In a typical strategy for single particle analysis, focal pairs of micrographs are taken, with the first micrograph close to focus, and the second micrograph further from focus. The following treatment assumes that this strategy has been followed.
The recommended sequence of micrograph processing is: (i) determine the CTF parameters, (ii) pick particles in the further from focus micrograph, and (iii) align the closer to focus micrograph with the further from focus micrograph and calculate the particle positions in the former.
To do the alignment, the following are required to be set in the parameter (STAR) file:
- All the micrograph data blocks for both close to focus and further from focus micrographs must be specified in the parameter file(s).
- Each pair of micrographs must be specified within the same field-of-view, with the closer to focus micrograph first.
- The sampling or pixel size must be correctly specified for each micrograph.
- There should be particle records for the further from focus micrographs.
Here is an example setup for micrograph alignment:
bmg -v 7 -number 2 -Pixel 2.4 -output mg_67xx.star 670*.star Project hierarchy: Field: 6701 Micrograph: 6701 Particles: 0 (0) Filaments: 0 (0) Micrograph: 6702 Particles: 27 (27) Filaments: 0 (0) Field: 6705 Micrograph: 6705 Particles: 0 (0) Filaments: 0 (0) Micrograph: 6706 Particles: 48 (48) Filaments: 0 (0) Field: 6707 Micrograph: 6707 Particles: 0 (0) Filaments: 0 (0) Micrograph: 6708 Particles: 43 (43) Filaments: 0 (0) Field: 6709 Micrograph: 6709 Particles: 0 (0) Filaments: 0 (0) Micrograph: 6710 Particles: 59 (59) Filaments: 0 (0) Totals: 177 particles 177 selected (100.0%) 0 filaments 0 filament nodes
bmgalign generates a set of panels or tiles for each micrograph, and then cross-correlates the corresponding tiles to produce a set of shift vectors. This set of shift vectors are then fit to a five-parameter model (xy shift, xy scale, and rotation angle) to determine the final alignment. The locations of the picked particles in the reference micrograph (the further from focus micrograph) is then transformed to predict the locations in the closer to focus micrograph.
bmgalign -v 7 -align mic -ref 2 -resol 5000,30 -correlate 2048,2048,1 -out mg_67xx_aln.star mg_67xx.star
- -resolution: The resolution limits are in angstrom and thus linked to the pixel size. Generally the idea is to cut out very low resolution features that does not contribute to alignment information, and high resolution information that is dominated by noise.
- -correlate: The default tile size of 512,512,1 for cross-correlation is usually sufficient to get a successful alignment. However, when the alignment fails, increasing the tile size often works.
The key lines generated in the ouput shows the two micrographs compared, the shift vector (3 values), the scale vector (3 values), the rotation angle (in degrees), and the residual (in pixels). The aim is to get a low residual, typically on the order of 1-3 pixels. Usually when the alignment fails, the residual is large (10-100 pixels) and the key options mentioned above should be modified.
Example output lines:
Best fit: 6702.tif 6701.tif 163.53 183.10 0.00 1.00228 1.00227 1.00000 0.06464 0.98 Best fit: 6706.tif 6705.tif -18.59 -107.90 0.00 1.00291 1.00315 1.00000 0.08264 1.29 Best fit: 6708.tif 6707.tif 238.96 74.60 0.00 1.00286 1.00299 1.00000 0.13664 0.99 Best fit: 6710.tif 6709.tif 47.01 146.23 0.00 1.00148 1.00299 1.00000 -0.09736 2.48