Whic mtz file to use and parameters for phenix.refine (GUI) to set?

Hermella Woldemdihin

19 Jul 2010 19 Jul '10

1:15 p.m.

Hi, I am new to phenix.refine. I'm using the graphical interface. I hav processed my dfiffraction data and used Phaser MR for molecular replacement. And I goyt a result: a solution pdb file and the associated mtz file. 1. So I use the mtz file from this Phaser MR solution or the original file from my data processing in phenix.refine? 2. I have set parameters for phenix.refine like this: Is there other settings for the parameter which I should do for a better result? PDB data: my solution from Phaser MR XRAY DATA= ???? Simulated Annealing=1000 Update waters Refine Target weights: Wxc=0.8 Wxu=1.6 Cycles=5 Fix rotamers Fix bad side chains

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Vellieux Frederic

19 Jul 19 Jul

12:13 p.m.

New subject: Whic mtz file to use and parameters for phenix.refine (GUI) to set?

Hi Hermella, The first thing (I think) you should do is to check your molecular replacement solution on the graphics station (use whichever software you feel most comfortable with): check the packing in particular, are there crystal-forming contacts that explain the crystal - if you have regions of void then it is likely that your solution is not a solution (you may have missing molecules for example). And you may also wish to model into the electron density coming out of Phaser (such as mutating residues, if the quality of the electron density map allows you to do that). For this, you use the mtz file coming out of phaser, that contains the electron density map coefficients (SigmaA). Also, if your search model is made up of several subunits, you will probably want to carry our rigid-body refinement (of the subunits independently) before carrying out any model building. For refinement, I suggest that you use the initial mtz file (containing FP, SIGFP, FREERFLAG), unless you want to have (after X refinement rounds) a single mtz file that contains all the history of the structure determination work. But as long as the input mtz file (for refinement) contains the FP, SIGFP, FREERFLAG columns UNMODIFIED then it's a question of taste. I keep everything in separate directories myself, I'm used to that... Fred. Hermella Woldemdihin wrote:

...

Hi,

I am new to phenix.refine. I'm using the graphical interface. I hav processed my dfiffraction data and used Phaser MR for molecular replacement. And I goyt a result: a solution pdb file and the associated mtz file. 1. So I use the mtz file from this Phaser MR solution or the original file from my data processing in phenix.refine? 2. I have set parameters for phenix.refine like this: Is there other settings for the parameter which I should do for a better result? PDB data: my solution from Phaser MR XRAY DATA= ???? Simulated Annealing=1000 Update waters Refine Target weights: Wxc=0.8 Wxu=1.6 Cycles=5 Fix rotamers Fix bad side chains

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Nathaniel Echols

1:41 p.m.

New subject: Whic mtz file to use and parameters for phenix.refine (GUI) to set?

On Mon, Jul 19, 2010 at 6:15 AM, Hermella Woldemdihin wrote:

...

I am new to phenix.refine. I'm using the graphical interface. I hav processed my dfiffraction data and used Phaser MR for molecular replacement. And I goyt a result: a solution pdb file and the associated mtz file. 1. So I use the mtz file from this Phaser MR solution or the original file from my data processing in phenix.refine?

The original file. I think Phaser outputs anisotropy-corrected data, and it may be at reduced resolution depending on how you run it. 2. I have set parameters for phenix.refine like this:

...

Is there other settings for the parameter which I should do for a better result? PDB data: my solution from Phaser MR XRAY DATA= ???? Simulated Annealing=1000 Update waters Refine Target weights: Wxc=0.8 Wxu=1.6 Cycles=5 Fix rotamers Fix bad side chains

Unless you've already run refinement with this dataset and are confident about the weights, I would not set wxc and wxu initially; automatic weight optimization is a better place to start. Otherwise, which refinement settings are most appropriate depends on the resolution and starting R-free. As Frederic points out, rigid-body refinement may be a good idea at this stage. (By the way, you list both "Fix rotamers" and "Fix bad side chains", but these are the same thing - unless I mislabeled something in the GUI...) -Nat

xaravich ivan

20 Jul 20 Jul

12:07 a.m.

New subject: Whic mtz file to use and parameters for phenix.refine (GUI) to set?

I thought you initially do not add waters until you have a reasonable density fit with the protein molecules. Is it ok to update waters right from the begining starting from an MR solution? Ivan On Mon, Jul 19, 2010 at 6:41 AM, Nathaniel Echols wrote:

...

On Mon, Jul 19, 2010 at 6:15 AM, Hermella Woldemdihin wrote:

...
I am new to phenix.refine. I'm using the graphical interface. I hav processed my dfiffraction data and used Phaser MR for molecular replacement. And I goyt a result: a solution pdb file and the associated mtz file. 1. So I use the mtz file from this Phaser MR solution or the original file from my data processing in phenix.refine?

The original file. I think Phaser outputs anisotropy-corrected data, and it may be at reduced resolution depending on how you run it.

2. I have set parameters for phenix.refine like this:

...
Is there other settings for the parameter which I should do for a better result? PDB data: my solution from Phaser MR XRAY DATA= ???? Simulated Annealing=1000 Update waters Refine Target weights: Wxc=0.8 Wxu=1.6 Cycles=5 Fix rotamers Fix bad side chains

Unless you've already run refinement with this dataset and are confident about the weights, I would not set wxc and wxu initially; automatic weight optimization is a better place to start. Otherwise, which refinement settings are most appropriate depends on the resolution and starting R-free. As Frederic points out, rigid-body refinement may be a good idea at this stage. (By the way, you list both "Fix rotamers" and "Fix bad side chains", but these are the same thing - unless I mislabeled something in the GUI...)

-Nat

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Pavel Afonine

12:27 a.m.

New subject: Whic mtz file to use and parameters for phenix.refine (GUI) to set?

Hi Ivan,

...

I thought you initially do not add waters until you have a reasonable density fit with the protein molecules. Is it ok to update waters right from the begining starting from an MR solution?

yes and no. Yes, it is bad to add waters into poor map and very partial model IF you and the software you use are careless (for example, treat once placed water is God given and never critically re-think it at all subsequent steps). This is because you will have to deal with model bias later on. No, it is good to add waters, because by adding waters (even temporarily into some residue side chain or ligand density), you in turn improve the map and the map improvement leads to a chance to build some more model and/or improve existing one in a next cycle, and building some model improves the map some more, and so on. The important thing is that you and the software you use should treat waters simply as density peaks (and not actual atoms) and do not hesitate to remove waters as necessary and build macromolecular into the density they are taking. I guess AutoBuild does exactly this. phenix.refine option "fix_rotamers" does this too. I'm not sure if all tools follow this yet, but I hope it will be the case sooner or later. Anyway, you can turn water recycling ON and remove all waters after the map is computed and before you go into manual model building in Coot or whatever program you use. Pavel.

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Hermella Woldemdihin
Nathaniel Echols
Pavel Afonine
Vellieux Frederic
xaravich ivan