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Gold Standard reconstruction

Starting with the version 4.05 of Auto3DEM, it is possible to follow what has been recently defined as the 'gold standard procedure' (called 'unbiased model reconstruction method' in the original paper on Auto3DEM by Yan et. al, 2007), in a way that is almost transparent to the user. The procedure consists of splitting the data into two half sets of particles, at the beginning of the iterative alignment: at each iteration each set generates an independent reconstruction, which is used as a reference, against the same set, for the next cycle of origin and orientation refinement. In this approach two independent maps are generated, and any possible bias introduced from the data by overfitting, will not contribute to overestimate the resolution of the final structure.

Generation of two independent stacks of data

The input particles need to be organized into two independent sets. This is achieved by generating two stack parameter files, each pointing to a different subset of particles. The actual files containing the images, i.e. the .pif files, don't need to be manipulated. The two stack parameter files can be generated by using the general purpose program autopp (option 6). If each stack of particles has a separate parameter file, it is recommended to consolidate them in one single file, still using autopp (option 5). The approach used to generate two independent sets of particles is to split them according to their sequential numbering in each stack: even-numbered particles are assigned to one set and odd-numbered to the other. The convention adopted for the stack parameter files is that their extension reflects the set their particles belong:

.dat_XXX   -> all particles from the stack
.date_XXX  -> only even-numbered particles from the stack
.dato_XXX  -> only odd-numbered particles from the stack

where XXX is the iteration number the parameters refer to. It is to remark that the strategy to split the data into two independent sets can actually be arbitrary, as long as the parameter files follow this convention. However, for different strategies the user needs to generate the .date and .dato files on his/her own, since autopp only splits by sequential numbering.

If you use the Random Model method implemented in Auto3DEM to generate an ab-initio reference map, the program takes care to generate the two independent stack parameter files, along with a _master file set for the gold standard procedure.

Reference maps

In the parameter file controlling the workflow of Auto3DEM (usually ending with _master or _continue) there is a new flag in the control parameters, that is 'auto gold_standard', which is set to 1 by default. Also, the reference maps for the two independent sets are specified in the fields 'auto start_map_even' and 'auto start_map_odd'. In order to succesfully run the procedure, an additional constraint is required:

As a result of the procedure, after each iteration three maps are actually generated: one from each set and one from all the particles. The last one is just for visualization purposes, and only its radial amplitude profile is utilized to re-calibrate some masking parameters.

Currently there are two caveats in the procedure:

Handedness
Currently, in case the two reconstructions converge to maps with opposite handedness, the program is not able to detect it, and therefore it will not be able to improve the reconstruction beyond the resolution where the handedness becomes evident. At the moment the simplest solution is to compare visually the two independent maps, and if the handedness is different then:

  1. modify the stack parameter files from the last iteration, for only one set, using the command handflip;
  2. change the handedness of the corresponding reconstruction. This can be done either recomputing the reconstruction from the modified parameter files (in the last _continue file, just set 'auto generate_map' to 'only' and launch it), or by modifying the map using an external program (e.g. using the program bimg from bsoft, with the option '-reslice xy-z').