difference map peaks
Hi everyone, I would like to have commnets from experts on the following issues. I am refining DNA-protein complex at 3 A resolution and my current Rfree is 21.5 % (phenix.refine). 1. I am getting difference map peaks (in Coot) of magnitude less than -5 sigmas (0.24 e/A3) (~10 or more peaks) and equal number of positive peaks of same magnitudes.The positive peaks hardly have 2fo-fc of more than 1.0 sigma level. I presumed this well could be due to bulk solvent or improper mask. Therefore I optimized the mask parameters (by giving option under "General refinement parameters" in phenix.refine GUI). I could get my R free lower as expected but I still got these peaks back. Although I am not absolutely sure, but positive peaks are more in polar pocket of protein and negative peaks are more in non-polar and aromatic pockets. I have PEG, Na acetate in my crystal soup. What these negative peaks represent for? 2. During addition of atoms like Na+, Cl- in map, do one need to careful about of the its coordination valency in surrounding pocket. ? I will be highly thankful to you for the same. Ravi Ravindra D. Makde, PhD Postdoctoral fellow, Tan lab, The Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, PA 16802 USA
Hi Ravi,
1. I am getting difference map peaks (in Coot) of magnitude less than -5 sigmas (0.24 e/A3) (~10 or more peaks) and equal number of positive peaks of same magnitudes. The positive peaks hardly have 2fo-fc of more than 1.0 sigma level. I presumed this well could be due to bulk solvent or improper mask. Therefore I optimized the mask parameters (by giving option under "General refinement parameters" in phenix.refine GUI). I could get my R free lower as expected but I still got these peaks back. Although I am not absolutely sure, but positive peaks are more in polar pocket of protein and negative peaks are more in non-polar and aromatic pockets. I have PEG, Na acetate in my crystal soup. What these negative peaks represent for?
Compute 2mFo-DFc and mFo-DFc Average Kick maps and see if these peaks are still there. If they disappear - lucky you, if not, then will think more. If you do not know what an Average Kick map is, check slide #20 here: http://cci.lbl.gov/~afonine/rsr/afonine_05OCT2009_.pdf
2. During addition of atoms like Na+, Cl- in map, do one need to careful about of the its coordination valency in surrounding pocket. ?
Not 100% sure, but at 3A resolution yes... Hope some one else comments on this. Pavel.
RAVINDRA D MAKDE wrote:
Hi everyone,
I would like to have commnets from experts on the following issues.
I am refining DNA-protein complex at 3 A resolution and my current Rfree is 21.5 % (phenix.refine).
1. I am getting difference map peaks (in Coot) of magnitude less than -5 sigmas (0.24 e/A3) (~10 or more peaks) and equal number of positive peaks of same magnitudes. The positive peaks hardly have 2fo-fc of more than 1.0 sigma level. I presumed this well could be due to bulk solvent or improper mask. Therefore I optimized the mask parameters (by giving option under "General refinement parameters" in phenix.refine GUI). I could get my R free lower as expected but I still got these peaks back. Although I am not absolutely sure, but positive peaks are more in polar pocket of protein and negative peaks are more in non-polar and aromatic pockets. I have PEG, Na acetate in my crystal soup. What these negative peaks represent for? I suspect that negative peaks are from the bulk-solvent mask, as you suspected. Some proteins have hydrophobic cavities that are big enough to make a small void that ends up with a bit of bulk solvent density. Optimizing bulk solvent parameters does not necessarily help, because similar sized cavities may in fact have a bit of actual solvent density.
It may be possible to put zero-occupancy dummy atoms on those sites to screen out the bulk solvent mask, but I don't know how the PHENIX bulk mask handles partial occupancy.
2. During addition of atoms like Na+, Cl- in map, do one need to careful about of the its coordination valency in surrounding pocket. ?
