In EMAN2, e2refine2d.py will compute and save Eigenimages from the aligned particle stack in each iteration as r2d_01/basis_XX.hdf http://eman2.org/Programs/e2refine2d or e2refine2d.py --help alternatively, if you want to just use a single alignment reference then compute Eigenimages: e2a2d_align.py ptclstack.hdf align_ref.hdf,3 --threads=8 --saveali where ptclstack.hdf contains the phase-flipped particle stack (note that you may still get Eigenimages related to defocus differences), align_ref.hdf is a particle stack (or single image) containing the alignment reference and "3" is the image number to use as a reference (0 is the first image), --threads is the number of cores on the machine e2msa.py m2d_01/aliptcls_01.hdf basis.hdf --nbasis 12 will compute the first 12 Eigenimages and save them in basis.hdf As with other EMAN2 programs you can use any file format you prefer for input... -------------------------------------------------------------------------------------- Steven Ludtke, Ph.D. <[log in to unmask]<mailto:[log in to unmask]>> Baylor College of Medicine Charles C. Bell Jr., Professor of Structural Biology Dept. of Biochemistry and Molecular Biology (www.bcm.edu/biochem<http://www.bcm.edu/biochem>) Academic Director, CryoEM Core (cryoem.bcm.edu<http://cryoem.bcm.edu>) Co-Director CIBR Center (www.bcm.edu/research/cibr<http://www.bcm.edu/research/cibr>) Co-Director National Center For Macromolecular Imaging (ncmi.bcm.edu<http://ncmi.bcm.edu>) On May 13, 2018, at 8:32 PM, Yangqi Gu <[log in to unmask]<mailto:[log in to unmask]>> wrote: Hi all, I am trying to compute the eigenimages of my particles and first I think I need to align my particles. Does anyone know which software will be the best to do the alignment? My particles are segmented helices and I do have some 2D class averages. Best, Yangqi