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首页> 外文期刊>Journal of Molecular Biology >Three-dimensional crystals of Ca2+-ATPase from sarcoplasmic reticulum: Merging electron diffraction tilt series and imaging the (h,k,0) projection
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Three-dimensional crystals of Ca2+-ATPase from sarcoplasmic reticulum: Merging electron diffraction tilt series and imaging the (h,k,0) projection

机译：肌浆网中Ca2 + -ATPase的三维晶体：合并电子衍射倾斜序列并成像（h，k，0）投影

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Electron crystallography offers an increasingly viable alternative to X-ray crystallography for structure determination, especially for membrane proteins. The methodology has been developed and successfully applied to 2D crystals; however, well-ordered thin, 3D crystals are often produced during crystallization trials and generally discarded due to complexities in structure analysis. To cope with these complexities, we have developed a general method for determining unit cell geometry and for merging electron diffraction data from tilt series. We have applied this method to thin, monoclinic crystals of Ca2+-ATPase from sarcoplasmic reticulum, thus characterizing the unit cell and generating a 3D set of electron diffraction amplitudes to 8 Angstrom resolution with tilt angles up to 30 degrees. The indexing of data from the tilt series has been verified by an analysis of Laue zones near the (h, k, 0) projection and the unit cell geometry is consistent with low-angle X-ray scattering from these crystals. Based on this unit cell geometry, we have systematically tilted crystals to record images of the (h, k, 0) projection After averaging the corresponding phases to 8 Angstrom resolution, an (h, k, 0) projection may has been calculated by combining image phases with electron diffraction amplitudes. This map contains discrete densities that most likely correspond to Ca2+-ATPase dimers, unlike previous maps of untilted crystals in which molecules from successive layers are not aligned. Comparison with a projection structure from tubular crystals reveals differences that are likely due to the conformational change accompanying calcium binding to Ca2+-ATPase. (C) 1998 Academic Press. [References: 40]
机译：电子晶体学提供了越来越多的替代X射线晶体学用于结构确定的方法，尤其是对于膜蛋白。该方法已经开发并成功应用于二维晶体。但是，有序的薄3D晶体通常在结晶试验期间产生，并且由于结构分析的复杂性而通常被丢弃。为了应对这些复杂性，我们开发了一种确定晶胞几何形状和合并倾斜序列电子衍射数据的通用方法。我们已经将该方法应用于来自肌浆网的Ca2 + -ATPase的单斜晶体，从而表征了晶胞并生成了3D电子衍射振幅，分辨率高达8 Ang，倾斜角高达30度。倾斜序列数据的索引已通过分析（h，k，0）投影附近的劳厄区进行了验证，并且晶胞几何形状与来自这些晶体的低角度X射线散射一致。基于此晶胞几何形状，我们已系统地倾斜了晶体以记录（h，k，0）投影的图像。在将相应相位平均为8埃分辨率后，可以通过组合计算出（h，k，0）投影电子衍射幅度的图像相位。该图包含离散密度，该密度最有可能对应于Ca2 + -ATPase二聚体，这与之前的不连续晶体的图（其中连续层的分子未对齐）不同。与管状晶体投影结构的比较表明，差异可能是由于钙与Ca2 + -ATPase结合引起的构象变化。 （C）1998年学术出版社。 [参考：40]

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