FREALIGN Using Samfrealign.py

(Visit FREALIGN website: http://emlab.rose2.brandeis.edu/frealign)

Procedure:

(1) Make a new working folder, for example, called "samfrealign", and go into it.
(2) Link or copy the particle stack in the current folder. Note that this stack must contain black particles on white background,
be normalized over variance and in MRC format. If not sure, please read about particle stack preparation.
(3) Make a folder called "cycle_000", and go into it. Link or copy the 3D initial model in MRC format, and name it as "3Dvol_000.mrc".
Link or copy the FREALIGN parameter file, and name it as "output_000.par". If not sure, please read about defocus determination.
(4) Go back into the main working folder, run "samfrealign.py setup". It prints out a list of default settings, and prompts for changes.
(To read the meaning of each parameter, type "samfrealign.py help", or read the original paper.)
Although the long list seems scary, usually you only need to make changes in the first 4 sections.

>>>> PLEASE CHECK GENERAL SETTINGS
===> How many jobs? (negative:QSUB) > 2
===> Particle MRC image stack (with extension) > image_stack.mrc
===> Total particle number > 10000

>>>> PLEASE CHECK EM SETTINGS
===> [AKV] Voltage of the microscope (kV) > 200
===> [CS] Cs of the microscope (mm) > 2.0
===> [WGH] Amplitude contrast for CTF correction > 0.07
===> [DSTEP] Densitometer step size (micron) > 30
===> [PSIZE] Pixel size (A) required for output map > 4.314

>>>> PLEASE CHECK ALIGNMENT SETTINGS
===> [IFLAG] 1/2/3:no/random/simple_Search & Refine; 4:Randomise > 4
===> [RO] Outer radius for refinement,reconstruction (A) > 100.0
===> [RI] Inner radius for refinement,reconstruction (A) > 0.0
===> [DANG] Angular step size for the angular search when IFLAG=3,4 > 100.0
===> [ITMAX] Number of cycles of randomised search/refine when IFLAG=2,4 > 40
===> [RMAX1] Low resolution limit for refinement (A) > 100.0
===> [RMAX2] High resolution limit for refinement (A) > 30.0
===> [ASYM] Symmetry for refinement (CnDn,T,O,I,I1,I2 or N (can be zero)) > 0

>>>> PLEASE CHECK REC SETTINGS
===> [ASYMR] Symmetry for reconstruction > C3
===> [THRESH] Particle phase residual cut-off for reconstruction > 55.0
===> [REC] Resolution to which the reconstruction is calculated > 15.0

>>>> PLEASE CHECK EXTRA_1
===> [CFORM] Input/Output image file format (!must use M) > M
===> [FMAG] Magnification refinement (T/F) > F
===> [FDEF] Defocus refinement (T/F) > F
===> [FASTIG] Defocus astigmatism refinement (T/F) > F
===> [FPART] Defocus refinement for individual particles (T/F) > F
===> [IEWALD] Ewald correction (0/1/2/-1/-2) > 0
===> [FBEAUT] Extra real space sym averaging/masking on final map (T/F) > F
===> [FCREF] FOM filter to final map using SQRT(2.0*FSC/(1.0+FSC)) (T/F) > F
===> [FMATCH] Write out matching projections after the refinement (T/F) > F
===> [IFSC] 0:two reconstruction odd/even;1/2/3:odd/even/all one rec > 0
===> [FSTAT] Calculate additional statistics in resolution table (T/F) > F
===> [IBLOW] 1/2/4. Padding factor for reference structure > 1

>>>> PLEASE CHECK EXTRA_2
===> [XSTD] 0/positive/negative. Positive: solvent flattening > 0
===> [PBC] Phase residual / pseudo-B-factor conversion Constant > 50
===> [BOFF] Average particle phase residual for weights to 3D map > 45.0
===> [IPMAX] Number of potential matches in a search for local refine > 1

>>>> PLEASE CHECK EXTRA_3
===> [MASK_psi] 0/1: exclude/include psi for refinement > 1
===> [MASK_theta] 0/1: exclude/include theta for refinement > 1
===> [MASK_phi] 0/1: exclude/include phi for refinement > 1
===> [MASK_dX] 0/1: exclude/include dX for refinement > 1
===> [MASK_dY] 0/1: exclude/include dY for refinement > 1

>>>> PLEASE CHECK EXTRA_4
===> [RELMAG] Relative magnification to apply to data set > 1.0
===> [TARGET] Target phase residual during search/refine > 5.0
===> [TX] Beam tilt [mrad] in X direction > 0.0
===> [TY] Beam tilt [mrad] in Y direction > 0.0

>>>> PLEASE CHECK EXTRA_5
===> [DFSTD] Defocus uncertainty > 0.0
===> [RBFACT] B-factor to particle images before search/refine > 0.0

(5) To start 5 cycles of FREALIGN refinement, run "samfrealign.py run 1 5 >& log &".
(6) The results can be analyzed by a python program called "pypar". To change the FREALIGN settings, run "samfrealign.py setup" again.
To continue, for example, another 5 cycles of refinement, run "samfrealign.py run 6 10 >& log &".