National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)
GM 58448
米国
引用
ジャーナル: Nature / 年: 2019 タイトル: Cryo-EM structure of the human α1β3γ2 GABA receptor in a lipid bilayer. 著者: Duncan Laverty / Rooma Desai / Tomasz Uchański / Simonas Masiulis / Wojciech J Stec / Tomas Malinauskas / Jasenko Zivanov / Els Pardon / Jan Steyaert / Keith W Miller / A Radu Aricescu / 要旨: Type A γ-aminobutyric acid (GABA) receptors are pentameric ligand-gated ion channels and the main drivers of fast inhibitory neurotransmission in the vertebrate nervous system. Their dysfunction is ...Type A γ-aminobutyric acid (GABA) receptors are pentameric ligand-gated ion channels and the main drivers of fast inhibitory neurotransmission in the vertebrate nervous system. Their dysfunction is implicated in a range of neurological disorders, including depression, epilepsy and schizophrenia. Among the numerous assemblies that are theoretically possible, the most prevalent in the brain are the α1β2/3γ2 GABA receptors. The β3 subunit has an important role in maintaining inhibitory tone, and the expression of this subunit alone is sufficient to rescue inhibitory synaptic transmission in β1-β3 triple knockout neurons. So far, efforts to generate accurate structural models for heteromeric GABA receptors have been hampered by the use of engineered receptors and the presence of detergents. Notably, some recent cryo-electron microscopy reconstructions have reported 'collapsed' conformations; however, these disagree with the structure of the prototypical pentameric ligand-gated ion channel the Torpedo nicotinic acetylcholine receptor, the large body of structural work on homologous homopentameric receptor variants and the logic of an ion-channel architecture. Here we present a high-resolution cryo-electron microscopy structure of the full-length human α1β3γ2L-a major synaptic GABA receptor isoform-that is functionally reconstituted in lipid nanodiscs. The receptor is bound to a positive allosteric modulator 'megabody' and is in a desensitized conformation. Each GABA receptor pentamer contains two phosphatidylinositol-4,5-bisphosphate molecules, the head groups of which occupy positively charged pockets in the intracellular juxtamembrane regions of α1 subunits. Beyond this level, the intracellular M3-M4 loops are largely disordered, possibly because interacting post-synaptic proteins are not present. This structure illustrates the molecular principles of heteromeric GABA receptor organization and provides a reference framework for future mechanistic investigations of GABAergic signalling and pharmacology.
モード: BRIGHT FIELD / 倍率(公称値): 75000 X / 倍率(補正後): 75000 X / 最大 デフォーカス(公称値): 700 nm / 最小 デフォーカス(公称値): 500 nm / Calibrated defocus min: 500 nm / 最大 デフォーカス(補正後): 700 nm / Cs: 2.7 mm / C2レンズ絞り径: 50 µm
試料ホルダ
試料ホルダーモデル: FEI TITAN KRIOS AUTOGRID HOLDER
撮影
平均露光時間: 60 sec. / 電子線照射量: 30.84 e/Å2 / 検出モード: COUNTING フィルム・検出器のモデル: FEI FALCON III (4k x 4k) 撮影したグリッド数: 1 / 実像数: 784
電子光学装置
位相板: VOLTA PHASE PLATE
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解析
ソフトウェア
名称: PHENIX / バージョン: 1.14rc2_3191: / 分類: 精密化
EMソフトウェア
ID
名称
バージョン
カテゴリ
5
Gctf
v1.18
CTF補正
10
RELION
初期オイラー角割当
11
RELION
最終オイラー角割当
12
RELION
v3.0
分類
13
RELION
3
3次元再構成
14
PHENIX
モデル精密化
CTF補正
詳細: CTF parameters were estimated using GCTF and CTF correction performed in RELION (full phase and amplitude correction). タイプ: PHASE FLIPPING AND AMPLITUDE CORRECTION
対称性
点対称性: C1 (非対称)
3次元再構成
解像度: 3.2 Å / 解像度の算出法: FSC 0.143 CUT-OFF / 粒子像の数: 55449 / アルゴリズム: FOURIER SPACE / クラス平均像の数: 1 / 対称性のタイプ: POINT