Single particle analysis of filamentous and highly elongated macromolecular assemblies
Single particle analysis of filamentous and highly elongated macromolecular assemblies
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DOI：
10.1016/j.jsb.2004.05.004
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发表时间：
2004-11-01
影响因子：
3
通讯作者：
Morris, EP
中科院分区：
文献类型：
--
作者：
Paul, D;Patwardhan, A;Morris, EP
以肌肉细丝为例，评价了单粒子技术在细长或丝状大分子组装体电子显微镜图像三维分析中的应用。尽管细丝包含局部螺旋对称性，但由于沿其固有的可变扭曲沿着，螺旋相干性不延伸足够大的距离以允许对称性用于沿细丝沿着的原肌球蛋白/肌钙蛋白重复的完全重建。因此，肌肉细细丝代表了丝状物体的一般情况，因为在全面分析中不可能利用对称性。由于电子显微镜中成像过程的性质，在不倾斜网格的情况下，只有细丝围绕其长轴的投影是可用的。至关重要的是，围绕单个轴的投影图像不提供足够的信息来使用当前方法从头分配欧拉角。用模型细丝结构进行的试验表明，近似20度的面外倾斜对于从头算角度分配需要足够的精度，以计算3D结构，其分辨率近似为25（A）。如果没有平面外视图可用，则替代方法是使用先前的3D模型作为初始角度分配的参考。测试与细丝模型表明，合理准确的角度分配可以使用含有肌动蛋白的参考，但缺乏调节蛋白原肌球蛋白和肌钙蛋白。我们还发现，需要精确的滤波反投影方法的适应，以允许正确加权的投影图像中的颗粒具有非常大的轴比。这一调整使重建工作有了重大改进。(C)2004年爱思唯尔公司All rights reserved.
The application of single particle techniques to the three-dimensional analysis of electron microscope images of elongated or filamentous macromolecular assemblies is evaluated, taking as an example the muscle thin filament. Although the thin filament contains local helical symmetry, because of the inherent variable twist along it, the helical coherence does not extend for large enough distances to allow the symmetry to be used for full reconstruction of the tropomyosin/troponin repeat along the filament. The muscle thin filament therefore represents a general case of a filamentous object in that it is not possible to exploit symmetry in a full analysis. Due to the nature of the imaging process in the electron microscope, only projections of the thin filament around its long axis are available without tilting the grid. Crucially, projection images around a single axis do not provide enough information to assign Euler angles ab initio using current methods. Tests with a model thin filament structure indicated that an out-of-plane tilt of similar to20degrees was needed for ab initio angular assignment of sufficient accuracy to calculate a 3D structure to a resolution of similar to25 (A) over circle. If no out-of-plane views are available, an alternative approach is to use a prior 3D model as a reference for the initial angle assignment. Tests with the thin filament model indicated that reasonably accurate angular assignment can be made using a reference containing actin, but lacking the regulatory proteins tropomyosin and troponin. We also found that an adaptation of the exact filtered back projection method is required to allow the correct weighting of projection images in which the particle has a very large axial ratio. This adaptation resulted in significant improvements in the reconstruction. (C) 2004 Elsevier Inc. All rights reserved.