EMDB/PDB/SASBDBから引用されている文献のデータベース

キーワード
構造解析手法	All X線回折 NMR 電子顕微鏡小角散乱中性子線回折線維回折粉末回折赤外分光 EPR FRET 理論モデルハイブリッド
著者・登録者
ジャーナル
IF	>30 >20 >10 >5 all

タイトル	Production and cryo-electron microscopy structure of an internally tagged SARS-CoV-2 spike ecto-domain construct.
ジャーナル・号・ページ	J Struct Biol X, Vol. 11, Page 100123, Year 2025
掲載日	2025年2月11日
著者	Suruchi Singh / Yi Liu / Meghan Burke / Vamseedhar Rayaprolu / Stephen E Stein / S Saif Hasan /
PubMed 要旨	The SARS-CoV-2 spike protein is synthesized in the endoplasmic reticulum of host cells, from where it undergoes export to the Golgi and the plasma membrane or retrieval from the Golgi to the ...The SARS-CoV-2 spike protein is synthesized in the endoplasmic reticulum of host cells, from where it undergoes export to the Golgi and the plasma membrane or retrieval from the Golgi to the endoplasmic reticulum. Elucidating the fundamental principles of this bidirectional secretion are pivotal to understanding virus assembly and designing the next generation of spike genetic vaccine with enhanced export properties. However, the widely used strategy of C-terminal affinity tagging of the spike cytosolic tail interferes with proper bidirectional trafficking. Hence, the structural and biophysical investigations of spike protein trafficking have been hindered by a lack of appropriate spike constructs. Here we describe a strategy for the internal tagging of the spike protein. Using sequence analyses and AlphaFold modeling, we identified a site down-stream of the signal sequence for the insertion of a twin-strep-tag, which facilitates purification of an ecto-domain construct from the extra-cellular medium of mammalian Expi293F cells. Mass spectrometry analyses show that the internal tag has minimal impact on -glycan modifications, which are pivotal for spike-host interactions. Single particle cryo-electron microscopy reconstructions of the spike ecto-domain reveal conformational states compatible for ACE2 receptor interactions, further solidifying the feasibility of the internal tagging strategy. Collectively, these results present a substantial advance towards reagent development for the investigations of spike protein trafficking during coronavirus infection and genetic vaccination.
リンク	J Struct Biol X / PubMed:40046771 / PubMed Central
手法	EM (単粒子)
解像度	2.32 - 2.99 Å
構造データ	44062 9b0y EMDB-44062, PDB-9b0y: SARS CoV-2 Spike protein Ectodomain with internal tag, all RBD-down conformation 手法: EM (単粒子) / 解像度: 2.32 Å 44116 9b2v EMDB-44116, PDB-9b2v: SARS CoV-2 Spike protein Ectodomain with internal tag, 1RBD-up conformation 手法: EM (単粒子) / 解像度: 2.55 Å 45893 9css EMDB-45893, PDB-9css: Cryo-EM structure of SARS-CoV-2 spike protein Ecto-domain with internal tag, 1UP RBD conformation 手法: EM (単粒子) / 解像度: 2.72 Å 45895 9ct2 EMDB-45895, PDB-9ct2: Cryo-EM structure of SARS-CoV-2 spike protein Ecto-domain with internal tag, All RBD down conformation, State-3 手法: EM (単粒子) / 解像度: 2.7 Å 45965 9cvh EMDB-45965, PDB-9cvh: Cryo-EM structure of SARS-CoV-2 spike protein Ecto-domain with internal tag, 1RBD UP, State-2 手法: EM (単粒子) / 解像度: 2.76 Å 45987 9cxe EMDB-45987, PDB-9cxe: SARS CoV-2 Spike protein Ectodomain with internal tag, all RBD-down conformation -C1 手法: EM (単粒子) / 解像度: 2.48 Å 48839 9n2l EMDB-48839, PDB-9n2l: Cryo-EM structure of locally refined up conformation of SARS-CoV-2 spike protein Receptor Binding Domain 手法: EM (単粒子) / 解像度: 2.99 Å
化合物	ChemComp-NAG: 2-acetamido-2-deoxy-beta-D-glucopyranose / N-アセチル-β-D-グルコサミン
由来	severe acute respiratory syndrome coronavirus 2 (ウイルス) severe acute respiratory syndrome coronavirus (SARS コロナウイルス)
キーワード	VIRAL PROTEIN / SARS CoV-2 Spike protein Ectodomain with internal tag / SARS-CoV-2 Spike protein Ectodomain with internal tag / SARS-CoV-2 Spike protein / Receptor Binding Domain