help with validating a very low res structure
Hi there-- Any old school experimental phasing experts want to help me out? I have an 8 angstrom structure solved using molecular replacement, refined to Rwork 0.38 and Rfree of 0.47. I also have a heavy atom (not SeMet) derivative data set to 10 angstroms. The idea is to get as much information from these data as possible so as to validate my current MR model. My approach is to calculate a difference map using the isomorphous difference Fouriers OR anomalous difference Fouriers as the amplitudes, with phases derived from the MR model-- then using the difference map to locate putative heavy atom sites. I would use various controls and experiments to (computationally) validate the credibility of these sites (e.g. cross-phasing with only iso/ano differences). Anyone have any suggestions for how to do this using Phenix? I use the GUI (generally the nightly builds), and am a newcomer to Phenix. Thanks!!
On Thu, Apr 5, 2012 at 5:55 PM, Yunji Wu
I have an 8 angstrom structure solved using molecular replacement, refined to Rwork 0.38 and Rfree of 0.47. I also have a heavy atom (not SeMet) derivative data set to 10 angstroms. The idea is to get as much information from these data as possible so as to validate my current MR model. My approach is to calculate a difference map using the isomorphous difference Fouriers OR anomalous difference Fouriers as the amplitudes, with phases derived from the MR model-- then using the difference map to locate putative heavy atom sites. I would use various controls and experiments to (computationally) validate the credibility of these sites (e.g. cross-phasing with only iso/ano differences). Anyone have any suggestions for how to do this using Phenix? I use the GUI (generally the nightly builds), and am a newcomer to Phenix.
I am neither old school nor an experimental phasing expert, but there are GUIs for creating anomalous difference maps (the standard "Create maps" interface, which will automatically generate an anomalous map if the data are anomalous) or isomorphous difference maps (further down the list). The other thing to try (in fact, the first thing I would try in this case) would be to run MR-SAD in the Phaser-EP GUI, using the refined model as input and telling it to complete the anomalous substructure with whatever heavy atom you used. The map it outputs will be phase-combined, so still very biased by the model, but being able to place the heavy atom is a good sign, and you can then take this and run simple SAD phasing AutoSol with this as input (with model-building disabled, obviously). Having done a little bit of very-low-resolution refinement recently, I think R-free of 0.47 is too high (the gap between R-factors is also very large). What refinement strategy were you using? If you're willing to share the data with us I'd be interested in taking a look, because I'd like to figure out exactly how much refinement we can get away with, and how well different strategies work. Pavel has suggested that individual B-factor refinement, if properly restrained, may work best even for data like this, and my experiences have tended to confirm this. -Nat
Yunji And I thought I had it tough interpreting 5A maps from experimental phases. I didn't think that anyone would be working at worse (ok except maybe Nenad Ban or Kiyoshi Nagai). And with molecular replacement even! What exactly are you trying to validate at this low resolution? That your MR solution isn't biased? F On Apr 5, 2012, at 6:55 PM, Yunji Wu wrote:
Hi there--
Any old school experimental phasing experts want to help me out?
I have an 8 angstrom structure solved using molecular replacement, refined to Rwork 0.38 and Rfree of 0.47. I also have a heavy atom (not SeMet) derivative data set to 10 angstroms. The idea is to get as much information from these data as possible so as to validate my current MR model. My approach is to calculate a difference map using the isomorphous difference Fouriers OR anomalous difference Fouriers as the amplitudes, with phases derived from the MR model-- then using the difference map to locate putative heavy atom sites. I would use various controls and experiments to (computationally) validate the credibility of these sites (e.g. cross-phasing with only iso/ano differences). Anyone have any suggestions for how to do this using Phenix? I use the GUI (generally the nightly builds), and am a newcomer to Phenix.
Thanks!!
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Dear All, There may be PDB files which shows different protonation state of HIS, at least there is PDB file shows double protonated HIS. At which resolution of the crystal can allow us to distinguish different protonation states of HIS? How do Phenix and Coot give different protonation states of HIS? I am looking forward to getting your reply. Cheers, Dialing
Hi Dialing,
Determining the protonation state of a HIS residue based on the diffraction
data would require very high resolution data (below 1.0A), if that is
possible at all.
However, it is possible to determine the protonation state of HIS residues
based on the local hydrogen-bonding network for a model at any resolution,
assuming it is well built. The program Reduce, which is the program used in
Phenix to add hydrogens to a model, does this automatically. Reduce looks
at the local hydrogen-bonding network created by neighboring residues and
other capable groups (waters, ligands, etc.) and then optimizes the HIS
orientation and protonation state. The three possibilities are delta
protonated (HD2), epsilon protonated (HE2), or both (HD2 and HE2).
You can read more about Reduce here:
http://kinemage.biochem.duke.edu/software/reduce.php
Jeff
On Fri, Apr 6, 2012 at 4:18 AM, Dialing Pretty
Dear All,
There may be PDB files which shows different protonation state of HIS, at least there is PDB file shows double protonated HIS. At which resolution of the crystal can allow us to distinguish different protonation states of HIS?
How do Phenix and Coot give different protonation states of HIS?
I am looking forward to getting your reply.
Cheers,
Dialing
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On Fri, Apr 6, 2012 at 7:06 AM, Jeff Headd
Determining the protonation state of a HIS residue based on the diffraction data would require very high resolution data (below 1.0A), if that is possible at all.
Or neutron diffraction (which doesn't require atomic resolution), for the people who really care. -Nat
Hi,
There may be PDB files which shows different protonation state of HIS, at least there is PDB file shows double protonated HIS. At which resolution of the crystalcan allow us to distinguish different protonation states of HIS?
an example: M. Blakeley, F. Ruiz, R. Cachau, I. Hazemann, F. Meilleur, A. Mitschler, S. Ginell, P. Afonine, O. Ventura, A. Cousido, M. Haertlein, A. Joachimiak, D. Myles & A. Podjarny. Quantum model of catalysis based on a mobile proton revealed by subatomic x-ray and neutron diffraction studies of h-aldose reductase. (2008). PNAS. 105, 1844-1848. Pavel
Sorry for the late posting on this subject, but I wanted to add that you might want to also use knowledge of the preferred tautomer of neutral histidine when assigning the ionization state and orientation of the imidazole. Hong's group has done some very nice work on this: Li, S., and Hong, M. (2011) Protonation, tautomerization, and rotameric structure of histidine: a comprehensive study by magic-angle-spinning solid-state NMR, J Am Chem Soc 133, 1534-1544. On Apr 6, 2012, at 10:05 AM, Pavel Afonine wrote: Hi, There may be PDB files which shows different protonation state of HIS, at least there is PDB file shows double protonated HIS. At which resolution of the crystal can allow us to distinguish different protonation states of HIS? an example: M. Blakeley, F. Ruiz, R. Cachau, I. Hazemann, F. Meilleur, A. Mitschler, S. Ginell, P. Afonine, O. Ventura, A. Cousido, M. Haertlein, A. Joachimiak, D. Myles & A. Podjarny. Quantum model of catalysis based on a mobile proton revealed by subatomic x-ray and neutron diffraction studies of h-aldose reductase. (2008). PNAS. 105, 1844-1848. Pavel _______________________________________________ phenixbb mailing list [email protected]mailto:[email protected] http://phenix-online.org/mailman/listinfo/phenixbb
participants (7)
-
Dialing Pretty -
Francis E Reyes -
Jeff Headd -
Nathaniel Echols -
Pavel Afonine -
Tanner, John J. -
Yunji Wu