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- EMDB-1044: Structure and gating mechanism of the acetylcholine receptor pore. -

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基本情報

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データベース: EMDB / ID: EMD-1044
タイトルStructure and gating mechanism of the acetylcholine receptor pore.
マップデータThe map is of the membrane-spanning domain of the nicotinic acetylcholine receptor in the closed state, viewed from the synaptic cleft. The arrangement of subunits around the central axis, clockwise beginning from the bottom (closest to 0 on the y-axis) ia alpha, gamma, beta, delta. The Fourier terms were derived from tubular crystals having helical symmetry. They are of higher quality along the meridional (y-axis) direction than the equatorial direction (where the diffraction is weaker and there is additional noise associated with layer-line overlap. This has resulted in some asymmetry in the map, with the best direction being along the axis of the tube (y-axis). The map was obtained by averaging data from four helical families in real space, weighting each family approximately according to the number of receptors analysed. The actual weights were: 0.70 (-16,6); 0.30 (-15,7); 0.30 (-17,5); 0.25 (-18,6). As explained in the Reference, the dominating low resolution terms were weakened by subtracting a map of the structure with terms extending to only 15 Angstroms. THe weight used for the subtraction map was -0.88. The terms along the equator have also been included with a weight of 0.04, so that the densities corresponding to the alpha-helical segments are represented at about the same level throughout the thickness of the bilayer.
試料
  • 試料: Crystalline postsynaptic membrane from Torpedo marmorata electric organ
  • 細胞器官・細胞要素: postsynaptic membrane lipids
  • タンパク質・ペプチド: acetylcholine receptor
機能・相同性
機能・相同性情報


acetylcholine-gated channel complex / acetylcholine receptor signaling pathway / acetylcholine-gated monoatomic cation-selective channel activity / transmitter-gated monoatomic ion channel activity involved in regulation of postsynaptic membrane potential / transmembrane signaling receptor activity / postsynaptic membrane / neuron projection
類似検索 - 分子機能
Nicotinic acetylcholine receptor / Neurotransmitter-gated ion-channel, conserved site / Neurotransmitter-gated ion-channels signature. / Neurotransmitter-gated ion-channel transmembrane domain / Neurotransmitter-gated ion-channel transmembrane region / Neurotransmitter-gated ion-channel transmembrane domain superfamily / Neuronal acetylcholine receptor / Neurotransmitter-gated ion-channel / Neurotransmitter-gated ion-channel ligand-binding domain / Neurotransmitter-gated ion-channel ligand-binding domain superfamily / Neurotransmitter-gated ion-channel ligand binding domain
類似検索 - ドメイン・相同性
Acetylcholine receptor subunit alpha / Acetylcholine receptor subunit beta / Acetylcholine receptor subunit gamma / Acetylcholine receptor subunit delta / Acetylcholine receptor subunit delta / Acetylcholine receptor gamma subunit / Acetylcholine receptor subunit beta
類似検索 - 構成要素
生物種Torpedo marmorata (エイ)
手法らせん対称体再構成法 / クライオ電子顕微鏡法 / ネガティブ染色法 / 解像度: 4.0 Å
データ登録者Miyazawa A / Fujiyoshi Y / Unwin N
引用ジャーナル: Nature / 年: 2003
タイトル: Structure and gating mechanism of the acetylcholine receptor pore.
著者: Atsuo Miyazawa / Yoshinori Fujiyoshi / Nigel Unwin /
要旨: The nicotinic acetylcholine receptor controls electrical signalling between nerve and muscle cells by opening and closing a gated, membrane-spanning pore. Here we present an atomic model of the ...The nicotinic acetylcholine receptor controls electrical signalling between nerve and muscle cells by opening and closing a gated, membrane-spanning pore. Here we present an atomic model of the closed pore, obtained by electron microscopy of crystalline postsynaptic membranes. The pore is shaped by an inner ring of 5 alpha-helices, which curve radially to create a tapering path for the ions, and an outer ring of 15 alpha-helices, which coil around each other and shield the inner ring from the lipids. The gate is a constricting hydrophobic girdle at the middle of the lipid bilayer, formed by weak interactions between neighbouring inner helices. When acetylcholine enters the ligand-binding domain, it triggers rotations of the protein chains on opposite sides of the entrance to the pore. These rotations are communicated through the inner helices, and open the pore by breaking the girdle apart.
履歴
登録2003年4月3日-
ヘッダ(付随情報) 公開2003年4月9日-
マップ公開2005年4月9日-
更新2014年6月18日-
現状2014年6月18日処理サイト: PDBe / 状態: 公開

