Stereology and EM Laboratory, Institute of Clinical Medicine, Aarhus University, DK-8000 Arhus C, Denmark.
Structure. 2010 Jun 9;18(6):667-76. doi: 10.1016/j.str.2010.05.001.
Single-particle electron cryomicroscopy is a powerful method for three-dimensional (3D) structure determination of macromolecular assemblies. Here we address the challenge of determining a 3D structure in the absence of reference models. The 3D structures are determined by alignment and weighted averaging of densities obtained by native cryo random conical tilt (RCT) reconstructions including consideration of missing data. Our weighted averaging scheme (wRCT) offers advantages for potentially heterogeneous 3D densities of low signal-to-noise ratios. Sets of aligned RCT structures can also be analyzed by multivariate statistical analysis (MSA) to provide insights into snapshots of the assemblies. The approach is used to compute 3D structures of the Escherichia coli 70S ribosome and the human U4/U6.U5 tri-snRNP under vitrified unstained cryo conditions, and to visualize by 3D MSA the L7/L12 stalk of the 70S ribosome and states of tri-snRNP. The approach thus combines de novo 3D structure determination with an analysis of compositional and conformational heterogeneity.
单颗粒电子冷冻显微镜是一种用于确定大分子组装体三维（3D）结构的强大方法。在这里，我们解决了在没有参考模型的情况下确定 3D 结构的挑战。通过对齐和加权平均密度来确定 3D 结构，这些密度是通过天然冷冻随机锥形倾斜（RCT）重建获得的，包括对缺失数据的考虑。我们的加权平均方案（wRCT）为低信噪比的潜在异质 3D 密度提供了优势。对齐的 RCT 结构集也可以通过多元统计分析（MSA）进行分析，以提供对组装体快照的洞察。该方法用于计算在玻璃化非染色冷冻条件下的大肠杆菌 70S 核糖体和人 U4/U6.U5 三 snRNP 的 3D 结构，并通过 3D MSA 可视化 70S 核糖体的 L7/L12 茎和三 snRNP 的状态。因此，该方法将从头确定 3D 结构与组成和构象异质性分析相结合。
