Frealign

Has anyone else encountered this error:

Entering HEXTEND with centre at 148.000 148.000 for map with NSAM = 294
Extending region between ZMIN,ZMAX = -95 388
FORTRAN STOP
TRILINMAP error X,Y or Z too small or too big
X,Y,Z = 145.39270 215.55147 294.56870 MIN,MAX = 1.00000 294.00009

Some details:
-I'm using the helical symmetry option (H)
-It only happens when FBEAUT is on (T)
-Changing the pixel size will change Z to some other fraction of 294 (such as 294.282, 294.672), but it was always be larger than 294.00009

Hi

Can you tell me whether Frealign would be suitable for refining a 3D model generated from negatively stained, tilted particles? The issue I see is in limiting the angles to be searched to those relevant to the tilt process.

Thanks!

The PFT3DR convention for origins is the pixel distance from the first pixel of the image. To get shiftx and shifty, do I simply subtract the "center" of the box from the origin? In other words, if my image box is 100 x 100 pixels ("center" at 51,51?, I assume from NSAMP/2 + 1) and the particle origin at 51.3,49.4, would the shifts be

51.3 - 51 = 0.3 and 49.4 - 50 = -1.6?

I thought I would try to write a little program to convert from PFT3DR (my adaptation of Tim Baker's PFT and EM3DR programs that uses the Bsoft format) to your convention and parameter files, so we can jump right into the refinement mode of FREALIGN. The Bsoft orientation convention is essentially the same as SPIDER, but I have a question about your icosahedral convention.

I'm getting the following error when running Frealign with IFLAG = -3:

MAGNIFICATION, STEPSIZE, TARGET & THRESH PHASE
RESIDUAL LIMIT, CS, KV, BEAM TILT X,Y ?
CARD 6: Error. Trying old CARD 6 input...
PGFIO-F-225/list-directed read/internal file/lexical error-- unknown token type.
In source file card6.f, at line number 32

My input line for Card 6 is the following:

1., 6.35, 25., 90., 2., 300., 0., 0. !RELMAG,DSTEP,TARGET,THRESH,CS,AKV,TX,TY

I'm getting the following error message:

PGFIO-F-225/list-directed read/unit=5/lexical error-- unknown token type.
File name = stdin formatted, sequential access record = 10
In source file card10.f, at line number 273

Looks like the problem might be in reading the image files. 2D Image files are MRC format, created with Bsoft package to convert from PIF to MRC format (specific Bsoft command is "bhead -verb 7 -info -slices A04815_particles.pif A04815_particles.mrc", "slices" option makes a "3D" file from a stack of 2D images). 3D model is also an MRC-format file.

Thanks.

Hello,

I made a script to convert my spider defocus and astigmatism values into the frealign convention.

The formula I use is:

df1 = spider.defocus - spider.magastig/2

df2 = spider.defocus + spider.magastig/2

angast = spider.angast + 45

Does that sound correct?

By advance, many thanks.

Hello,

In mode 4 we can set the angular step size for the systematic search with DANG, and the number of starting angles to be tested with ITMAX.
In your hands, what is the most successful combination? ITMAX 100, DANG 100, as in your 2007 jsb paper? Or some other variations of the two? (I was thinking about trying DANG 15, ITMAX 25)
Also, should we use different strategies for symmetric and asymmetric particles?

Many thanks,

Hello,

Doing a refinement on a small dataset, I see that the most time consuming step is the reconstruction, as it is done on one single node. For 18 000 particles, 350x350, it takes about 5 hours. For 100 000 particles, I guess that it's going to be ~28 hours. It will cut that time a little bit once I get the multicore to work, but is there parameters that could be set to speed up the process?

Hello,

I'm trying to figure out the most efficient way to split my jobs on the cluster. I have 8 core nodes with 32 Gb of ram.
How can I calculate how much memory I need for a given job?
Right now, when submitting 200 images 350x350 to one core, I use about 10 gb of RAM. But I don't know what takes the space.

By advance, thank you.

Amedee