Assembly of macromolecular complexes by satisfaction of spatial restraints from electron microscopy images

To obtain a structural model of a macromolecular assembly by single-particle EM, a large number of particle images need to be collected, aligned, clustered, averaged, and finally assembled via reconstruction into a 3D density map. This process is limited by the number and quality of the particle images, the accuracy of the initial model, and the compositional and conformational heterogeneity. Here, we describe a structure determination method that avoids the reconstruction procedure. The atomic structures of the individual complex components are assembled by optimizing a match against 2D EM class-average images, an excluded volume criterion, geometric complementarity, and optional restraints from proteomics and chemical cross-linking experiments. The optimization relies on a simulated annealing Monte Carlo search and a divide-and-conquer message-passing algorithm. Using simulated and experimentally determined EM class averages for 12 and 4 protein assemblies, respectively, we show that a few class averages can indeed result in accurate models for complexes of as many as five subunits. Thus, integrative structural biology can now benefit from the relative ease with which the EM class averages are determined

The molecular architecture of the yeast spindle pole body core determined by Bayesian integrative modeling

Microtubule-organizing centers (MTOCs) form, anchor, and stabilize the polarized network of microtubules in a cell. The central MTOC is the centrosome that duplicates during the cell cycle and assembles a bipolar spindle during mitosis to capture and segregate sister chromatids. Yet, despite their importance in cell biology, the physical structure of MTOCs is poorly understood. Here we determine the molecular architecture of the core of the yeast spindle pole body (SPB) by Bayesian integrative structure modeling based on in vivo fluorescence resonance energy transfer (FRET), small-angle x-ray scattering (SAXS), x-ray crystallography, electron microscopy, and two-hybrid analysis. The model is validated by several methods that include a genetic analysis of the conserved PACT domain that recruits Spc110, a protein related to pericentrin, to the SPB. The model suggests that calmodulin can act as a protein cross-linker and Spc29 is an extended, flexible protein. The model led to the identification of a single, essential heptad in the coiled-coil of Spc110 and a minimal PACT domain. It also led to a proposed pathway for the integration of Spc110 into the SPB