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構造の表示

ムービー
  • 表面図（断面を密度値に従い着色）
  • 表面レベル: 2.2
  • UCSF Chimeraによる作画
  • ダウンロード
  • 表面図（円筒半径に従い着色）
  • 表面レベル: 2.2
  • UCSF Chimeraによる作画
  • ダウンロード
  • あてはめたモデルとの重ね合わせ
  • 原子モデル: PDB-1oed
  • 表面レベル: 2.2
  • UCSF Chimeraによる作画
  • ダウンロード
ムービービューア
構造ビューアEMマップ:
SurfViewMolmilJmol/JSmol
添付画像

1044.gif

ダウンロードとリンク

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マップ

ファイルダウンロード / ファイル: emd_1044.map.gz / 形式: CCP4 / 大きさ: 3.4 MB / タイプ: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
注釈The map is of the membrane-spanning domain of the nicotinic acetylcholine receptor in the closed state, viewed from the synaptic cleft. The arrangement of subunits around the central axis, clockwise beginning from the bottom (closest to 0 on the y-axis) ia alpha, gamma, beta, delta. The Fourier terms were derived from tubular crystals having helical symmetry. They are of higher quality along the meridional (y-axis) direction than the equatorial direction (where the diffraction is weaker and there is additional noise associated with layer-line overlap. This has resulted in some asymmetry in the map, with the best direction being along the axis of the tube (y-axis). The map was obtained by averaging data from four helical families in real space, weighting each family approximately according to the number of receptors analysed. The actual weights were: 0.70 (-16,6); 0.30 (-15,7); 0.30 (-17,5); 0.25 (-18,6). As explained in the Reference, the dominating low resolution terms were weakened by subtracting a map of the structure with terms extending to only 15 Angstroms. THe weight used for the subtraction map was -0.88. The terms along the equator have also been included with a weight of 0.04, so that the densities corresponding to the alpha-helical segments are represented at about the same level throughout the thickness of the bilayer.
ボクセルのサイズX=Y=Z: 1 Å
密度
表面レベル1: 2.18 / ムービー #1: 2.2
最小 - 最大-2.43914 - 6.31988
平均 (標準偏差)-0.000000169242 (±1.0)
対称性空間群: 1
詳細

EMDB XML:

マップ形状
Axis orderYXZ
Origin0052
サイズ12812855
Spacing12812855
セルA: 128 Å / B: 128 Å / C: 168 Å
α=β=γ: 90 °

CCP4マップ ヘッダ情報:

modeImage stored as Reals
Å/pix. X/Y/Z111
M x/y/z128128168
origin x/y/z0.0000.0000.000
length x/y/z128.000128.000168.000
α/β/γ90.00090.00090.000
start NX/NY/NZ0052
NX/NY/NZ12812855
MAP C/R/S213
start NC/NR/NS0052
NC/NR/NS12812855
D min/max/mean-2.4396.320-0.000

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添付データ

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試料の構成要素

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全体 : Crystalline postsynaptic membrane from Torpedo marmorata electric...

全体名称: Crystalline postsynaptic membrane from Torpedo marmorata electric organ
要素
  • 試料: Crystalline postsynaptic membrane from Torpedo marmorata electric organ
  • 細胞器官・細胞要素: postsynaptic membrane lipids
  • タンパク質・ペプチド: acetylcholine receptor

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超分子 #1000: Crystalline postsynaptic membrane from Torpedo marmorata electric...

超分子名称: Crystalline postsynaptic membrane from Torpedo marmorata electric organ
タイプ: sample / ID: 1000
集合状態: The acetylcholine receptors are hetero-pentamers composed of 2 alpha 1 beta 1 gamma and 1 delta subunit
Number unique components: 2

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超分子 #1: postsynaptic membrane lipids

超分子名称: postsynaptic membrane lipids / タイプ: organelle_or_cellular_component / ID: 1 / 組換発現: No / データベース: NCBI
由来(天然)生物種: Torpedo marmorata (エイ) / 別称: Torpedo marmorata / Organelle: electric organ / 細胞中の位置: plasma membrane

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分子 #1: acetylcholine receptor

分子名称: acetylcholine receptor / タイプ: protein_or_peptide / ID: 1
詳細: This is the MW of the glycosylated protein. The protein itself accounts for 258kD
集合状態: pentamer / 組換発現: No / データベース: NCBI
由来(天然)生物種: Torpedo marmorata (エイ) / 別称: marbled electric ray / Organelle: electric organ / 細胞中の位置: plasma membrane
分子量実験値: 290 KDa

