Alejandro Peña / Tiago R D Costa / Bach Pham / Min Chen / Edward H Egelman / Konstantinos Beis / Gad Frankel / ![]() ![]() 要旨: Bacterial conjugation mediates contact-dependent transfer of DNA from donor to recipient bacteria, thus facilitating the spread of virulence and resistance plasmids. Here we describe how variants of ...Bacterial conjugation mediates contact-dependent transfer of DNA from donor to recipient bacteria, thus facilitating the spread of virulence and resistance plasmids. Here we describe how variants of the plasmid-encoded donor outer membrane (OM) protein TraN cooperate with distinct OM receptors in recipients to mediate mating pair stabilization and efficient DNA transfer. We show that TraN from the plasmid pKpQIL (Klebsiella pneumoniae) interacts with OmpK36, plasmids from R100-1 (Shigella flexneri) and pSLT (Salmonella Typhimurium) interact with OmpW, and the prototypical F plasmid (Escherichia coli) interacts with OmpA. Cryo-EM analysis revealed that TraN interacts with OmpK36 through the insertion of a β-hairpin in the tip of TraN into a monomer of the OmpK36 porin trimer. Combining bioinformatic analysis with AlphaFold structural predictions, we identified a fourth TraN structural variant that mediates mating pair stabilization by binding OmpF. Accordingly, we devised a classification scheme for TraN homologues on the basis of structural similarity and their associated receptors: TraNα (OmpW), TraNβ (OmpK36), TraNγ (OmpA), TraNδ (OmpF). These TraN-OM receptor pairings have real-world implications as they reflect the distribution of resistance plasmids within clinical Enterobacteriaceae isolates, demonstrating the importance of mating pair stabilization in mediating conjugation species specificity. These findings will allow us to predict the distribution of emerging resistance plasmids in high-risk bacterial pathogens. | |||||||||
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構造ビューア | 分子: ![]() ![]() |
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-関連構造データ
関連構造データ | ![]() 25567MC 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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集合体
登録構造単位 | ![]()
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要素
#1: タンパク質 | 分子量: 38058.918 Da / 分子数: 3 / 由来タイプ: 天然 / 由来: (天然) ![]() #2: タンパク質・ペプチド | | 分子量: 906.964 Da / 分子数: 1 / 由来タイプ: 組換発現 / 詳細: pKpQIL / 由来: (組換発現) ![]() 詳細 (発現宿主): TraN gene (D28 to Q651)cells and expressed in Terrific Broth (TB)restriction enzyme sites. The construct was transformed into E. coli C43 (DE3) competent His7-tag and a tobacco ...詳細 (発現宿主): TraN gene (D28 to Q651)cells and expressed in Terrific Broth (TB)restriction enzyme sites. The construct was transformed into E. coli C43 (DE3) competent His7-tag and a tobacco etch virus (TEV) cleavage site using the NcoI and XhoIfrom pKpQIL was subcloned into the pTAMAHISTEV vector with an N- 発現宿主: ![]() ![]() |
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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: Conjugal mating pair stabilizing complex OmpK36 and TraN タイプ: COMPLEX 詳細: OmpK36WT ATCC43816 serially passaged in vitro on Rifampicin containing LB plates to 100mcg/ml followed by two passages in BALB/c mice. Mature TraN gene D28 to Q651 from pKpQIL was sub-cloned ...詳細: OmpK36WT ATCC43816 serially passaged in vitro on Rifampicin containing LB plates to 100mcg/ml followed by two passages in BALB/c mice. Mature TraN gene D28 to Q651 from pKpQIL was sub-cloned into the pTAMAHISTEV vector with an N-terminal His7-tag and a tobacco etch virus TEV cleavage site using the NcoI and XhoI restriction enzyme sites. The construct was transformed into E. coli C43 DE3 competent cells and expressed in Terrific Broth TB medium Formedium supplemented with 100 microgram/mL ampicillin. Entity ID: all / 由来: RECOMBINANT | ||||||||||||||||||||
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分子量 | 値: 0.12 MDa / 実験値: YES | ||||||||||||||||||||
由来(天然) | 生物種: ![]() | ||||||||||||||||||||
由来(組換発現) | 生物種: ![]() ![]() | ||||||||||||||||||||
緩衝液 | pH: 7.8 / 詳細: SEC Buffer | ||||||||||||||||||||
緩衝液成分 |
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試料 | 濃度: 0.033 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: 200 divisions/in. / グリッドのタイプ: C-flat-1.2/1.3 | ||||||||||||||||||||
急速凍結 | 装置: LEICA EM GP / 凍結剤: ETHANE / 湿度: 90 % |
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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): 81000 X / 最大 デフォーカス(公称値): 2200 nm / 最小 デフォーカス(公称値): 1200 nm / Cs: 2.7 mm |
撮影 | 平均露光時間: 4.78 sec. / 電子線照射量: 50 e/Å2 / フィルム・検出器のモデル: GATAN K3 (6k x 4k) / 実像数: 13688 |
電子光学装置 | エネルギーフィルタースリット幅: 10 eV |
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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 | ||||||||||||||||||||||||
粒子像の選択 | 選択した粒子像数: 13780567 | ||||||||||||||||||||||||
対称性 | 点対称性: C1 (非対称) | ||||||||||||||||||||||||
3次元再構成 | 解像度: 2.7 Å / 解像度の算出法: FSC 0.5 CUT-OFF / 粒子像の数: 598537 / 対称性のタイプ: POINT | ||||||||||||||||||||||||
原子モデル構築 | 空間: REAL |