A: Upload a nice micrograph and inform the pixel size (in Angstroms) and the aproximate particle size (in pixels).

• Q: What happens to the images I submit and with the resulting files ?
  A: They will be accessible through a secret URL that gives you a unique address to share with other people so they can access the page, but keeps your results secret from anyone who does not have the address. The secret URL (optionally sent to you by e-mail) and all the related files might be deleted after 7 days to save disk space.
 
• Q: If I use your web server, what is your policy regarding confidentiality of images that I submit ?
  A: Because there is no data encryption, we cannot necessarily guarantee confidentiality of your images submitted via the web server. Use it at your own risk.
 
• Q: Is this a common-lines⁶ based method ?
  A: No. The algorithms implemented here are based on Symmetry Adapted Functions¹ and the only similarity to the common-lines method is the use of the projection theorem (also known as Fourier slice theorem).
 
• Q: How does it work ?
  A: It is based on the work of Jorge Navaza¹. In short, it tests for all the combinations of the unknown parameters (views and centers) by making thousands of possible 3D reconstructions based on a single particle. The best match between the image and the correponding re-projection of the 3D reconstructions indicates the best set of parameters. Afterwards, 3D reconstructions based on two or more particles are made and the parameters are refined.
  In a more procedural way:
  • Pre-processing:
    1. : Calculation of Fourier-Bessel transform of the particles' images.
    2. : Initial center assignment by the SCA⁷ method.
  • Views/centers assignment:
    1. : Exhaustive assessment of particles' orientations and refinement of centers (one particle at a time).
    2. : Search for the best agreement between all the independently obtained 3D reconstructions to elect the best particle as a reference.
    3. : Exhaustive re-assessment of particles' orientations (two particles at a time, particle+reference).
    4. : Estimation of the maximum resolution of the 3D reconstruction according to the accuracy of the numerical invertion. This takes into account the number of particles and the respective coverage of the asymmetric unit by the views distribution.
  • Views/centers refinement:
    1. : Refinement of the orientations and centers (all particles at once).
 
• Q: What do to if it doesn't work ?
  A: There is a "Troubleshooting" section in the particle picking page where you can try:
  1. : To switch-off the SCA centering (step II above) if you see that it doesn't give improved centers or if too many particles are discarded.
  2. : To choose the reference manually based on what you obtained during a previous orientation assessment (step III, one particle at a time).
  3. : Alternatively, you can try to use a more defocalised (higher contrast) micrograph.

1. NAVAZA, J. ; "On the three-dimensional reconstruction of icosahedral particles.", Journal of Structural Biology, v. 144, p.13-23, 2003.
2. ESTROZI, L. F. ; NAVAZA, J. "Fast projection matching for Cryo-electron Microscopy Image Reconstruction", Journal of Structural Biology, 162, p.324-334, 2008.

*** If this server has been useful to you, please cite this reference:

3. ESTROZI, L. F. ; NAVAZA, J. "Ab initio high-resolution single-particle 3D reconstructions: the Symmetry-Adapted-Functions way", Journal of Structural Biology, 172, p.253-260, 2010. [PDF]
 
4. GRIGORIEFF, N. "FREALIGN: high-resolution refinement of single particle structures", Journal of Structural Biology, 157, p.117–125, 2007.
5. BAKER, T.S. ; CHENG, R.H. "A model-based approach for determining orientations of biological macromolecules imaged by cryoelectron microscopy", Journal of Structural Biololy, 116(1), p.120-130, 1996.
6. CROWTHER, R.A., "Procedures for Three-Dimensional Reconstruction of Spherical Viruses by Fourier Synthesis from Electron Micrographs", Phylosophical Transactions of the Royal Society B, v. 261, no.837, p. 221-230, 1971. (doi: 10.1098/rstb.1971.0054). [PDF]
7. ESTROZI, L. F. ; TRAPANI, S. ; NAVAZA, J. "SCA: symmetry-based center assignment of 2D projections of symmetric 3D objects", Journal of Structural Biology, v. 157, p. 339-347, 2007.