ジャーナル: Cell Rep / 年: 2016 タイトル: Heterogeneous MAC Initiator and Pore Structures in a Lipid Bilayer by Phase-Plate Cryo-electron Tomography. 著者: Thomas H Sharp / Abraham J Koster / Piet Gros / 要旨: Pore formation in membranes is important for mammalian immune defense against invading bacteria. Induced by complement activation, the membrane attack complex (MAC) forms through sequential binding ...Pore formation in membranes is important for mammalian immune defense against invading bacteria. Induced by complement activation, the membrane attack complex (MAC) forms through sequential binding and membrane insertion of C5b6, C7, C8, and C9. Using cryo-electron tomography with a Volta phase plate and subtomogram averaging, we imaged C5b-7, C5b-8, and C5b-9 complexes and determined the C5b-9 pore structure in lipid bilayers. The in situ C5b-9 pore structure at 2.3-nm resolution reveals a 10- to 11.5-nm cone-shaped pore starting with C5b678 and multiple copies of C9 that is poorly closed, yielding a seam between C9 and C6 substituting for the shorter β strands in C6 and C7. However, large variations of composite pore complexes are apparent in subtomograms. Oligomerized initiator complexes C5b-7 and C5b-8 show stages of membrane binding, deformation, and perforation that yield ∼3.5-nm-wide pores. These data indicate a dynamic process of pore formation that likely adapts to biological membranes under attack.
ダウンロード / ファイル: emd_3289.map.gz / 形式: CCP4 / 大きさ: 51.5 MB / タイプ: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
注釈
Subtomogram average of the complement membrane attack complex
ボクセルのサイズ
X=Y=Z: 2.861 Å
密度
表面レベル
登録者による: 0.11 / ムービー #1: 0.11
最小 - 最大
-0.1858658 - 0.41718206
平均 (標準偏差)
0.00101197 (±0.0297654)
対称性
空間群: 1
詳細
EMDB XML:
マップ形状
Axis order
X
Y
Z
Origin
-120
-120
-120
サイズ
240
240
240
Spacing
240
240
240
セル
A=B=C: 686.64 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
2.861
2.861
2.861
M x/y/z
240
240
240
origin x/y/z
0.000
0.000
0.000
length x/y/z
686.640
686.640
686.640
α/β/γ
90.000
90.000
90.000
MAP C/R/S
1
2
3
start NC/NR/NS
-120
-120
-120
NC/NR/NS
240
240
240
D min/max/mean
-0.186
0.417
0.001
-
添付データ
-
試料の構成要素
-
全体 : Subtomogram average of the membrane attack complex pore in a lipi...
全体
名称: Subtomogram average of the membrane attack complex pore in a lipid bilayer
要素
試料: Subtomogram average of the membrane attack complex pore in a lipid bilayer
タンパク質・ペプチド: Complement component C5b6
タンパク質・ペプチド: Complement component C7
タンパク質・ペプチド: Complement component C8-alpha chain
タンパク質・ペプチド: Complement component C8-beta chain
タンパク質・ペプチド: Complement component C8-gamma chain
タンパク質・ペプチド: Complement component C9 chain
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超分子 #1000: Subtomogram average of the membrane attack complex pore in a lipi...
超分子
名称: Subtomogram average of the membrane attack complex pore in a lipid bilayer タイプ: sample / ID: 1000 集合状態: One copy of C5b, C6 and C7; one C8 heterotrimer; multiple copies of C9 Number unique components: 6
詳細: 300 mesh copper grids with lacey-carbon support, glow discharged.
凍結
凍結剤: ETHANE / チャンバー内湿度: 95 % / チャンバー内温度: 90.15 K / 装置: LEICA EM GP 詳細: 6 ul sample pipetted onto freshly plasma-cleaned 300 mesh copper grids with lacey-carbon support 手法: Blot for 6 seconds before plunging
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電子顕微鏡法
顕微鏡
FEI TITAN KRIOS
詳細
Low-dose protocol used. Volta phase plate used
日付
2015年3月10日
撮影
カテゴリ: CCD フィルム・検出器のモデル: FEI FALCON II (4k x 4k) 平均電子線量: 1 e/Å2 / 詳細: Volta phase plate used
試料ホルダーモデル: FEI TITAN KRIOS AUTOGRID HOLDER Tilt series - Axis1 - Min angle: 60 ° / Tilt series - Axis1 - Max angle: 60 °
実験機器
モデル: Titan Krios / 画像提供: FEI Company
-
画像解析
詳細
Subtomograms were picked by hand
最終 再構成
想定した対称性 - 点群: C1 (非対称) / アルゴリズム: OTHER / 解像度のタイプ: BY AUTHOR / 解像度: 23.0 Å / 解像度の算出法: OTHER / ソフトウェア - 名称: EMAN2 詳細: Initial model was phase-randomized beyond 6 nm. Refinement was against a rotationally-averaged map. 使用したサブトモグラム数: 986