Computational Technology for High-Throughput Cryo-EM

描述(由申请人提供)：我们的目标是开发所需的软件技术，以常规和快速的方式使用电子显微镜来确定分离的大分子组件的原子结构。为了达到这一目标所需的高分辨率的信噪比，需要大量孤立的(单个)大分子颗粒的图像。一个大约100,000个非对称单位的数据集可以产生8-12埃分辨率的三维重建。这一分辨率足以插入并在必要时调整组成大分子的原子分辨率模型，这些大分子的结构以前是通过其他方法(X射线或电子结晶学或核磁共振光谱学)确定的。甚至需要更大的数据集，由至少100万个不对称单位组成，才能达到优于5埃的分辨率，并最终获得约3.5埃的三维重建，这是从头确定原子结构所需的分辨率水平。对于这种规模的数据集，必须创建新的软件工具，以便快速完成计算工作。我们在该计划项目中确定的工作步骤是：(1)单粒子软件将被优化为在高度并行的计算机上运行，因为在单节点工作站上处理如此庞大的数据集变得不切实际。(2)对单个粒子的计算机辅助“装箱”(识别)将得到改进，以至于不再需要对候选粒子图库进行繁琐的人工编辑。(3)发展更好的算法来估计单个粒子的排列参数。此外，该计划项目将包括核心研究和基础设施的关键要素。将获得两个已知原子结构的大分子组件的实验图像。这些参考数据是开发和验证本方案将创建的新软件技术所需的。

DESCRIPTION (provided by applicant): Our goal is to develop the software technology required to use electron microscopy to determine the atomic structures of isolated macromolecular assemblies in a routine and rapid fashion. Images of a very large number of isolated (single) macromolecular particles are needed in order to achieve the signal-to-noise ratio at high resolution that is required for this goal. A data set of approximately 100,000 asymmetric units can produce a three-dimensional reconstruction at 8-12Angstroms resolution. This resolution is sufficient to insert, and adjust if necessary, atomic-resolution models of component macromolecules, whose structure would have been determined previously by other methods (X-ray or electron crystallography, or NMR spectroscopy). Even larger data sets, consisting of at least one million asymmetric units, are needed to achieve resolutions better than 5Angstroms, and ultimately to obtain 3-D reconstructions at about 3.5Angstroms, a level of resolution which is needed for de novo determination of an atomic structure. With data sets of this size, new software tools must be created in order to complete the computational work in a rapid fashion. The steps that we have identified for work within this Program Project are: (1) Single-particle software will be optimized to run on highly parallel computers, since processing of such large data sets on a single-node workstation becomes unrealistically long. (2) Computer-assisted "boxing" (identification) of single particles will be improved to such an extent that tedious, human editing of galleries of candidate particles is no longer necessary. (3) Better algorithms will be developed to estimate the alignment-parameters of single particles. In addition, the Program Project will include key elements of core research and infrastructure. Experimental images will be obtained for two macromolecular assemblies whose atomic structures are already known. These reference-data are needed for the development and validation of the new software technology that the Program will create.