Difficulty Solving Fab-Antigen Complex

Troubleshooting
1

Hi All,

I am trying to solve the structure of a Fab-antigen complex. It has a relatively complex quaternary structure (two chains in Fab, three chains in antigen), and the particles are not particularly large (only 50 and 130 Angstrom diameters at minimum and maximum, respectively). We know the structures of the antigen and of the Fab independently, but would like to characterize the structure of the complex, to ascertain the Fab epitope.

After pre-processing (motion correction + CTF estimation) and curation of exposures, 2,008 exposures were used for (1) manual picking followed by (2) template picking. Template picks were then used to train a Topaz model. Topaz selected 193,353 particles; however, 2D classes have a pixelated appearance (see image below), and Ab Initio models (4 classes) are not producing a structure that appears close to what I expect.

How could I improve my output? Thank you all for your time and guidance.

Best Regards,

J204_2d_classes_for_iteration_26
J204_2d_classes_for_iteration_261920×3837 914 KB

2

How big is the box you have used for extraction here (in px and Å)? I suspect it is too small.

3

Thank you for your quick response. 300 pixels (for the Extract from Micrographs job, after Topaz Extract). I’m not sure how large that is in Angstroms. We have generally been using default parameters for all jobs.

Best Regards,

4

What is your pixel size?

5

Raw pixel size is 0.73 Å. (300 pixels = 219 Å ?)