ANALYSIS OF TRANSIENT STRUCTURES BY CRYOMICROSCOPY COMBINED WITH RAPID MIXING OF SPRAY DROPLETS
ANALYSIS OF TRANSIENT STRUCTURES BY CRYOMICROSCOPY COMBINED WITH RAPID MIXING OF SPRAY DROPLETS
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DOI：
10.1016/0304-3991(94)90012-4
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发表时间：
1994-12-01
期刊：
影响因子：
2.2
通讯作者：
UNWIN, N
中科院分区：
文献类型：
--
作者：
BERRIMAN, J;UNWIN, N
本文介绍了一种测定生物分子瞬时构象的简单方法。两种反应物（例如蛋白质复合物和配体）通过含有一种组分的喷雾液滴与含有另一种组分的薄的网格支撑的水膜的聚结而快速混合。然后通过快速冷冻捕获瞬态，并随后通过冷冻显微镜进行研究。与1-100 ms的反应时间相关联的构象的图像可以通过调节液滴撞击和冻结之间的延迟来实现。液滴（直径通常为1 μ m）通过雾化器喷射到网格上。它示出的液滴撞击在液膜上迅速蔓延在其表面上的表面张力的影响下，只有微弱的干扰下的公司，部分取代其内容远离点的影响。喷雾盐溶液的实验，使用来自红细胞的囊泡作为微渗透压计，表明通过膜和横向扩散发生快速混合。喷雾过程不会产生任何可检测的浓度变化，由于干燥的液滴或公司，该方法适用于高分辨率成像。
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 mu m 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 firm, 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 firm, and the method is applicable to high-resolution imaging.