First do the Le Bail fitting as per normalRefer to the tutorials described in the GSAS Hints/Resources area. Note the following screen dump that depending on the data and its source (in this case from ESRF), only four parameters are needed here (using Pseudo-Voight with FCJ asymmetry correction) - GV width, LX shape and the two asymmetry parameters.
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Set the phase type as macromolecularAssuming you have not done so already, set the phase type to macromolecular. You may have to start using the traditional GSAS interface at this point if EXPGUI complains it cannot handle macromolecular GSAS files.
Y P P M 1 !modify phase type for phase 1 D !macromolecular structure
Note from Bob von Dreele: One important point is that in a multiphase mixture the protein must be phase #1. That is the only one that can be "macromolecular". Current limits are 500 atoms for "ordinary" phases and 5000 atoms for macromolecular ones.
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Insert the PDB File atom co-ordinates
Y L A I B (input the PDB file and answer the questions - go for Matrix (not Cell))
Note from Bob von Dreele: >Would you normally also import the water/HOH molecules, >disordered atoms and hydrogens? >(though the PDB file I seem to have does not include the >hydrogens) "Normally" not for powder data. Single crystal protein folks seem only to add waters with higher resolution data (dmin ~ 2.5A or better). Some PDB files do have H-atoms. I change the UISO's for all the atoms to 0.3 which is about average for protein atoms. Powder data isn't good enough to do UISO's in a refinement even when all constrained to be identical.
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Set the Solvent scattering
Y L !Least Squares Setup F !Edit Atom form factors S !Edit solvent Scattering info C !Change solvent scattering factors 5 1 !Set Asolvent and Usolvent to 5 and 1 (As Bob von Dreele mentions, these can be refined)
Note from Bob von Dreele: >Next query if there is the tolerance for it - how do you >enter the "solvent scattering"? Solvent scattering is set in the form factor menu under "S". Start with A=5 & U=1. These can be refined.
Subject: GSAS paramenters From: bond@img.cryst.bbk.ac.uk Date: Wed, 18 Dec 2002 16:32:28 +0000 (GMT) To: RIETVELD_L@ill.fr Hello, I am working powder diffraction of proteins. Recently there has been a lot of help on the list with regard to this. In 1 message, Bob suggests setting solvent scattering as follows: set Asolvent to 5 and Usolvent to 1. My question is what is Asolvent? and what is Usolvent? Is this ordered solvent intrinsic within the crystal structure and extrinsic solvent? Thanks for your help & happy Christmas to all! Cheers john b
Subject: Re: GSAS paramenters From: "Bob Von Dreele" [vondreele@lanl.gov] To: rietveld_l@ill.fr Date: Wed, 18 Dec 2002 09:46:27 -0800 Hi John, Happy to see someone is trying this. The A & U (solvent) are coefficients for a Babinet's Principle modification to the scattering factors. See GSAS Manual for function details. Obviously this models contribution from "unstructured" water within the protein crystal structure at very low scattering angles. Localized water molecules might be found in difference density maps. I've seen'em but have chosen to ignore them so far. Bob
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Set the Least Squares Controls as per advice from Bob von Dreele
Y L !Least Squares Setup B 300 !band width of 300 D 1.2 !Marquardt damping factor of 1.2 V 1.7 !Set the convergence criterial P !Select options for output listing S !Toggle print of summary shift/esd data after last cycle
Note from Bob von Dreele: >And are there any other >settings that are important - that could be missed by someone more >used to refining inorganics? Yes, one needs to go to the least squares controls menu & pick a band width for the LS matrix; I use 300 for my work & I know Jon Wright has used 50. Also pick a Marquardt damping factor; try 1.20 to start & adjust as needed. Try to make small if possible. Finally, change the convergence factor; you enter this as the log, so -2.0 is the default of 0.01. For a 3000 parameter protein refinement 1.7 (~50 for sum shift/esd) is more reasonable. I also set the print option for shift/esd summary table and do 9 cycles at a time.
