Multivariate statistical analysis (MSA) improves azimuthal alignment along the axis of helical objects and results in higher resolution for reconstruction using a single particle approach

To obtain high structural resolution for reconstruction of helical objects by single particle methods a highly accurate alignment along the helical axis is necessary. In case of the actin-myosin-complex a typical misalignment on the order of the helical symmetry (±12.7°) limits resolution to 1-2.5nm depending on radius. Applying MSA to conventionally aligned 2D-projections different projection angles are distinguished. If a low resolution reconstructed density is available 2D-projections of that density along different azimuthal angles can be used to define a multi-dimensional factor space. MSA then defines a 1D-sub-space including its scale for the projection angle. Matching model data to experimental data for signal strength, resolution, and CTF, these experimental data can then directly be mapped into the given factor space. The experimental data shows a close distribution around the sub-space. An azimuthal angle can be assigned by determining the smallest distance to the sub-space. In a first application using the actin-myosin- complex it is possible to reduce the error in azimuthal alignment to less than ±3°. The effect on the final reconstruction and further refinement strategies are discussed