Abstract:
Atomic force microscopy (AFM) and high-speed AFM allow direct observation of biomolecular structures and their functional dynamics. Based on scanning the molecular surface of a sample deposited on a supporting substrate by a probing tip, topographic images of its dynamic shape are obtained. Critical to successful AFM observations is a balance between immobilization of the sample while avoiding too strong perturbations of its functional conformational dynamics. Since the sample placement on the supporting substrate cannot be directly controlled in experiments, the relative orientation is a priori unknown, and, due to limitations in the spatial resolution of images, difficult to infer from a posteriori analysis, thus hampering the interpretation of measurements. We present a method to predict the macromolecular placement of samples based on electrostatic interactions with the AFM substrate and demonstrate applications to HS-AFM observations of the Cas9 endonuclease, an aptamer-protein complex, the Monalysin protein, and the ClpB molecular chaperone. The model also allows predictions of imaging stability taking into account buffer conditions. We implemented the developed method within the freely available BioAFMviewer software package. Predictions based on available structural data can therefore be made even prior to an actual experiment, and the method can be applied for post-experimental analysis of AFM imaging data.
摘要:
原子力显微镜(AFM)和高速AFM允许直接观察生物分子结构及其功能动力学。基于探针扫描沉积在支撑基板上的样品的分子表面,获得其动态形状的地形图像。成功的AFM观察的关键是样品的固定与避免其功能构象动力学的过强扰动之间的平衡。由于在实验中不能直接控制样品在支撑基板上的放置,相对方向是先验未知的,and,由于图像空间分辨率的限制,很难从后验分析中推断,从而阻碍了测量的解释。我们提出了一种基于与AFM底物的静电相互作用来预测样品的大分子放置的方法,并证明了Cas9内切核酸酶在HS-AFM观察中的应用。适体-蛋白质复合物,Monalysin蛋白,和ClpB分子伴侣.该模型还允许在考虑缓冲条件的情况下预测成像稳定性。我们在免费提供的BioAFMviewer软件包中实施了开发的方法。因此,即使在实际实验之前,也可以根据可用的结构数据进行预测。该方法可用于AFM成像数据的实验后分析。