Single-particle selection and alignment with heavy atom cluster-antibody conjugates
Single-particle selection and alignment with heavy atom cluster-antibody conjugates
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DOI：
10.1073/pnas.95.16.9262
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发表时间：
1998-08-04
影响因子：
11.1
通讯作者：
Kornberg, RD
中科院分区：
文献类型：
--
作者：
Jensen, GJ;Kornberg, RD
提出了一种利用低温电子显微镜对单个生物颗粒图像进行选择和对齐以获得高分辨率结构信息的方法。这些粒子将被标记有多个重原子簇，以允许精确确定粒子的位置和相对取向，即使在低电子剂量下靠近焦点成像时，也是记录高分辨率细节的最佳条件。重原子团簇也应该允许选择的图像从许多hinds的缺陷，包括试样运动和粒子不均匀性。可以通过构建基于单链Fv抗体片段的“衔接子”分子以一般方式引入重原子簇，所述衔接子分子由工程化用于最佳簇结合的恒定框架区和针对特定靶标选择的可变抗原结合区组成。该方法的成功取决于重原子簇在颗粒上的移动性，在图像中可以定位簇的精度，以及簇投影单独定向和选择用于平均的粒子的充分性。开发了必要的计算算法，并在模拟中实施，以解决该方法的可行性。
A method is proposed for selecting and aligning images of single biological particles to obtain high-resolution structural information by cryoelectron microscopy. The particles will be labeled with multiple heavy atom clusters to permit the precise determination of particle locations and relative orientations even when imaged close to focus with a low electron dose, conditions optimal for recording high-resolution detail. Heavy atom clusters should also allow selection of images free from many hinds Of defects, including specimen movement and particle inhomogeneity. Heavy atom clusters may be introduced in a general way by the construction of "adaptor" molecules based on single-chain Fv antibody fragments, consisting of a constant framework region engineered for optimal cluster binding and a variable antigen binding region selected for a specific target, The success of the method depends on the mobility of the heavy atom cluster on the particle, an the precision to which clusters can be located in an image, and on the sufficiency of cluster projections alone to orient and select particles for averaging. The necessary computational algorithms were developed and implemented in simulations that address the feasibility of the method.