- EMDB-3877: Inner membrane Bcs complex (E. coli) -
+
データを開く
IDまたはキーワード:
読み込み中...
-
基本情報
登録情報
データベース: EMDB / ID: EMD-3877
タイトル
Inner membrane Bcs complex (E. coli)
マップデータ
An inner membrane subcomplex of the E. coli cellulose secretion system (Bcs). The complex was purified from solubilised membranes of Bl21(DE3) cells over-expressing the BcsRQAB subunits.
ジャーナル: Nat Commun / 年: 2017 タイトル: Insights into the structure and assembly of a bacterial cellulose secretion system. 著者: Petya Violinova Krasteva / Joaquin Bernal-Bayard / Laetitia Travier / Fernando Ariel Martin / Pierre-Alexandre Kaminski / Gouzel Karimova / Rémi Fronzes / Jean-Marc Ghigo / 要旨: Secreted exopolysaccharides present important determinants for bacterial biofilm formation, survival, and virulence. Cellulose secretion typically requires the concerted action of a c-di-GMP- ...Secreted exopolysaccharides present important determinants for bacterial biofilm formation, survival, and virulence. Cellulose secretion typically requires the concerted action of a c-di-GMP-responsive inner membrane synthase (BcsA), an accessory membrane-anchored protein (BcsB), and several additional Bcs components. Although the BcsAB catalytic duo has been studied in great detail, its interplay with co-expressed subunits remains enigmatic. Here we show that E. coli Bcs proteins partake in a complex protein interaction network. Electron microscopy reveals a stable, megadalton-sized macromolecular assembly, which encompasses most of the inner membrane and cytosolic Bcs components and features a previously unobserved asymmetric architecture. Heterologous reconstitution and mutational analyses point toward a structure-function model, where accessory proteins regulate secretion by affecting both the assembly and stability of the system. Altogether, these results lay the foundation for more comprehensive models of synthase-dependent exopolysaccharide secretion in biofilms and add a sophisticated secretory nanomachine to the diverse bacterial arsenal for virulence and adaptation.
ダウンロード / ファイル: emd_3877.map.gz / 形式: CCP4 / 大きさ: 22.2 MB / タイプ: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
注釈
An inner membrane subcomplex of the E. coli cellulose secretion system (Bcs). The complex was purified from solubilised membranes of Bl21(DE3) cells over-expressing the BcsRQAB subunits.
ボクセルのサイズ
X=Y=Z: 1.9 Å
密度
表面レベル
登録者による: 0.015 / ムービー #1: 0.015
最小 - 最大
-0.09078771 - 0.12465287
平均 (標準偏差)
0.00024227778 (±0.0091167325)
対称性
空間群: 1
詳細
EMDB XML:
マップ形状
Axis order
X
Y
Z
Origin
0
0
0
サイズ
180
180
180
Spacing
180
180
180
セル
A=B=C: 342.0 Å α=β=γ: 90.0 °
CCP4マップ ヘッダ情報:
mode
Image stored as Reals
Å/pix. X/Y/Z
1.9
1.9
1.9
M x/y/z
180
180
180
origin x/y/z
0.000
0.000
0.000
length x/y/z
342.000
342.000
342.000
α/β/γ
90.000
90.000
90.000
MAP C/R/S
1
2
3
start NC/NR/NS
0
0
0
NC/NR/NS
180
180
180
D min/max/mean
-0.091
0.125
0.000
-
添付データ
-
試料の構成要素
-
全体 : Bcs inner membrane sub-complex.
全体
名称: Bcs inner membrane sub-complex.
要素
複合体: Bcs inner membrane sub-complex.
-
超分子 #1: Bcs inner membrane sub-complex.
超分子
名称: Bcs inner membrane sub-complex. / タイプ: complex / ID: 1 / 親要素: 0 詳細: Sample purified using BcsA as bait from solubilised E. coli Bl21(DE3) cells over-expressing the BcsRQAB sub-units of cellulose-secreting E. coli 1094.
pH: 8 詳細: 20 mM HEPES 8.0 120 mM NaCl 10 glycerol 5 mM MgCl2 10 uM AppCp 2 uM c-di-GMP 0.008% LM-NPG cOmplete protease inhibitors
染色
タイプ: NEGATIVE / 材質: uranyl acetate 詳細: 5 ul of sample were spotted on glow-discharged carbon-coated copper grids and incubated for 1 minute. Liquid was blotted off and the sample was passed through 3 drops of 10 ul 2% uranyl ...詳細: 5 ul of sample were spotted on glow-discharged carbon-coated copper grids and incubated for 1 minute. Liquid was blotted off and the sample was passed through 3 drops of 10 ul 2% uranyl acetate with a 30-second incubation over the third. After that the stain was blotted off and the grid was air-dried.
Samples were purified using anti-FLAG affinity gel from detergent-solubilised membranes of E. coli(DE3) cells over-expressing the BcsRQAB subunits of an E. coli 1094-derived strain (pCDF vector, IPTG-inducible overnight expression in TB, affinity tag on BcsA). Eluted samples were further purified by centrifugation over a 10-40% glycerol density gradient.
-
電子顕微鏡法
顕微鏡
FEI TECNAI F20
撮影
フィルム・検出器のモデル: FEI FALCON II (4k x 4k) 撮影したグリッド数: 2 / 平均電子線量: 12.0 e/Å2
電子線
加速電圧: 200 kV / 電子線源: FIELD EMISSION GUN
電子光学系
最大 デフォーカス(補正後): 3.5 µm / 照射モード: OTHER / 撮影モード: BRIGHT FIELD / 最大 デフォーカス(公称値): 0.5 µm / 倍率(公称値): 50000
試料ステージ
ホルダー冷却材: NITROGEN
実験機器
モデル: Tecnai F20 / 画像提供: FEI Company
+
画像解析
粒子像選択
選択した数: 127400 詳細: Particles were picked using the automated picking function of EMAN2.0/Boxer.
CTF補正
ソフトウェア - 名称: EMAN (ver. 2.0) 詳細: CTF correction was carried out in EMAN2.0 (e2ctf) in two rounds, before and after structure factor generation
初期モデル
モデルのタイプ: OTHER 詳細: Initial model was generated using EMAN2.0 (e2initialmodel) using a subset of high contrast class averages following 2D classification in Relion1.4.
タイプ: NOT APPLICABLE / ソフトウェア - 名称: EMAN (ver. 2.0)
最終 角度割当
タイプ: NOT APPLICABLE
最終 3次元分類
クラス数: 200 / ソフトウェア - 名称: RELION (ver. 1.4) 詳細: Particles were saved as an .mrcs stack and subjected to two rounds of 2D classification in Relion. Only high-contrast classes (20182 particles in total) were used for 3D classification with ...詳細: Particles were saved as an .mrcs stack and subjected to two rounds of 2D classification in Relion. Only high-contrast classes (20182 particles in total) were used for 3D classification with three classes. Two of the classes converged to the same 3D model and the corresponding particles (14362) were used for 3D refinement of the EMAN2.0-generated initial model.