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Frealign: Multi-Reference Refinement & Classification

To start a multi-reference refinement, the parameter "nclasses" in the mparameters file has to be set to a number larger than 1. In addition, if desired, a number of initial 3D references can be supplied, together with their Frealign alignment parameter files. The reference and related parameter files must be numbered in sequence using the integers N that are part of Frealign’s file naming scheme (see Getting Started). Typically, however, no 3D reference files for different classes are available and, instead, a set of 3D references is generated using randomly selected subsets of the particle stack. To generate these random subsets and 3D reconstructions, simply set the value for "nclasses" to the desired number of classes and start a Frealign refinement run for the next cycle. Frealign will generate random seed reconstructions using the parameter file from the previous cycle and will then carry on with refinement and classification.

Frealign performs alignment and classification using data from different resolution intervals. The resolution used for particle alignment is specified by "res_high_refinement" in mparameters, while "res_high_class" species the resolution used for classification. Depending on the size of the expected differences between classes, it is often useful to limit the resolution used for classification to a somewhat lower value that the resolution used for alignment. For example, alignment can be carried out with data out to 6 Angstrom resolution while data used for classification is limited to 8 Angstrom resolution.

Convergence of the classification can be improved by alternating different types of refinement cycles. Frealign can refine class membership, particle Euler angles and x,y shifts simultaneously. Alternatively, class membership can be refined together with the Euler angles while leaving x,y shifts unchanged, or only class membership is refined while keeping all alignment parameters unchanged. The parameters "refineangleinc" and "refineshiftinc" in mparameters determine how frequent each refinement type occurs when multiple cycles are run. For example if "refineangleinc" is set to 3 and "refineshiftinc" set to 6, Frealign will perform the following sequence of cycles:

Cycle #   class membership (OCC)   Euler angles   x,y, shifts
1         yes                      no             no
2         yes                      no             no
3         yes                      yes            no
4         yes                      no             no
5         yes                      no             no
6         yes                      yes            yes
7         yes                      no             no
8         yes                      no             no
9         yes                      yes            no
10        yes                      no             no
11        yes                      no             no
12        yes                      yes            yes

After convergence, it is sometimes useful to do further classification of just one of the classes (sometimes referred as "sub-classification"). To extract one class (or merge multiple classes), the program "merge_classes.exe", which is supplied together with Frealign, can be used. It generates a new parameter file containing only the parameters of the particles from the selected classes, as well as a new particle image stack. These files can then be used in further refinement cycles, as well as sub-classification.