“The Orthogonal Tilt Reconstruction method: an approach to generating single-class volumes with no missing cone for ab initio reconstruction of asymmetric particles”
Andres Laschziner – University of California Berkeley
Generating reliable initial models is a critical step in the reconstruction of asymmetric single particles by three-dimensional electron microscopy and is particularly difficult when heterogeneity is present in the sample. The Random Conical Tilt (RCT) method, arguably the most robust presently to accomplish this task, requires significant user intervention to solve the missing cone problem. We have developed a novel approach, termed the Orthogonal Tilt Reconstruction method (OTR), that eliminates the missing cone altogether, making it possible for single-class volumes to be used directly as initial references in refinement without further processing. The method involves collecting data at +45o and -45o tilts and only requires that particles adopt a relatively large number of orientations on the grid. We have tested this method with synthetic data and compared its performance to that of the RCT method. We also propose a way of increasing the level of homogeneity in individual 2-D classes (and volumes) in a heterogeneous data set and identifying the most homogeneous volumes.