Dear Sjors

I have been getting that same error consistently with some very anisotropic maps. Specifying a fully "white" input mask or masks that work fine on other (less anisotropic) reconstructions of the same particle does not resolve the issue. Neither does specifying a lower threshold for mask calculation. Calculation of the FSC in SPIDER on converted maps yields entirely normal FSC curves indicating sub-nanometer resolution on average (the well and poorly resolved directions of the map probably have very different information content).

Therefore, anisotropy of the data - to which Relion high resolution refinement seems to be particularly sensitive, much more so than the 3D classifications - may also play a role here.

Best regards,

Basil Greber

Ban Lab
ETH Zurich


Am 03.05.2014 um 09:11 schrieb Sjors Scheres:

> Hi Zhao,
> Please use the cppem list (CC) for these questions. I think the answer is
> described there already. You might be using the wrong threshold for your
> auto mask calculation. Check whether your auto mask is all-zero: if so
> that would explain the result. Then just just re-run with a lower
> ini-threshold.
> We always check our map in Chimera and make sure the ini_threshold for the
> auto mask is around the value we use to display the map where we don't see
> any noisy blobs in the solvent region.
> HTH, S
> 
> 
>> Hi, Dr, Scheres,
>> I got this error message when I run postprocess for my refinement ,
>> do you have any idea  why it report error message.  FSC curve never
>> drops below randomize_fsc_at
>> 
>> elion_postprocess --i run03 --o postprocess_run1 --angpix 1.81 --mtf
>> mtf-3200-ddd-new.star  --auto_bfac --skip_fsc_weighting --auto_mask
>> == Reading input half-reconstructions:
>>  + half1-map:                run03_half1_class001_unfil.mrc
>>  + half2-map:                run03_half2_class001_unfil.mrc
>> == Perform auto-masking ...
>>  + density threshold:        0.02
>>  + extend ini mask:          3 pixels
>>  + width soft edge:          6 pixels
>> == Extending initial binary mask ...
>> 000/??? sec ~~(,_,">
>>       [o  11/  11 sec
>> ...........................................................~~(,_,"
>> 11/  11 sec
>> ...........................................................~~(,_,"
>> 12/  12 sec
>> ............................................................~~(,_,">
>> == Making a soft edge on the extended mask ...
>> 000/??? sec ~~(,_,">
>>       [o  41/  41 sec
>> ...........................................................~~(,_,"
>> 42/  42 sec
>> ...........................................................~~(,_,"
>> 42/  42 sec
>> ............................................................~~(,_,">
>> == Masking input maps ...
>>  + randomize phases beyond:  inf Angstroms
>> +++ RELION: command line arguments (with defaults for optional ones
>> between parantheses) +++
>> ====== General options =====
>>                                --i : Input rootname, e.g. run1
>>                           --angpix : Pixel size in Angstroms
>>                  --o (postprocess) : Output rootname
>> ====== Masking options =====
>>                --auto_mask (false) : Perform automated masking, based
>> on a density threshold
>>         --inimask_threshold (0.02) : Density at which to threshold
>> the map for the initial seed mask
>>              --extend_inimask (3.) : Number of pixels to extend the
>> initial seed mask
>>             --width_mask_edge (6.) : Width for the raised cosine soft
>> mask edge (in pixels)
>>                          --mask () : Filename of a user-provided mask
>> (1=protein, 0=solvent, all values in range [0,1])
>> ====== Sharpening options =====
>>                           --mtf () : User-provided STAR-file with the
>> MTF-curve of the detector
>>                --auto_bfac (false) : Perform automated B-factor
>> determination (Rosenthal and Henderson, 2003)
>>               --autob_lowres (10.) : Lowest resolution (in A) to
>> include in fitting of the B-factor
>>               --autob_highres (0.) : Highest resolution (in A) to
>> include in fitting of the B-factor
>>                  --adhoc_bfac (0.) : User-provided B-factor (in A^2)
>> for map sharpening, e.g. -400
>> ====== Filtering options =====
>>       --skip_fsc_weighting (false) : Do not use FSC-weighting
>> (Rosenthal and Henderson, 2003) in the sharpening process
>>                    --low_pass (0.) : Resolution (in Angstroms) at
>> which to low-pass filter the final map (by default at final
>> resolution)
>> ====== Expert options =====
>>           --randomize_at_fsc (0.8) : Randomize phases from the
>> resolution where FSC drops below this value
>>            --filter_edge_width (2) : Width of the raised cosine on
>> the low-pass filter edge (in resolution shells)
>>                         --verb (1) : Verbosity
>> Postprocessing::run ERROR: FSC curve never drops below randomize_fsc_at
>> File: src/postprocessing.cpp line: 744
>> 
>> --
>> Baylor College of Medicine
>> One Baylor Plaza, N421, houston ,TX,77030
>> 
> 
> 
> -- 
> Sjors Scheres
> MRC Laboratory of Molecular Biology
> Francis Crick Avenue, Cambridge Biomedical Campus
> Cambridge CB2 0QH, U.K.
> tel: +44 (0)1223 267061
> http://www2.mrc-lmb.cam.ac.uk/groups/scheres