Hi Anne,
This may also happen when you downscale the particles upon extraction. 
In that case, use additional option --dont_check_norm.
HTH,
Sjors

On 02/23/2017 05:00 PM, Ries, Anne wrote:
> Hi,
>
> we are having a problem with normalization during particle extraction. We are working with a dataset of an icosahedral virus.
> Extraction yields several particle images that are very white and not properly normalized. This causes an error in subsequent 2D classification:
>
>   fn_img= [log in to unmask] bg_avg= -0.63104 bg_stddev= 0.705528 bg_radius= 138.365
> ERROR: It appears that these images have not been normalised to an average background value of 0 and a stddev value of 1.
>
>
> We had run the extraction with the following parameters:
>
> `which relion_preprocess_mpi` --i Select/job065/micrographs.star --coord_dir Import/job086/ --coord_suffix .box --part_star Extract/job113/particles.star --part_dir Extract/job113/ --extract --extract_size 888 --scale 296 --norm --bg_radius 138 --white_dust -1 --black_dust -1 --invert_contrast
>
> We tried to solve the problem by using white dust removal values > 0. By eye, the images look better, especially with high values (10). I put three .png’s in the attachments, with some particles without and with values of 3.5 and 10 for white dust removal.
> Unfortunately, we find hot pixels on the affected particle images and 2D classification gives us the same error, no matter if we remove white dust with values of 1.5, 3.5, 10 or 100. We also tried to reduce the diameter background circle to half the size (did not help).
> For the time being, we are doing the 2D classification by switching off the normalization flag.
>
> How can we solve this? Is turning the normalization off in that case an option?
>
> Best regards,
> Anne
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> [cid:6B93971E-74A5-44BD-9963-1FC93C0B9884@biocenter.helsinki.fi][cid:5A2BD057-AC4B-4D9E-B98C-7CE6BB67A44E@biocenter.helsinki.fi][cid:AD9F81D7-58B9-4519-87B7-94BD30681372@biocenter.helsinki.fi]

-- 
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