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実験情報

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構造解析

手法ネガティブ染色法, クライオ電子顕微鏡法
解析らせん対称体再構成法
試料の集合状態filament

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試料調製

緩衝液pH: 6.8 / 詳細: 100mM sodium cacodylate, 1mM CaCl2
染色タイプ: NEGATIVE / 詳細: no stains or fixatives used
グリッド詳細: holey carbon film made over 300 mesh copper grids. To minimise beam movement at the 4K imaging temperature, it was essential that the carbon films had a high electrical conductivity - ...詳細: holey carbon film made over 300 mesh copper grids. To minimise beam movement at the 4K imaging temperature, it was essential that the carbon films had a high electrical conductivity - achieved by evaporation of carbon in a high vacuum and pre-irradiation of the grids.
凍結凍結剤: ETHANE / チャンバー内湿度: 90 % / チャンバー内温度: 100 K / 装置: HOMEMADE PLUNGER
詳細: Vitrification instrument: Home-built model. The plunging apparatus was contained in a bench-top fridge having a window made in the door. Wet air was continually bubbled into the fridge, which ...詳細: Vitrification instrument: Home-built model. The plunging apparatus was contained in a bench-top fridge having a window made in the door. Wet air was continually bubbled into the fridge, which was maintained at 4-8 deg. centigrade.
手法: The grid was first glow-discharged in the presence of amyl amine. The specimen was applied to the carbon-film side in 4.2ul droplets. Blotting was done from the other side, removing the ...手法: The grid was first glow-discharged in the presence of amyl amine. The specimen was applied to the carbon-film side in 4.2ul droplets. Blotting was done from the other side, removing the filter paper and plunging as soon as the paper and grid were observed to lose water-contact with each other - typically after 6 seconds.

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電子顕微鏡法

顕微鏡JEOL KYOTO-3000SFF
温度最低: 4.2 K / 最高: 4.2 K / 平均: 4.2 K
アライメント法Legacy - 非点収差: correction on carbon film at 250,000
撮影カテゴリ: FILM / フィルム・検出器のモデル: KODAK SO-163 FILM / デジタル化 - スキャナー: OTHER / デジタル化 - サンプリング間隔: 5 µm / 実像数: 359 / 平均電子線量: 20 e/Å2
詳細: Scanning done with a point-source, flat-bed Joyce-Loebl microdensitometer, modified in-house
Od range: 1 / ビット/ピクセル: 10
電子線加速電圧: 300 kV / 電子線源: FIELD EMISSION GUN
電子光学系倍率（補正後）: 36800 / 照射モード: FLOOD BEAM / 撮影モード: BRIGHT FIELD / Cs: 1.3 mm / 最大 デフォーカス（公称値）: 1.8 µm / 最小 デフォーカス（公称値）: 0.8 µm / 倍率（公称値）: 40000
試料ステージ試料ホルダー: top-entry / 試料ホルダーモデル: OTHER

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画像解析

詳細The specimens were tubular crystals forming a range of helical families, with the receptors in each case being organised on a p2 surface lattice.
最終 再構成解像度のタイプ: BY AUTHOR / 解像度: 4.0 Å / 解像度の算出法: FSC 0.5 CUT-OFF
ソフトウェア - 名称: In-house software based on MRC system
詳細: Layer-line data were collected from 4 helical families of tubes - (-16,6),(-15,7),(-17,5),(-18,6) - after dividing the tubes into short segments to correct for distortions. The maps ...詳細: Layer-line data were collected from 4 helical families of tubes - (-16,6),(-15,7),(-17,5),(-18,6) - after dividing the tubes into short segments to correct for distortions. The maps calculated from each of the families were then averaged in real space to derive the final three-dimensional densities.
CTF補正詳細: Measurement of positions of Thon rings from area of tube that was processed
FSC曲線 (解像度の算出)

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原子モデル構築 1

詳細Interpretation of the experimental density map and model building into the densities were performed using O. The helical segments were fitted individually, using the protruding regions along the helical densities to identify the largest side chains. This allowed tentative assignments to be made of each amino acid according to the sequence, both along the helices and along the short connecting loops. These assignments were then validated for each subunit by checking their consistency with residues in equivalent positions around the pentamer.
得られたモデル

PDB-1oed:
STRUCTURE OF ACETYLCHOLINE RECEPTOR PORE FROM ELECTRON IMAGES

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