====== 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. <wrap hi>The actual files containing the images, i.e. the .pif files, 
don't need to be manipulated.</wrap> 
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. 

===== Reference maps =====
Each half set of particles needs to be aligned against a different reference.
In the parameter file controlling the workflow of Auto3DEM (usually
ending with _master or _continue) the reference maps for the two independent sets are specified in
the fields
  auto  start_map_even
  auto  start_map_odd
When starting the iterative procedure, the same map can be used as initial reference for both the two sets, as long as only the low resolution features are used determining the alignment parameters. This is achieved by setting either the parameter ''ppft resolution_high'' or ''po2r res_max'', depending if ''auto mode'' is set to ''search'' or ''refine'', respectively, to 30 Å or higher.

===== Aligning the particles =====
In the parameter file controlling the workflow of Auto3DEM a flag, ''auto gold_standard'', controls the use of the gold standard procedure. If set to 1, the fields ''data'' can only contain a list of stack parameter files properly split, that is only //.dato// and //.date// files are accepted. If the flag is set to 0, no gold standard procedure is followed, all the stack parameter files are treated as one single data set, and their particles are aligned against the map listed in the parameter ''auto start_map''.
In the gold standard 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.

<WRAP center round info 85%>
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 properly set for the gold standard procedure.</WRAP>

===== Limitations =====
Currently there are two caveats in the procedure:
<WRAP center round important 85%>
**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:
  - modify the stack parameter files from the last iteration, for only one set, using the command //handflip//;
  - change the handedness of the corresponding reconstruction. This can be done either by recomputing the reconstruction from the modified parameter files (open the last  _continue file, set ''auto generate_map only'', save it and run it), or by modifying the map using Robem (open the map, go to the menu ''Manipulate/Invert 3D Map'', select ''Reflect in xy plane (z -> -z)'', and save the modified map).
</WRAP>