Thank you guys for the suggestions! For Dr Bosch's question, every monomer binds two ligands, and the first binding site is different from the second binding site. The ligand is placed differently for the two binding sites for each monomer, but for the symmetrical sites in the dimer protein, the ligand is placed in the same conformation, even though the density of the ligand in the second monomer is not good enough to place the ligand. Thank you so much! Best, Wei On Thu, Oct 24, 2013 at 10:31 PM, Bosch, Juergen wrote:

Hi Wei, have you considered modeling two conformations of your ligand in the four sites ? Jürgen

On Oct 24, 2013, at 10:19 PM, Wei Shi wrote:

Hi all, I am working on a structure of protein-ligand complex. Four ligands are placed for the dimer protein and the density for the two ligands of the first monomer is better than the density for the other two ligands of the second monomer. Ligand is moved to fit density better in Coot (and for two ligands of the first monomer, they fits the density almost perfectly), but after a refinement in phenix (default settings + NCS restraints + Secondary structural restraints + Optimize X-ray/stereochemistry weight +Optimize X-ray/ADP weight), some part of the ligand which fits density good before moved out of the density again...Besides, it always shows green density in Coot for the ligand region even in places where the ligand is in density... Any suggestions or ideas about how to fit the ligand better and why the density for ligands of the second monomer is worse than that for the first monomer and why the ligands would move out of density after refinement? Is it because the protein model is not good enough to get the ligand density good? There is a conformational change upon ligand binding, and I rebuilt some part of the protein manually, and the density for a region of about 30 residues is not very good, and I tried to mutate those to alanies and refine, but it didn't help me see the density better... Any suggestions or ideas on how to improve this protein-complex structural model? Thank you so much! The statistics for the current best model is as follows, and the resolution of the dataset is 2.8Å.

start final ------------------------------ ---------------- R-work: 0.3359 0.2993 R-free: 0.3619 0.3558 RMS(angles): 1.03 1.55 RMS(bonds): 0.006 0.007

MolProbity validation Ramachandran outliers: 4.7% (Goal: < 0.2%) Ramachandran favored: 85.3% (Goal: > 98%) Rotamer outliers: 4.5% (Goal: 1%) C-beta outliers: 0 (Goal: 0) Clashscore: 7.43 Overall score: 2.56

Thank you so much!

Best, Wei _______________________________________________ phenixbb mailing list [email protected] http://phenix-online.org/mailman/listinfo/phenixbb

...................... Jürgen Bosch Johns Hopkins University Bloomberg School of Public Health Department of Biochemistry & Molecular Biology Johns Hopkins Malaria Research Institute 615 North Wolfe Street, W8708 Baltimore, MD 21205 Office: +1-410-614-4742 Lab: +1-410-614-4894 Fax: +1-410-955-2926 http://lupo.jhsph.edu

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Hi Wei, it's difficult to tell what's happening without looking at files. First, I would compute ligand OMIT map. Then fit ligand into it and refined ligands coordinates and ADPs. If you see negative density around ligand after refinement among other reasons this may indicate that the ligand or parts of it have multiple conformations in which case you need to model them as such or at least refine group occupancy of the one copy. If ligand crosses a special position then it becomes a different story. If you send me files (data, model, cifs if any) and indicate which ligands are in question I will have a look. Please send files to my email directly, not the entire mailing list. Pavel On 10/25/13 9:28 AM, Wei Shi wrote:

Also, as suggested by Dr Terwilliger, I deleted the ligands and refine only the protein structure. When I go over the protein structure, I could see some region didn't fit the density well, so I am thinking of mutating those regions to alanines and do a refinement in phenix (default settings + NCS restraints + Secondary structural restraints + Optimize X-ray/stereochemistry weight +Optimize X-ray/ADP weight), and see whether I could see better density. Any suggestions or ideas about how to improve the protein structural model? Thank you so much!

Best, Wei

On Fri, Oct 25, 2013 at 12:15 PM, Wei Shi mailto:[email protected]> wrote:

Thank you guys for the suggestions! For Dr Bosch's question, every monomer binds two ligands, and the first binding site is different from the second binding site. The ligand is placed differently for the two binding sites for each monomer, but for the symmetrical sites in the dimer protein, the ligand is placed in the same conformation, even though the density of the ligand in the second monomer is not good enough to place the ligand. Thank you so much!

Best, Wei

On Thu, Oct 24, 2013 at 10:31 PM, Bosch, Juergen mailto:[email protected]> wrote:

Hi Wei, have you considered modeling two conformations of your ligand in the four sites ? Jürgen

On Oct 24, 2013, at 10:19 PM, Wei Shi wrote:

...

Hi all, I am working on a structure of protein-ligand complex. Four ligands are placed for the dimer protein and the density for the two ligands of the first monomer is better than the density for the other two ligands of the second monomer. Ligand is moved to fit density better in Coot (and for two ligands of the first monomer, they fits the density almost perfectly), but after a refinement in phenix (default settings + NCS restraints + Secondary structural restraints + Optimize X-ray/stereochemistry weight +Optimize X-ray/ADP weight), some part of the ligand which fits density good before moved out of the density again...Besides, it always shows green density in Coot for the ligand region even in places where the ligand is in density... Any suggestions or ideas about how to fit the ligand better and why the density for ligands of the second monomer is worse than that for the first monomer and why the ligands would move out of density after refinement? Is it because the protein model is not good enough to get the ligand density good? There is a conformational change upon ligand binding, and I rebuilt some part of the protein manually, and the density for a region of about 30 residues is not very good, and I tried to mutate those to alanies and refine, but it didn't help me see the density better... Any suggestions or ideas on how to improve this protein-complex structural model? Thank you so much! The statistics for the current best model is as follows, and the resolution of the dataset is 2.8Å.

start final ------------------------------ ---------------- R-work: 0.3359 0.2993 R-free: 0.3619 0.3558 RMS(angles): 1.03 1.55 RMS(bonds): 0.006 0.007

MolProbity validation Ramachandran outliers: 4.7% (Goal: < 0.2%) Ramachandran favored: 85.3% (Goal: > 98%) Rotamer outliers: 4.5% (Goal: 1%) C-beta outliers: 0 (Goal: 0) Clashscore: 7.43 Overall score: 2.56

Thank you so much!

Best, Wei _______________________________________________ phenixbb mailing list [email protected] mailto:[email protected] http://phenix-online.org/mailman/listinfo/phenixbb

...................... Jürgen Bosch Johns Hopkins University Bloomberg School of Public Health Department of Biochemistry & Molecular Biology Johns Hopkins Malaria Research Institute 615 North Wolfe Street, W8708 Baltimore, MD 21205 Office: +1-410-614-4742 tel:%2B1-410-614-4742 Lab: +1-410-614-4894 tel:%2B1-410-614-4894 Fax: +1-410-955-2926 tel:%2B1-410-955-2926 http://lupo.jhsph.edu

_______________________________________________ phenixbb mailing list [email protected] mailto:[email protected] http://phenix-online.org/mailman/listinfo/phenixbb

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