Frealign

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


Hi,

I try to launch frealign 8.08 on my cluster with this attribute :
1 stack dal_stack.mrc size : 530 Mo and more than 13000 particles
I set the first, last and increment : 1,1000,100 to use 10 core with small stack
I use Torque system (PBS and MAUI) and I had modified your examples script.

The script PBS run but the third job always stop
I have a error message in the Third log file : dal_mrefine_n.log_201_300

Opening MRC/CCP4 file for READ...
112 File : dal_2.mrc_201_300
113 NX, NY, NZ: 100 100 100
114 MODE : real

I am writing to get some advice on what frealign parameters I should vary for trying to go to high (better than 7A) resolution. I have a data set that will go to 6A with EMAN right now, and it only goes to 8A with frealign. Some of that resolution in EMAN is noise bias, I'm sure, but the details with the EMAN reconstruction are better than what I'm currently getting with frealign, even after tweaking the B factor. Should I try different RREC, RMAX1, and RMAX2? If so, do you have recommendations for those values? Any other suggestions?

Thanks,
Scott

Hello,
I am wondering if I have to compile frealign in order to process images bigger than 256. The current documentation suggests that I do, but I would prefer to avoid compiling if I don't have to.

Thanks,
Scott

Hello Niko,

What is the difference is between gather_spi.com and gather_spi_boot.com?
And I have used spider to calculate my defocus instead of ctftilt. Is there still an easy way to retrieve my spider defocus values ? Or do you strongly recommend to use ctftilt instead of spider for defocus estimation?

By advance, many thanks,

I'm running into a strange problem with Frealign. It seems that the SHX and SHY parameters have no effect on moving the reference projection from the center of the box. Is that correct? If so, what do I do if my particles are not perfectly centered? I checked the matching projections generated by setting FMATCH = T for two different parameter files (see below). In both runs I do not refine any of the parameters, hence I'm only calculating reference projections. In both cases, the matching projections are centered, even though the shifts are changing.

Hi,

Is there a way to generate a reconstruction from a particular set of particles in the parameter file (.par). For example, I'd like to generate a reconstruction using particles that come from film number 23. I realize that I can create distinct parameter and stack files for each film number to do this, but was wondering if there is another way. Thanks.

We are getting our initial particle orientations and shifts from Spider projection matching (CTF parameters have been determined using CTFFIND3). We then want to do the final refinement and CTF correction in FREALIGN. However we usually end up with the same resolution after the refinement in FREALIGN as before. We seem to be stuck in the 30-20A resolution range. Is there a particular way to go about the FREALIGN refinement (incrementally changing the resolution of the refinement, etc)? Should we get our initial alignment parameters a different way? Or are we just limited by our data?

I've done some bench-marking of the various pre-compiled frealign versions (v8) for the search example using the 200 particle PDH data set. frealign_v8.exe and frealign_v8_fftw.exe seem to give essentially the same results, with some minor numerical differences (seen in the final values for things like FSPR, FSC, CC, SIG C and the ERFC. On my Ubuntu x86_64 machine, the fftw version runs about 8% faster. I will do some real stats (multiple runs) on this to see whether it is a real difference.