ジャーナル: EMBO J / 年: 2013 タイトル: Structure of a bacterial type IV secretion core complex at subnanometre resolution. 著者: Angel Rivera-Calzada / Rémi Fronzes / Christos G Savva / Vidya Chandran / Pei W Lian / Toon Laeremans / Els Pardon / Jan Steyaert / Han Remaut / Gabriel Waksman / Elena V Orlova / 要旨: Type IV secretion (T4S) systems are able to transport DNAs and/or proteins through the membranes of bacteria. They form large multiprotein complexes consisting of 12 proteins termed VirB1-11 and ...Type IV secretion (T4S) systems are able to transport DNAs and/or proteins through the membranes of bacteria. They form large multiprotein complexes consisting of 12 proteins termed VirB1-11 and VirD4. VirB7, 9 and 10 assemble into a 1.07 MegaDalton membrane-spanning core complex (CC), around which all other components assemble. This complex is made of two parts, the O-layer inserted in the outer membrane and the I-layer inserted in the inner membrane. While the structure of the O-layer has been solved by X-ray crystallography, there is no detailed structural information on the I-layer. Using high-resolution cryo-electron microscopy and molecular modelling combined with biochemical approaches, we determined the I-layer structure and located its various components in the electron density. Our results provide new structural insights on the CC, from which the essential features of T4S system mechanisms can be derived.
A: TRAO PROTEIN B: TRAO PROTEIN C: TRAO PROTEIN D: TRAO PROTEIN E: TRAO PROTEIN F: TRAO PROTEIN G: TRAO PROTEIN H: TRAO PROTEIN I: TRAO PROTEIN J: TRAO PROTEIN K: TRAO PROTEIN L: TRAO PROTEIN M: TRAO PROTEIN N: TRAO PROTEIN
名称: TRAN, TRAO AND TRAF COMPLEX ENCODED BY PKM101 DIGESTED WITH 0.02 MG ML- 1 OF ELASTASE FOR 40 MIN AT ROOM TEMPERATURE タイプ: COMPLEX
緩衝液
名称: 50 MM TRIS-HCL, 200 MM NACL, 10 MM LDAO / pH: 8 / 詳細: 50 MM TRIS-HCL, 200 MM NACL, 10 MM LDAO
試料
濃度: 0.01 mg/ml / 包埋: NO / シャドウイング: NO / 染色: NO / 凍結: YES
試料支持
詳細: CARBON
急速凍結
装置: FEI VITROBOT MARK III / 凍結剤: ETHANE 詳細: VITRIFICATION 1 -- CRYOGEN- ETHANE, HUMIDITY- 100, TEMPERATURE- 92, INSTRUMENT- FEI VITROBOT MARK III, METHOD- BLOT 4 SECONDS BEFORE PLUNGING,
モード: BRIGHT FIELD / 倍率(公称値): 66000 X / 倍率(補正後): 68100 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): 1500 nm / Cs: 2.1 mm
試料ホルダ
温度: 95 K
撮影
電子線照射量: 20 e/Å2 / フィルム・検出器のモデル: GENERIC GATAN
画像スキャン
デジタル画像の数: 262
放射波長
相対比: 1
-
解析
EMソフトウェア
ID
名称
カテゴリ
1
Flex-EM
モデルフィッティング
2
Sculptor
モデルフィッティング
3
Situs
モデルフィッティング
4
UCSF Chimera
モデルフィッティング
5
IMAGIC
3次元再構成
CTF補正
詳細: PHASE FLIPPING, EACH CCD IMAGE
対称性
点対称性: C14 (14回回転対称)
3次元再構成
手法: COMMON LINES / 解像度: 8.5 Å / 粒子像の数: 5430 / ピクセルサイズ(公称値): 2.2 Å / ピクセルサイズ(実測値): 2.08 Å 詳細: SUBMISSION BASED ON EXPERIMENTAL DATA FROM EMDB EMD -2233. (DEPOSITION ID: 11228). 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 / Target criteria: Cross-correlation coefficient 詳細: METHOD--RIGID BODY FOLLOWED BY FLEXIBLE FITTING REFINEMENT PROTOCOL--MODELLED