SARS-CoV-2 infection is controlled by the opening of the spike protein receptor binding domain (RBD), which transitions from a glycan-shielded ‘down’ to an exposed ‘up’ state to bind the human angiotensin-converting enzyme 2 receptor and infect cells. While snapshots of the ‘up’ and ‘down’ states have been obtained by cryo-electron microscopy and cryo-electron tomagraphy, details of the RBD-opening transition evade experimental characterization. Here over 130 µs of weighted ensemble simulations of the fully glycosylated spike ectodomain allow us to characterize more than 300 continuous, kinetically unbiased RBD-opening pathways. Together with ManifoldEM analysis of cryo-electron microscopy data and biolayer interferometry experiments, we reveal a gating role for the N-glycan at position N343, which facilitates RBD opening. Residues D405, R408 and D427 also participate. The atomic-level characterization of the glycosylated spike activation mechanism provided herein represents a landmark study for ensemble pathway simulations and offers a foundation for understanding the fundamental mechanisms of SARS-CoV-2 viral entry and infection.

SARS-CoV-2 感染由刺突蛋白受体结合域 (RBD) 的开放控制，RBD 从聚糖屏蔽的“向下”转变为暴露的“向上”状态以结合人血管紧张素转换酶 2 受体和感染细胞。虽然已经通过冷冻电子显微镜和冷冻电子断层扫描获得了“向上”和“向下”状态的快照，但 RBD 开放跃迁的细节避开了实验表征。在这里，超过 130 µs 的完全糖基化尖峰胞外域的加权集合模拟使我们能够表征 300 多个连续的、动力学上无偏的 RBD 开放途径。连同对低温电子显微镜数据和生物层干涉测量实验的 ManifoldEM 分析，我们揭示了N的门控作用-N343 位的聚糖，促进 RBD 打开。残基 D405、R408 和 D427 也参与其中。本文提供的糖基化尖峰激活机制的原子级表征代表了集合通路模拟的里程碑式研究，并为理解 SARS-CoV-2 病毒进入和感染的基本机制提供了基础。