标题： 可溶性蛋白在软着陆ESIBD后的高分辨率冷冻电镜结构确定 显示英文标题

标题： High-resolution cryoEM structure determination of soluble proteins after soft-landing ESIBD

摘要： 软着陆电喷雾离子束沉积（ESIBD），即在真空下对大型和复杂分子进行化学选择性、完整沉积的方法，是一种用于扫描探针显微镜（SPM）的成熟样品制备方法，它有助于对大型和复杂分子进行高分辨率成像。 继承自质谱分析，ESIBD的优势特性，如化学纯度和选择性，以及在真空环境下对样品环境的精确控制，与其他分析方法结合时也具有吸引力。 因此，最近ESIBD被用于通过天然电喷雾电离（天然ESIBD）将蛋白质引入气相来制备电子冷冻显微镜（cryoEM）的样品。 然而，这种方法的普遍适用性和基于ESIBD的cryoEM工作流程的最佳条件仍不清楚。 在此，我们介绍了专为ESIBD+cryoEM工作流程设计的仪器和方法，该方法基于对沉积条件和样品环境的精确控制。 我们确定了蛋白质温和沉积的最优条件，均质超薄非晶冰的生长条件以及避免污染的条件，从而实现了多种蛋白质系统的原子级分辨率cryoEM成像。 我们的结果证实了蛋白质的天然二级结构和总体形状的保留。 ESIBD+cryoEM直接将天然MS的化学洞察与cryoEM的结构精度联系起来，为结构生物学应用提供了一个多功能平台。 显示英文摘要

摘要： Soft-landing electrospray ion beam deposition (ESIBD), i.e. the chemically selective, intact deposition of large and complex molecules on surfaces in vacuum, is a well-established sample preparation method for scanning probe microscopy (SPM), where it facilitates the high-resolution imaging of large and complex molecules. Inherited from mass spectrometry, the advantageous properties of ESIBD, chemical purity and selectivity, as well as precise control over the sample environment in vacuum, are also desirable in combination with other analytical methods. Therefore, recently ESIBD has been pursued for the fabrication of samples for electron cryo-microscopy (cryoEM) with proteins introduced into the gas phase via native electrospray ionisation (native ESIBD). However, the general applicability of this approach and the optimal conditions for an ESIBD-based cryoEM workflow remain unclear. Here, we present instrumentation and methods for a dedicated ESIBD+cryoEM workflow, based on precise control over deposition conditions and sample environment. We establish optimal conditions for gentle deposition of the proteins, for growing homogeneous, ultra-thin, amorphous ice, and for avoiding contamination, which allows for atomic-resolution cryoEM imaging of multiple protein systems. Our results confirm the retention of the protein's native secondary structure and general shape. ESIBD+cryoEM directly links native MS chemical insights with cryoEM's structural precision, offering a versatile platform for structural biology applications.