Inverted density in recons

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I am trying to run Frealign with CTF correction. I start with uncorrected (black) cryoEM particles and an initial model with positive density. My WGH value is set to 0.07, so I would expect to get CTF correction.

The resulting model has negative density and is of poor quality. My question is:

* Should the models have negative density?
* Are there any glowing errors in my parameter file/frealign parameters?

Thanks,
Neil Voss

Parameter file:
Iteration 0:
C PSI THETA PHI SHX SHY MAG FILM DF1 DF2 ANGAST PRESA
1 36.29 121.37 76.23 -0.25 -0.31 10000. 1 21272.5 19350.6 -12.55 0.00 0.00
2 308.27 60.01 255.49 -1.21 -1.17 10000. 1 21272.5 19350.6 -12.55 0.00 0.00
3 84.71 132.35 83.18 0.79 -0.93 10000. 1 21272.5 19350.6 -12.55 0.00 0.00
Iteration 1:
1 36.29 120.11 76.23 0.16 -0.64 10000. 1 21272.5 19350.6 -12.55 72.63 72.63
2 308.27 58.88 256.69 -2.29 -2.56 10000. 1 21272.5 19350.6 -12.55 73.43 73.43
3 84.15 133.22 81.68 -0.48 -0.52 10000. 1 21272.5 19350.6 -12.55 73.46 73.46

Job file:
frealign.exe << EOF
I,0,F,F,F,F,0,T,F,F,0,T,1
197,0,2.640,0.07,0.00,100,70,5,10,0
1 1 1 1 1
1, 500
I
1.00,2.64,15.00,85.00,2.00,120.00,0.00,0.00
5.30,50.00,10.60,100.00,0.00
stack
match
params.iter001.par
outparams.par
shift.par
0.0,0.0,0.0,0.0,0.0,0.0,0.0,0.0
volume
weights
odd
even
phasediffs
pointspread
EOF

The model should always have positive density. Your job file looks good. The only somewhat unusual parameter I see is the FSTAT flag, which is set to T (to enable calculation of more statistical arrays during reconstruction). However this should only lead to higher memory requirements and not to the problem you are describing.

I know that the IMAGIC format has recently changed somewhat. I have just checked with one of the example data sets included in the Frealign distribution if these changes also affect the header values read by Frealign. Converting the files from MRC to IMAGIC format did not break the Frealign routines, however. Our IMAGIC installation was updated on May 12, 2010 (EM2EM version 10-Mar-2010). I seem to remember that you use IMAGIC files that are generated by EMAN. Is it possible that EMAN's files differ slightly from the native IMAGIC files? To make sure your problem is not related to the image format, would it be possible to convert the files to CCP4/MRC and try again? If this fixes the problem we know it is related to the image format.

In reply to by niko

I figured it out. There was an error in my script and I accidentally provided a CTF corrected stack as input. Which would explain the inverted density on the recon. I should have probably slept on it first.

We do use EMAN to convert it, but I had to add an additional step to set the z-slice size to 1 and set the machine stamp to the intel endian value. I set the FSTAT to true, because I like the extra statistics and we have plenty of memory (48GB) at this time.

Anyway, thanks for the help.