+データを開く
-基本情報
登録情報 | データベース: PDB / ID: 5wln | |||||||||
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タイトル | Cryo-EM structure of the T2SS secretin XcpQ from Pseudomonas aeruginosa | |||||||||
要素 | Type II secretion system protein DType II secretion system | |||||||||
キーワード | MEMBRANE PROTEIN (膜タンパク質) / T2SS / Secretin (セクレチン) / Type 2 secretion system / Pentadecamer / GspD / XcpQ | |||||||||
機能・相同性 | 機能・相同性情報 protein secretion by the type II secretion system / type II protein secretion system complex / protein secretion / cell outer membrane / identical protein binding 類似検索 - 分子機能 | |||||||||
生物種 | Pseudomonas aeruginosa (緑膿菌) | |||||||||
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: 3.57 Å | |||||||||
データ登録者 | Hay, I.D. / Belousoff, M.J. / Lithgow, T.J. | |||||||||
資金援助 | オーストラリア, 2件
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引用 | ジャーナル: mBio / 年: 2017 タイトル: Structural Basis of Type 2 Secretion System Engagement between the Inner and Outer Bacterial Membranes. 著者: Iain D Hay / Matthew J Belousoff / Trevor Lithgow / 要旨: Sophisticated nanomachines are used by bacteria for protein secretion. In Gram-negative bacteria, the type 2 secretion system (T2SS) is composed of a pseudopilus assembly platform in the inner ...Sophisticated nanomachines are used by bacteria for protein secretion. In Gram-negative bacteria, the type 2 secretion system (T2SS) is composed of a pseudopilus assembly platform in the inner membrane and a secretin complex in the outer membrane. The engagement of these two megadalton-sized complexes is required in order to secrete toxins, effectors, and hydrolytic enzymes. has at least two T2SSs, with the ancestral nanomachine having a secretin complex composed of XcpQ. Until now, no high-resolution structural information was available to distinguish the features of this -type secretin, which varies greatly in sequence from the well-characterized -type and -type secretins. We have purified the ~1-MDa secretin complex and analyzed it by cryo-electron microscopy. Structural comparisons with the -type secretin complex revealed a striking structural homology despite the differences in their sequence characteristics. At 3.6-Å resolution, the secretin complex was found to have 15-fold symmetry throughout the membrane-embedded region and through most of the domains in the periplasm. However, the N1 domain and N0 domain were not well ordered into this 15-fold symmetry. We suggest a model wherein this disordering of the subunit symmetry for the periplasmic N domains provides a means to engage with the 6-fold symmetry in the inner membrane platform, with a metastable engagement that can be disrupted by substrate proteins binding to the region between XcpP, in the assembly platform, and the XcpQ secretin. How the outer membrane and inner membrane components of the T2SS engage each other and yet can allow for substrate uptake into the secretin chamber has challenged the protein transport field for some time. This vexing question is of significance because the T2SS collects folded protein substrates in the periplasm for transport out of the bacterium and yet must discriminate these few substrate proteins from all the other hundred or so folded proteins in the periplasm. The structural analysis here supports a model wherein substrates must compete against a metastable interaction between XcpP in the assembly platform and the XcpQ secretin, wherein only structurally encoded features in the T2SS substrates compete well enough to disrupt XcpQ-XcpP for entry into the XcpQ chamber, for secretion across the outer membrane. | |||||||||
履歴 |
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-構造の表示
ムービー |
ムービービューア |
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構造ビューア | 分子: MolmilJmol/JSmol |
-ダウンロードとリンク
-ダウンロード
PDBx/mmCIF形式 | 5wln.cif.gz | 978.4 KB | 表示 | PDBx/mmCIF形式 |
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PDB形式 | pdb5wln.ent.gz | 810.3 KB | 表示 | PDB形式 |
PDBx/mmJSON形式 | 5wln.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/wl/5wln ftp://data.pdbj.org/pub/pdb/validation_reports/wl/5wln | HTTPS FTP |
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-関連構造データ
-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: 66485.656 Da / 分子数: 15 / 断片: UNP residues 35-658 / 由来タイプ: 組換発現 由来: (組換発現) Pseudomonas aeruginosa (strain ATCC 15692 / DSM 22644 / CIP 104116 / JCM 14847 / LMG 12228 / 1C / PRS 101 / PAO1) (緑膿菌) 株: ATCC 15692 / DSM 22644 / CIP 104116 / JCM 14847 / LMG 12228 / 1C / PRS 101 / PAO1 遺伝子: xcpQ, PA3105 / 発現宿主: Escherichia coli (大腸菌) / 株 (発現宿主): C43 / 参照: UniProt: P35818 |
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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: Type 2 secretion system outer membrane secretin XcpQType II secretion system タイプ: COMPLEX / Entity ID: all / 由来: RECOMBINANT | |||||||||||||||
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分子量 | 値: 1 MDa | |||||||||||||||
由来(天然) | 生物種: Pseudomonas aeruginosa PAO1 (緑膿菌) | |||||||||||||||
由来(組換発現) | 生物種: Escherichia coli (大腸菌) / 株: C43 / プラスミド: pET20b | |||||||||||||||
緩衝液 | pH: 8 | |||||||||||||||
緩衝液成分 |
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試料 | 濃度: 0.5 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 R1.2/1.3 | |||||||||||||||
急速凍結 | 装置: FEI VITROBOT MARK II / 凍結剤: ETHANE / 湿度: 100 % / 凍結前の試料温度: 277 K |
-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: 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(補正後): 130000 X / Calibrated defocus min: 600 nm / 最大 デフォーカス(補正後): 2500 nm / Cs: 2.7 mm |
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: 40 e/Å2 フィルム・検出器のモデル: GATAN K2 SUMMIT (4k x 4k) |
電子光学装置 | エネルギーフィルター名称: GIF |
-解析
ソフトウェア | 名称: PHENIX / バージョン: 1.12_2829: / 分類: 精密化 | ||||||||||||||||||||||||||||||||
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EMソフトウェア |
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画像処理 | 詳細: MotionCorr 2.1 | ||||||||||||||||||||||||||||||||
CTF補正 | 詳細: CTFFIND4 / タイプ: PHASE FLIPPING AND AMPLITUDE CORRECTION | ||||||||||||||||||||||||||||||||
粒子像の選択 | 選択した粒子像数: 18005 | ||||||||||||||||||||||||||||||||
対称性 | 点対称性: C15 (15回回転対称) | ||||||||||||||||||||||||||||||||
3次元再構成 | 解像度: 3.57 Å / 解像度の算出法: FSC 0.143 CUT-OFF / 粒子像の数: 18005 / クラス平均像の数: 1 / 対称性のタイプ: POINT | ||||||||||||||||||||||||||||||||
原子モデル構築 | プロトコル: FLEXIBLE FIT | ||||||||||||||||||||||||||||||||
拘束条件 |
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