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While in the relevant menus, apply all the macro files in the c:\gsas\macros directory (except c60,mac)
Run angles.mac for setting the bond angle restraintsY L !Least Squares Setup S !Edit soft constraints data A !Edit bond angle restraints @r angles.mac
Run bonds.mac for setting the bond length restraintsY L !Least Squares Setup S !Edit soft constraints data D !Edit bond length restraints @r bonds.mac
Run chiral.mac for setting chiral volume restraintsY L !Least Squares Setup S !Edit soft constraints data K !Edit chiral volume restraints @r chiral.mac
Run planes.mac for setting chiral volume restraintsY L !Least Squares Setup S !Edit soft constraints data P !Edit planar restraints @r planes.mac
Run Rama.mac for setting Phi/Psi psuedopotential restraintsY L !Least Squares Setup S !Edit soft constraints data R !Edit Phi/Psi psuedopotential restraints @r Rama.mac
Run Torsion.mac for setting Torsion angle restraintsY L !Least Squares Setup S !Edit soft constraints data T !Edit Torsion angle restraints @r Torsion.mac
Note from Bob von Dreele: >I take it after the structure is happily imported, it is >just a matter of running all the restraints macros in >the c:\gsas\macros directory (except C60) Yes, these will go through the protein structure & build all the needed restraints. This is pretty quick. All the amino acids in the protein must be one of the standard 20. You'll have to do other molecules (ligands, etc.) by hand. A few other pointers: I use Swiss PDB Viewer to look at the resulting crystal structure. It reads PDB files - these are made by gsas2pdb. SPDBV can then be used to fix up "bad" bits in the structure - "mutate" side chains, etc., save the result and GSAS can then read back in the new coordinates. One careful point is to change all the UISO's back to 0.3 (or whatever you pick) as SPDBV tends to change them to nonsense if the "mutate" is used.
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Now read the E-mails on the subject via the Rietveld Users' Mailing List to get a good feel for what is going and some issues you can expect when doing this type of refinementDate: Mon, 02 Dec 2002 17:29:27 +0000 To: Rietveld_l@ill.fr From: Lachlan Cranswick [l.m.d.cranswick@dl.ac.uk] Subject: RIET: GSAS query on importing Protein PDB atom positions? For importing a protein PDB file into GSAS via EXPEDT. How is this done? There is a menu option for importing BNL PDB format in EXPEDT via K L A I B - but there seems to be no prompt for the filename - and the following error is given if you try using the I B menu option? Give atom editing command (>,$,I,S,X) >i Command structure for inserting an atom I s - enter atom with sequence number "s" I N - enter atom with next sequence number I B - Read atoms from BNL PDB format I R - Read atoms from non-GSAS file or from another EXP file Phase No. 1; Phase has 0 atoms; Title: blah Give atom editing command (>,$,I,S,X) >i B PDB format only allowed for protein phase type *** No atoms found for B - ERROR - *** Phase No. 1; Phase has 0 atoms; Title: blah Give atom editing command (>,$,I,S,X) > ================== If you type I R - a GSAS EXP file is prompted for. Thanks in advance, Lachlan. ----------------------- Lachlan M. D. Cranswick Collaborative Computational Project No 14 (CCP14) for Single Crystal and Powder Diffraction Birkbeck University of London and Daresbury Synchrotron Laboratory Postal Address: CCP14 - School of Crystallography, Birkbeck College, Malet Street, Bloomsbury, WC1E 7HX, London, UK Tel: (+44) 020 7631 6850 Fax: (+44) 020 7631 6803 E-mail: l.m.d.cranswick@dl.ac.uk Room: B091 WWW: http://www.ccp14.ac.uk/
Date: Mon, 02 Dec 2002 18:55:26 +0100 To: rietveld_l@ill.fr From: Jonathan WRIGHT [wright@esrf.fr] Subject: Re: RIET: GSAS query on importing Protein PDB atom positions? Lachlan, Did you toggle the phase flags to say this is macromolecule (p p m 1 d) ? The hint was in you mail ;-) >PDB format only allowed for protein phase type Although I thought I read somewhere that recent versions had this restriction removed... wishful thinking perhaps? Cheers, Jon
From: "Bob Von Dreele" [vondreele@lanl.gov] Subject: Re: RIET: GSAS query on importing Protein PDB atom positions? To: rietveld_l@ill.fr Date: Mon, 02 Dec 2002 10:44:40 -0800 Hi Lachlan, The phase type must be "macromolecular" for the "I B" option to work. Sometime I'll fix this so nonmacromolecular structures can be read too. Bob
Date: Mon, 02 Dec 2002 18:12:26 +0000 To: rietveld_l@ill.fr From: Lachlan Cranswick