Definitely. The PDB is full of misidentified ions, even in active sites at resolutions much better than 3.0. Many Polymerase Beta structures have an Mg++ ion in the model, where the true identity is Na+. It is easy for ions to have partial occupancy with waters or other ions, or some disorder due to the water ligands. Of course, there are probably even more invalid waters in the PDB than anything else. My opinion is that it is better to make your best guess rather than just leave in phony waters, and put remarks to the deposited PDB file for anything that is uncertain. Unfortunately, most PDB consumers don't read remarks, so we would benefit from a standard way to flag qualitative, author-defined uncertainties. Joe Krahn
I suspect that negative peaks are from the bulk-solvent mask, as you suspected.
The bulk solvent is flat... at least phenix.refine uses Flat Bulk solvent model (mask based). If bulk solvent is guilty for this, why would you observe peaks ? I repeat once again: looking at Average Kick map may resolve this puzzle. Pavel.
Dear Phenix-Gurus, running Phenix-1.5-2 under RHEL 5, 64 bit I get the following error message -- this appears to occur after the actual torsion angle refinement. Hydrogens were added, custom-defined bonds excluded. Could anybody help me out, please? Thanks, Ulrich merge clusters with multiple connections: 1 pass suppressing allowed origin shifts: True new target temperature: 2500.00 K step= 1 temperature= 2500.0 rmsd=0.0459 r_work=0.2537 r_free=0.3224 step= 25 temperature= 2394.4 rmsd=0.3481 r_work=0.2575 r_free=0.3316 step= 50 temperature= 2284.4 rmsd=0.3254 r_work=0.2505 r_free=0.3287 step= 75 temperature= 2174.4 rmsd=0.3205 r_work=0.2476 r_free=0.3271 step= 100 temperature= 2064.4 rmsd=0.3189 r_work=0.2461 r_free=0.3261 step= 125 temperature= 1954.4 rmsd=0.3205 r_work=0.2451 r_free=0.3255 step= 150 temperature= 1844.4 rmsd=0.3254 r_work=0.2443 r_free=0.3252 step= 175 temperature= 1734.4 rmsd=0.3287 r_work=0.2437 r_free=0.3250 step= 200 temperature= 1624.4 rmsd=0.3329 r_work=0.2432 r_free=0.3250 step= 225 temperature= 1514.4 rmsd=0.3393 r_work=0.2427 r_free=0.3250 step= 250 temperature= 1404.4 rmsd=0.3433 r_work=0.2424 r_free=0.3249 step= 275 temperature= 1294.4 rmsd=0.3479 r_work=0.2421 r_free=0.3250 step= 300 temperature= 1184.4 rmsd=0.3543 r_work=0.2419 r_free=0.3251 step= 325 temperature= 1074.4 rmsd=0.3581 r_work=0.2416 r_free=0.3250 step= 350 temperature= 964.4 rmsd=0.3629 r_work=0.2414 r_free=0.3251 step= 375 temperature= 854.4 rmsd=0.3687 r_work=0.2413 r_free=0.3251 step= 400 temperature= 744.4 rmsd=0.3722 r_work=0.2411 r_free=0.3252 step= 425 temperature= 634.4 rmsd=0.3771 r_work=0.2409 r_free=0.3252 step= 450 temperature= 524.4 rmsd=0.3819 r_work=0.2408 r_free=0.3252 step= 475 temperature= 414.4 rmsd=0.3854 r_work=0.2407 r_free=0.3253 step= 500 temperature= 304.4 rmsd=0.3906 r_work=0.2406 r_free=0.3254 allowed origin shift velocity corrections applied (magnitudes): n: 501 min: 0 max: 1.30923e-16 mean: 1.24537e-17 sum of allowed origin shift velocity corrections (vectors): (0.00000, 0.00000, -0.00000) ================================ xyz refinement =============================== Traceback (most recent call last): File "/usr/local/phenix-1.5-2/phenix/phenix/command_line/refine.py", line 11, in <module> command_line.run(command_name="phenix.refine", args=sys.argv[1:]) File "/usr/local/phenix-1.5-2/phenix/phenix/refinement/command_line.py", line 89, in run call_back_handler=call_back_handler) File "/usr/local/phenix-1.5-2/phenix/phenix/refinement/driver.py", line 1121, in run call_back_handler = call_back_handler) File "/usr/local/phenix-1.5-2/phenix/phenix/refinement/strategies.py", line 552, in refinement_machine target_weights_params, main, h_params, log, params) File "/usr/local/phenix-1.5-2/phenix/phenix/refinement/strategies.py", line 839, in __init__ minimized = self.minimize() File "/usr/local/phenix-1.5-2/phenix/phenix/refinement/strategies.py", line 918, in minimize h_params = self.h_params) File "/usr/local/phenix-1.5-2/cctbx_project/mmtbx/refinement/minimization.py", line 39, in __init__ self.regularize_h_and_update_xray_structure() File "/usr/local/phenix-1.5-2/cctbx_project/mmtbx/refinement/minimization.py", line 222, in regularize_h_and_update_xray_structure self.h_params.xh_bond_distance_deviation_limit) File "/usr/local/phenix-1.5-2/cctbx_project/mmtbx/model.py", line 167, in idealize_h for t in self.xh_connectivity_table(): File "/usr/local/phenix-1.5-2/cctbx_project/mmtbx/model.py", line 118, in xh_connectivity_table xray_structure = xray_structure).table File "/usr/local/phenix-1.5-2/cctbx_project/mmtbx/model.py", line 47, in __init__ xray_structure.sites_cart()).bond_proxies.simple AttributeError: 'NoneType' object has no attribute 'simple'
Hi Ulrich, sorry for the problem! It is a known bug and I notified Ralf about it on 8/19/09. My guess is that it has something to do with torsion angle dynamics code that modifies the geometry restraints manager in-place making it invalid for the rest of refinement machinery. I'm afraid that the only solution for now is to turn the torsion angle dynamics off. Pavel. On 10/7/09 5:26 AM, Ulrich Baumann wrote:
Dear Phenix-Gurus,
running Phenix-1.5-2 under RHEL 5, 64 bit I get the following error message -- this appears to occur after the actual torsion angle refinement. Hydrogens were added, custom-defined bonds excluded. Could anybody help me out, please? Thanks, Ulrich
merge clusters with multiple connections: 1 pass
suppressing allowed origin shifts: True new target temperature: 2500.00 K step= 1 temperature= 2500.0 rmsd=0.0459 r_work=0.2537 r_free=0.3224 step= 25 temperature= 2394.4 rmsd=0.3481 r_work=0.2575 r_free=0.3316 step= 50 temperature= 2284.4 rmsd=0.3254 r_work=0.2505 r_free=0.3287 step= 75 temperature= 2174.4 rmsd=0.3205 r_work=0.2476 r_free=0.3271 step= 100 temperature= 2064.4 rmsd=0.3189 r_work=0.2461 r_free=0.3261 step= 125 temperature= 1954.4 rmsd=0.3205 r_work=0.2451 r_free=0.3255 step= 150 temperature= 1844.4 rmsd=0.3254 r_work=0.2443 r_free=0.3252 step= 175 temperature= 1734.4 rmsd=0.3287 r_work=0.2437 r_free=0.3250 step= 200 temperature= 1624.4 rmsd=0.3329 r_work=0.2432 r_free=0.3250 step= 225 temperature= 1514.4 rmsd=0.3393 r_work=0.2427 r_free=0.3250 step= 250 temperature= 1404.4 rmsd=0.3433 r_work=0.2424 r_free=0.3249 step= 275 temperature= 1294.4 rmsd=0.3479 r_work=0.2421 r_free=0.3250 step= 300 temperature= 1184.4 rmsd=0.3543 r_work=0.2419 r_free=0.3251 step= 325 temperature= 1074.4 rmsd=0.3581 r_work=0.2416 r_free=0.3250 step= 350 temperature= 964.4 rmsd=0.3629 r_work=0.2414 r_free=0.3251 step= 375 temperature= 854.4 rmsd=0.3687 r_work=0.2413 r_free=0.3251 step= 400 temperature= 744.4 rmsd=0.3722 r_work=0.2411 r_free=0.3252 step= 425 temperature= 634.4 rmsd=0.3771 r_work=0.2409 r_free=0.3252 step= 450 temperature= 524.4 rmsd=0.3819 r_work=0.2408 r_free=0.3252 step= 475 temperature= 414.4 rmsd=0.3854 r_work=0.2407 r_free=0.3253 step= 500 temperature= 304.4 rmsd=0.3906 r_work=0.2406 r_free=0.3254 allowed origin shift velocity corrections applied (magnitudes): n: 501 min: 0 max: 1.30923e-16 mean: 1.24537e-17 sum of allowed origin shift velocity corrections (vectors): (0.00000, 0.00000, -0.00000)
