Dear Rajesh, We had a similar problem with a yeast glutaminyl-tRNA synthetase. In eukaryotic systems these have an appended N-terminal domain not present in prokaryotes. When the full length protein was crystallized the last ~200 residues were missing from the structure. A packing diagram (highly recommended) showed large solvent channels. We re-expressed the full length protein with a His tag on the N-terminus, purified with a Ni affinity column, and then recrystallized, re-determined the structure and missing residues, followed by anti-his antibody detection on dissolved crystals – the resides were in the crystal but not structurally resolved. We ran a lot of gels which gave similar data but not as conclusive as the antibody approach. The N terminal was present but flexible in the large solvent channels. We used SAXS to look at the full length protein and detected the N-terminal determining that it was globular with a flexible linker. We cut the linker off and the N-terminal domain was crystallized on its own resulting in crystals within minutes of setting up. We used the SAXS envelope to position the various components of the full length protein and determined that the N-terminal domain is structurally similar to the GAtB subunit of GatCAB.
The full story is described in two papers, Nucleic Acids Res. 2012 Apr;40(8):3723-31. doi: 10.1093/nar/gkr1223 and J Mol Biol. 2013 Jul 24;425(14):2480-93. doi: 10.1016/j.jmb.2013.03.043. It took a while but provided a very biologically interesting result. I would note a remarkable development in SAXS analysis related to these early studies that now allows the direct determination of electron density from a scattering curve rather than bead models or envelopes that were originally used: Grant, Thomas D. (2018). Ab initio electron density determination directly from solution scattering data. Nature Methods. http://dx.doi.org/10.1038/nmeth.4581. And shamelessly, also note the crystallization facility involved, http://getacrystal.org. Yours is a smaller protein, but I hope this example might be of some use to answering your question, Best wishes, Eddie Edward Snell Ph.D. President and CEO Hauptman-Woodward Medical Research Institute BioInnovations Chaired Professorship, University at Buffalo, SUNY 700 Ellicott Street, Buffalo, NY 14203-1102 hwi.buffalo.edu Phone: (716) 898 8631 Fax: (716) 898 8660 Skype: eddie.snell Email: esn...@hwi.buffalo.edu [cid:image001.png@01D3B7BC.B5187550] Heisenberg was probably here! From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Rajesh Sent: Friday, March 9, 2018 3:26 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Structure with missing density Dear BB, I apologize for the off topic. But I strongly believe this is the right place to ask my question. We are working on a protein that is 300 amino acids in length. After many efforts, we could solve the structure at 1.60 Å resolution. It took almost 10 months to get this crystal and we could not reproduce it. The maps are interpretable and we could model only till residue 190 and we could not see any density for the rest of the molecule. My question is- Does anyone has encountered such a structure with lots of missing density? I would appreciate your efforts if someone can send me few references describing such type of structures. Is there any chance that microbes can cleave the protein in the crystal drops? Thanks, Rajesh..