From: "Bob Von Dreele" [vondreele@lanl.gov] Subject: Re: RIET: GSAS query on importing Protein PDB atom positions? To: rietveld_l@ill.fr Date: Mon, 02 Dec 2002 12:26:13 -0800 Hi Lachlan (& everyone else!), See below... Bob On Mon, 02 Dec 2002 18:12:26 +0000 Lachlan Cranswick
To: rietveld_l@ill.fr From: Jonathan WRIGHT [wright@esrf.fr] Subject: Re: RIET: GSAS query on importing Protein PDB atom positions? > >Would you normally also import the water/HOH molecules, > >disordered > >atoms and hydrogens? > >(though the PDB file I seem to have does not include the > >hydrogens) > >"Normally" not for powder data. Single crystal protein >folks seem only to add waters with higher resolution data >(dmin ~ 2.5A or better). Some PDB files do have H-atoms. I >change the UISO's for all the atoms to 0.3 which is about >average for protein atoms. Powder data isn't good enough >to do UISO's in a refinement even when all constrained to >be identical. In the case which I think Lachlan is looking at - you have a ~1.5 angstrom (=very good) single crystal structure, which matches the powder data. Importing or not the water molecules should only really impact on the solvent scattering factors, but if you want to refine the structure it will be something of a challenge to get the waters to keep to sensible positions. If you delete the the H2O/H parts of the structure then in theory you'll just put them back into the structure via the "solvent scattering", but not know exactly where they are any more. For neutron data (if you have any), it ought to make a much bigger difference, as the simplistic solvent scattering model has been reported to break down with single crystal data. Remember that refining a high resolution single crystal structure against powder data is only going to make it worse, whether it's a protein, inorganic structure or small organic molecule, assuming the sample is the same. A question for Bob, have you (or anyone else) tried fitting any single crystal datasets using GSAS? In theory it ought to be an alternative to shelx, refmac etc? Cheers, Jon
From: "Bob Von Dreele" [vondreele@lanl.gov] Subject: Re: RIET: GSAS query on importing Protein PDB atom positions? To: rietveld_l@ill.fr Date: Tue, 03 Dec 2002 08:28:20 -0800 On Tue, 03 Dec 2002 13:54:53 +0100 Jonathan WRIGHT [wright@esrf.fr] wrote: > >>>Would you normally also import the water/HOH molecules, >>>disordered >>>atoms and hydrogens? >>>(though the PDB file I seem to have does not include the >>>hydrogens) >> >>"Normally" not for powder data. Single crystal protein >>folks seem only to add waters with higher resolution data >>(dmin ~ 2.5A or better). Some PDB files do have H-atoms. >>I change the UISO's for all the atoms to 0.3 which is about >>average for protein atoms. Powder data isn't good enough >>to do UISO's in a refinement even when all constrained to >>be identical. > >In the case which I think Lachlan is looking at - you >have a ~1.5 angstrom (=very good) single crystal >structure, which matches the powder data. Importing or >not the water molecules should only really impact on the >solvent scattering factors, but if you want to refine the >structure it will be something of a challenge to get the >waters to keep to sensible positions. If you delete the >the H2O/H parts of the structure then in theory you'll >just put them back into the structure via the "solvent >scattering", but not know exactly where they are any >more. For neutron data (if you have any), it ought to >make a much bigger difference, as the simplistic solvent >scattering model has been reported to break down with >single crystal data. Remember that refining a high >resolution single crystal structure against powder data >is only going to make it worse, whether it's a protein, >inorganic structure or small organic molecule, assuming >the sample is the same. This is an interesting problem (single crystal vs powder). I've not found that a calculated powder pattern from a protein single crystal structure can actually reproduce the observed powder diffraction pattern. The discrepancies are most apparent in the low angle part (>8A d-spacing) and are quite large. Because the size of the d-spacings (10-30A) for these reflections and the magnitude of the differences, they are associated with very large scale features in the crystal structure and not just the placement of a hundred or so water molecules. Consequently, Rietveld refinement of the protein structure starting with a "good" single crystal structure will show shifts in atom positions of 1-2A. This also means