strategy suggestion ???
Hi, I just started learning crystallography months ago, and the software I used most is XDS, HKL2000, phenix and coot. Now I am working on a series of datasets-----the same target protein with various ligands. The following is my refinement workflow: 1, Processing data with XDS (or HKL2000 for some of the datasets) 2,Molecule replacement with phaser. 3,Check and modify the structure residue by residue with COOT, 4,phenix.refine with the following command line: phenix.refine protein.1.mtz protein.pdb \ strategy=individual_sites+individual_sites_real_space+individual_adp+tls \ simulated_annealing=true simulated_annealing.start_temperature=1000 \ simulated_annealing.cool_rate=10 main.number_of_macro_cycles=5 \ ordered_solvent=true refinement.input.xray_data.labels="F,SIGF" \ output.prefix=myprotein 5,Fit the ligand and redo step 3&4; stop refining when the Rwork and Rfree looks reasonable. 6,Chage the strategy line to strategy=individual_adp adp.individual.isotropic=all \ to remove the ANISOU lines in pdb file. Any suggestion for this workflow ? or how do you always deal with the similar case ? cause I'm new about this field,maybe I did something stupid. Ps, the value of Rwork and Rfree are too close, like, Final: r_work = 0.1741 r_free = 0.1817 bonds = 0.036 angles = 1.818 Is that reasonable ? -- Drug Discovery and Design Center, Shanghai Institute of Materia Medica, Chinese Academy of Sciences Address: Room 101, 646 Songtao Road, Zhangjiang Hi-Tech Park, Pudong New Area, Shanghai, 201203, P.R. China
You don't always need to carry out simulated annealing. Typically, simulated annealing will be carried out for a "de novo" structure determination; for molecular replacement I hardly ever use simulated annealing (or not at all). However, if the initial electron density maps after molecular replacement are of insufficient quality, and if the resolution is high enough, then I send the starting model and mtz file to the Arp / Warp server for automatic model rebuilding. Fred. ChenTiantian wrote:
Hi, I just started learning crystallography months ago, and the software I used most is XDS, HKL2000, phenix and coot. Now I am working on a series of datasets-----the same target protein with various ligands. The following is my refinement workflow: 1, Processing data with XDS (or HKL2000 for some of the datasets) 2,Molecule replacement with phaser. 3,Check and modify the structure residue by residue with COOT, 4,phenix.refine with the following command line:
phenix.refine protein.1.mtz protein.pdb \ strategy=individual_sites+individual_sites_real_space+individual_adp+tls \ simulated_annealing=true simulated_annealing.start_temperature=1000 \ simulated_annealing.cool_rate=10 main.number_of_macro_cycles=5 \ ordered_solvent=true refinement.input.xray_data.labels="F,SIGF" \ output.prefix=myprotein
5,Fit the ligand and redo step 3&4; stop refining when the Rwork and Rfree looks reasonable. 6,Chage the strategy line to strategy=individual_adp adp.individual.isotropic=all \ to remove the ANISOU lines in pdb file.
Any suggestion for this workflow ? or how do you always deal with the similar case ? cause I'm new about this field,maybe I did something stupid. Ps, the value of Rwork and Rfree are too close, like, Final: r_work = 0.1741 r_free = 0.1817 bonds = 0.036 angles = 1.818 Is that reasonable ?
-- Drug Discovery and Design Center, Shanghai Institute of Materia Medica, Chinese Academy of Sciences Address: Room 101, 646 Songtao Road, Zhangjiang Hi-Tech Park, Pudong New Area, Shanghai, 201203, P.R. China ------------------------------------------------------------------------
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Hi,
4,phenix.refine with the following command line:
phenix.refine protein.1.mtz protein.pdb \ strategy=individual_sites+individual_sites_real_space+individual_adp+tls \ simulated_annealing=true simulated_annealing.start_temperature=1000 \ simulated_annealing.cool_rate=10 main.number_of_macro_cycles=5 \ ordered_solvent=true refinement.input.xray_data.labels="F,SIGF" \ output.prefix=myprotein
- as Fred mentioned, SA is typically done at the beginning of structure refinement when the structure is supposed to have gross errors. At final stages of refinement SA may be rather harmful than helpful. - you did not define TLS groups which means the whole molecule is treated as one TLS group. Most of the time it is suboptimal. Use phenix.find_tls_groups to determine TLS groups automatically. You need to re-run this command every so often to update TLS selections as your model improves. - what is the resolution? Typically, refinement strategy is a function of both: data quality (resolution & completeness) and current model quality. This may significantly change refinement strategy. - customization for SA seems a bit odd to me (cool_rate=10 & start_temperature=1000). - water picking should probably be not run at early stages of refinement if you do not use fix_rotamers option (if you use fix_rotamers then it is safer to use ordered_solvent=true).
6,Chage the strategy line to strategy=individual_adp adp.individual.isotropic=all \ to remove the ANISOU lines in pdb file.
This seems TOTALLY WRONG. To understand why, see: http://www.phenix-online.org/presentations/latest/pavel_validation.pdf http://www.phenix-online.org/presentations/latest/pavel_refinement_general.p...
Any suggestion for this workflow ? or how do you always deal with the similar case ? cause I'm new about this field,maybe I did something stupid.
See above.
Ps, the value of Rwork and Rfree are too close, like, Final: r_work = 0.1741 r_free = 0.1817 bonds = 0.036 angles = 1.818 Is that reasonable ?
Pages #36-42 here: http://www.phenix-online.org/presentations/latest/pavel_validation.pdf contain the answer to this question. Also, you may want to run AutoBuild (phenix.autobuild) to automatically rebuild your model which should reduce the amount of manual model building. Cheers, Pavel.
On Mon, May 30, 2011 at 6:31 PM, ChenTiantian
1, Processing data with XDS (or HKL2000 for some of the datasets) 2,Molecule replacement with phaser. 3,Check and modify the structure residue by residue with COOT, 4,phenix.refine with the following command line:
Step 3 is probably redundant - you can usually go straight to refinement, and afterwards the maps will be much clearer and easier to correct manually. Add the rigid_body strategy if you're worried about the overall orientation of domains, and fix_rotamers=True to correct individual residues. -Nat
participants (4)
-
ChenTiantian -
Nathaniel Echols -
Pavel Afonine -
Vellieux Frederic