Hi Katrin, First, I think you meant that the green density disappears after contouring at 6 Sigma and not 6 A? That you ligand is only partly visible due to disorder and has partial occupancy happens often and is no reason for concern. Of course, you could try soaking with a 10-fold higher ligand concentration of that would be possible. The approach you use: fit would you see and don’t fit what you don’t see is also correct. However, as you come closer to the noise level of your electron density map, you have to be more careful not to introduce model bias and artefacts. What I would to copy your current pdb file to another pdb file, delete the ligand you fitted and any water molecules near the ligand and run a few rounds of refinement to get an unbiased omit map. Then view this map together with your refined ligand a see if it makes sense.
Good luck and happy holidays! Herman Von: CCP4 bulletin board <[email protected]> Im Auftrag von Katherine Lim Gesendet: Freitag, 20. Dezember 2019 05:57 An: [email protected] Betreff: [EXTERNAL] [ccp4bb] Potential weak binding ligand in the active site EXTERNAL : Real sender is [email protected]<mailto:[email protected]> Hi all, I apologise in advance for the long post. I am working on solving a structure that looks like it could have a ligand bound in the active site. My data was obtained from a crystal of just the soluble domain of my protein that had been soaked overnight in the ligand solution. The apo crystal structure is already known and so I have solved my structure using phaser MR. I have attached the Aimless report output of my structure at the end of this email. The current R values I have after refinement and adding in all the waters are Rfree: 0.2413 and Rwork: 0.1897. I can clearly see green density that is much larger (only disappears when I contour the Fo-Fc map to about 6 A) than in my control crystal that had been soaked in the same concentration of just solvent (I had used DMF). I am struggling to add in my ligand as it doesn't seem like the entire ligand can fit in the green density. We think it may be because we have only used the soluble domain and so the ligand isn't held very securely since the transmembrane domain is missing. I have been trying to fit in smaller sections of it and doing an occupancy refinement. So far I have been able to get part of the ligand in with an occupancy of about 0.6 but after the refinement run, phenix.refine seems to move this part of the ligand slightly out of the area where I had tried to fit it into the green density. Interestingly, there isn't a big red density in the area that this ligand section has moved to. I would appreciate any advice on how I should proceed with trying to figure out if I have tried the correct section of the ligand and the kind of refinement settings to use with a weak binder. Space Group P212121 Unit cell abc 84.76, 89.84, 91.55 unit cell alpha beta gamma 90, 90, 90 OVERALL LOW RES HIGH RES Low res limit 45.78 45.78 1.94 High res limit 1.9 9.11 1.9 Rmerge 0.234 0.052 1.684 Rmeas 0.244 0.054 1.768 Rpim 0.069 0.016 0.527 Total # observations 663073 6282 36851 Total # unique 55174 579 3359 I/sigma 7.5 27.9 1.4 CC ½ 0.997 0.999 0.747 Completeness % 99 98.7 94.7 Multiplicity 12 10.8 11 Katherine Lim PhD Candidate School of Biomedical Sciences; School of Molecular Sciences Marshall Centre for Infectious Disease Research and Training The University of Western Australia ________________________________ To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB&A=1<https://urldefense.proofpoint.com/v2/url?u=https-3A__www.jiscmail.ac.uk_cgi-2Dbin_webadmin-3FSUBED1-3DCCP4BB-26A-3D1&d=DwMFaQ&c=Dbf9zoswcQ-CRvvI7VX5j3HvibIuT3ZiarcKl5qtMPo&r=HK-CY_tL8CLLA93vdywyu3qI70R4H8oHzZyRHMQu1AQ&m=7_VYfH8Ol87eqoDkMqPGum6JMrkXaDZxWlhebE8lJRY&s=lLLEY5jR53T_a8okR4d7jd4jhwO7--B0zCRTBBMzbmo&e=> ######################################################################## To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB&A=1