that trying to do joint single crystal-powder refinements don't seem to work very well. I've often wondered if the powder material (1 micron crystals) may in fact be inherently different from the usual single crystals used for protein work (100+ micron crystals). I can't tell from comparisons of lattice parameters because the precision of the single crystal values isn't really good enough to make any useful comparison to the powder ones. Any comments? >A question for Bob, have you (or anyone else) tried >fitting any single crystal datasets using GSAS? In theory >it ought to be an alternative to shelx, refmac etc? I have. It works just fine. The PDB (www.rcsb.org) does have structure factors deposited for many of the protein structures. These can be read into GSAS in the usual way and the protein structure refined with as many restraints as one wishes. However, GSAS doesn't "do" R(free), etc. familiar to the protein folks. Results aren't any different that those obtained by conventional protein programs (TNT, O, etc.). Bob
Date: Tue, 03 Dec 2002 18:38:51 +0100 To: rietveld_l@ill.fr From: Jonathan WRIGHT [wright@esrf.fr] Subject: Re: RIET: GSAS query on importing Protein PDB atom positions? >This is an interesting problem (single crystal vs powder). >I've not found that a calculated powder pattern from a >protein single crystal structure can actually reproduce >the observed powder diffraction pattern. The discrepancies >are most apparent in the low angle part (>8A d-spacing) >and are quite large.... Any comments? For the myoglobin I found that the deposited observed structure factors match the powder pattern fairly well (but not exactly). Most of the problems were due to the dataset being incomplete below about 10 angstrom, so any comparison for those peaks was not possible. I seem to remember that the deposited data did a better job than the deposited structure for the low resolution data. There is some neutron single crystal work on myoglobin which developed a more sophisticated model for the solvent scattering than the Babinet's principle model (x-ray models were really hopeless for neutron data at low resolution). I have the impression that being lousy at low resolution is a known problem with many x-ray models of protein structure, which various people have improved by modelling the solvent in more sophisticated ways. Unfortunately it's the intensities which are easiest to resolve with a powder which are the hardest to account for! Cheers, Jon
From: "Bob Von Dreele" [vondreele@lanl.gov] Subject: Re: RIET: GSAS query on importing Protein PDB atom positions? To: rietveld_l@ill.fr Date: Tue, 03 Dec 2002 10:43:02 -0800 On Tue, 03 Dec 2002 18:38:51 +0100 Jonathan WRIGHT [wright@esrf.fr] wrote: >>This is an interesting problem (single crystal vs >>powder). >>I've not found that a calculated powder pattern from a >>protein single crystal structure can actually reproduce >>the observed powder diffraction pattern. The >>discrepancies >>are most apparent in the low angle part (>8A d-spacing) >>and are quite large.... Any comments? > >For the myoglobin I found that the deposited observed >structure factors match the powder pattern fairly well >(but not exactly). Most of the problems were due to the >dataset being incomplete below about 10 angstrom, so any >comparison for those peaks was not possible. I seem to >remember that the deposited data did a better job than >the deposited structure for the low resolution data. >There is some neutron single crystal work on myoglobin >which developed a more sophisticated model for the >solvent scattering than the Babinet's principle model >(x-ray models were really hopeless for neutron data at >low resolution). I have the impression that being lousy >at low resolution is a known problem with many x-ray >models of protein structure, which various people have >improved by modelling the solvent in more sophisticated >ways. Unfortunately it's the intensities which are >easiest to resolve with a powder which are the hardest to >account for! Jon, It's also my understanding that these reflections are also the most difficult to measure accurately by the single crystal techniques used in protein data collection. I also wonder if the hydration models used for protein structure analysis take on the same role as anisotropic thermal parameters do in small molecule work (i.e. as a "catch all" for systematic errors!). I also see that the calculated powder pattern from a single crystal protein structure does match the powder pattern for higher 2-theta (d<6A). Bob |