Berriman J, Unwin N
MRC Laboratory of Molecular Biology, Cambridge, UK.
Ultramicroscopy. 1994 Dec;56(4):241-52. doi: 10.1016/0304-3991(94)90012-4.
A simple method to determine transient conformations of biological molecules is described. The two reactants (e.g. protein complex and ligand) are mixed rapidly by the coalescence of spray droplets containing one component, with a thin, grid-supported aqueous film containing the other. The transient state is then trapped by rapid freezing, and investigated later by cryo-microscopy. Images of conformations associated with reaction times of 1-100 ms can be achieved by adjusting the delay between the droplet impact and freezing. The droplets (typically 1 micron in diameter) are propelled onto the grid by an atomizer spray. It is shown that the droplets impinging on the liquid film spread rapidly over its surface under the influence of surface tension, and only weakly disturb the underlying film, partially displacing its contents away from the point of impact. Experiments with sprayed salt solutions, using vesicles derived from erythrocytes as micro-osmometers, indicate that rapid mixing occurs both through the film and laterally, by diffusion. The spraying process does not produce any detectable concentration changes due to drying in either the droplets or the film, and the method is applicable to high-resolution imaging.
本文描述了一种确定生物分子瞬态构象的简单方法。两种反应物（如蛋白质复合物和配体）通过含有一种成分的喷雾液滴与含有另一种成分的薄的、网格支撑的水膜合并而快速混合。然后通过快速冷冻捕获瞬态状态，随后通过冷冻显微镜进行研究。通过调整液滴撞击和冷冻之间的延迟，可以获得与1-100毫秒反应时间相关的构象图像。液滴（通常直径为1微米）由雾化器喷雾喷射到网格上。结果表明，撞击液膜的液滴在表面张力的影响下在其表面迅速扩散，仅对下层液膜产生微弱扰动，将其内容物部分地从撞击点移开。使用源自红细胞的囊泡作为微渗透计对喷雾盐溶液进行的实验表明，快速混合通过液膜并通过扩散横向发生。喷雾过程不会因液滴或液膜中的干燥而产生任何可检测到的浓度变化，并且该方法适用于高分辨率成像。