================================ xyz refinement ===============================
Traceback (most recent call last): File "/usr/local/phenix-1.5-2/phenix/phenix/command_line/refine.py", line 11, in <module> command_line.run(command_name="phenix.refine", args=sys.argv[1:]) File "/usr/local/phenix-1.5-2/phenix/phenix/refinement/command_line.py", line 89, in run call_back_handler=call_back_handler) File "/usr/local/phenix-1.5-2/phenix/phenix/refinement/driver.py", line 1121, in run call_back_handler = call_back_handler) File "/usr/local/phenix-1.5-2/phenix/phenix/refinement/strategies.py", line 552, in refinement_machine target_weights_params, main, h_params, log, params) File "/usr/local/phenix-1.5-2/phenix/phenix/refinement/strategies.py", line 839, in __init__ minimized = self.minimize() File "/usr/local/phenix-1.5-2/phenix/phenix/refinement/strategies.py", line 918, in minimize h_params = self.h_params) File "/usr/local/phenix-1.5-2/cctbx_project/mmtbx/refinement/minimization.py", line 39, in __init__ self.regularize_h_and_update_xray_structure() File "/usr/local/phenix-1.5-2/cctbx_project/mmtbx/refinement/minimization.py", line 222, in regularize_h_and_update_xray_structure self.h_params.xh_bond_distance_deviation_limit) File "/usr/local/phenix-1.5-2/cctbx_project/mmtbx/model.py", line 167, in idealize_h for t in self.xh_connectivity_table(): File "/usr/local/phenix-1.5-2/cctbx_project/mmtbx/model.py", line 118, in xh_connectivity_table xray_structure = xray_structure).table File "/usr/local/phenix-1.5-2/cctbx_project/mmtbx/model.py", line 47, in __init__ xray_structure.sites_cart()).bond_proxies.simple AttributeError: 'NoneType' object has no attribute 'simple'
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Pavel Afonine wrote:
I suspect that negative peaks are from the bulk-solvent mask, as you suspected.
The bulk solvent is flat... at least phenix.refine uses Flat Bulk solvent model (mask based). If bulk solvent is guilty for this, why would you observe peaks ?
I repeat once again: looking at Average Kick map may resolve this puzzle.
Pavel.
Sometimes there are voids just big enough for a tiny bit of solvent mask to fill. This results in negative difference density. Even though the mask is flat, the B-factor adjustment will convert small , isolated fragments of bulk mask into an approximately gaussian sphere, producing a well-defined negative peak. However, this situation is fairly rare. Voids big enough for the bulk mask to enter are usually big enough for a water. A true hydrophobic void deters from protein folding stability, and normally only occur if it pertains to the protein's function. So, a simple noise peak probably is more likely. The only way to know for sure is to make a bulk-solvent map. Is it possible to write out bulk solvent coefficients? Joe Krahn
participants (4)
-
Joe Krahn -
Pavel Afonine -
RAVINDRA D MAKDE -
Ulrich Baumann