Re: [ccp4bb] Criteria for Ligand fitting
While I'm quite happy with all the responses this question has provoked there is an additional point I would like to contribute. It is not enough to say that you can interpret your map with a model based on what you expect. You have to also show that you can't interpret your map with any other reasonable model. Saying that my map is consistent with my model is a very weak statement in the absence of exclusivity. A recent example of this sort of problem can be read about at (warning: tooting my own horn) http://www.springerlink.com/content/b8h6lg138635380v/?MUD=MP Dale Tronrud On 04/23/12 21:02, Naveed A Nadvi wrote: Dear Crystallographers, We have obtained a 1.7 A dataset for a crystal harvested from crystallization drop after 2 weeks of soaking with inhibitor. The inhibitor has an aromatic ring and also an acidic tail derived from other known inhibitors. The active site hydrophobic crown had been reported to re-orient and a charged residue is known to position for forming a salt-bridge with similar ligands. When compared to apo strucutres, we can clearly see the re-orientation of these protein residues. However, there are no clear density visible for the ligand in the Fo-Fc map. Some density is visible in the 2Fo-Fc map with default settings in COOT. We were expecting co-valent modifcations between the inhbitor, co-factor and protein residues. In fact, the Fo-Fc map suggested the protein residue is no longer bonded to the co-factor (red negative density) and a green positive density is observed nearby for the protein residue. These observations, along with the extended soaking and the pre-determined potency convince us that the inhibitor is present in the complex. When I lower the threshold of the blue 2Fo-Fc map (0.0779 e/A^3; 0.2 rmsd) we can see the densities for the aromatic ring and the overall structural features. These densities were observed without the cofactor and the inhibtor in the initial MR search model. The R/Rfree for this dataset without inhibitor was 0.20/0.24 (overall Bfactor 17.4 A^2). At 50% occupancy, modeling the inhibtor showed no negative desities upon subsequent refinement. With the inhibtor, the R/Rfree was 0.18/0.22 (overall Bfactor 18.8 A^2). The temp factors of the inhibitor atoms (50% occ) were 15-26 A^2. My understanding is phase from the MR search model may influence Fo-Fc maps, and the 2Fo-Fc map minimizes phase bias. Since the inhibitor was absent from the MR search model, can these observations be used to justify the fitting of the ligand in the map? Given the low map-level used to 'see' the ligand, would this be considered noise? Can I justfiy the subsequent fall in R/Rfree and the absence of negative density upon ligand fitting as proof of correct inhibtor modeling? I would appreciate if you could comment on this issue. Or tell me that I'm dying to see the inhibitor and hence imagining things! Kind Regards, Naveed Nadvi.
Re: [ccp4bb] Criteria for Ligand fitting
Dear Naveed, From your description, I get the impression that indeed you have a partially bound inhibitor. However, I do have some comments: At 1.7 Å, you should refine a group-occupancy for your inhibitor. With a partially bound inhibitor, the density in your active site will be a superposition of bound and unbound conformations and you should look if you could model both using alternative conformations and if that better explains the electron density maps. The proof of the pudding is the electron density: Does the electron density of your inhibitor after refinement look convincing? Many inhibitors are in fact slow substrates and still get converted if you incubate them long enough with the enzyme. If the inhibitor is expected to react covalently with the protein and the covalent cofactor is not covalently bound any more to the protein, this looks to me like you have incubated too long. If you did not try already, I would really recommend shorter soaks (1 hour, 1 day). Another thing to consider is to add fresh inhibitor as concentrated as possible prior to freezing. Especially if you do not add enough inhibitor to your cryprotectant solution, you may loose your inhibitor in seconds. Best regards, Herman -Original Message- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Naveed A Nadvi Sent: Tuesday, April 24, 2012 6:02 AM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Criteria for Ligand fitting Dear Crystallographers, We have obtained a 1.7 A dataset for a crystal harvested from crystallization drop after 2 weeks of soaking with inhibitor. The inhibitor has an aromatic ring and also an acidic tail derived from other known inhibitors. The active site hydrophobic crown had been reported to re-orient and a charged residue is known to position for forming a salt-bridge with similar ligands. When compared to apo strucutres, we can clearly see the re-orientation of these protein residues. However, there are no clear density visible for the ligand in the Fo-Fc map. Some density is visible in the 2Fo-Fc map with default settings in COOT. We were expecting co-valent modifcations between the inhbitor, co-factor and protein residues. In fact, the Fo-Fc map suggested the protein residue is no longer bonded to the co-factor (red negative density) and a green positive density is observed nearby for the protein residue. These observations, along with the extended soaking and the pre-determined potency convince us that the inhibitor is present in the complex. When I lower the threshold of the blue 2Fo-Fc map (0.0779 e/A^3; 0.2 rmsd) we can see the densities for the aromatic ring and the overall structural features. These densities were observed without the cofactor and the inhibtor in the initial MR search model. The R/Rfree for this dataset without inhibitor was 0.20/0.24 (overall Bfactor 17.4 A^2). At 50% occupancy, modeling the inhibtor showed no negative desities upon subsequent refinement. With the inhibtor, the R/Rfree was 0.18/0.22 (overall Bfactor 18.8 A^2). The temp factors of the inhibitor atoms (50% occ) were 15-26 A^2. My understanding is phase from the MR search model may influence Fo-Fc maps, and the 2Fo-Fc map minimizes phase bias. Since the inhibitor was absent from the MR search model, can these observations be used to justify the fitting of the ligand in the map? Given the low map-level used to 'see' the ligand, would this be considered noise? Can I justfiy the subsequent fall in R/Rfree and the absence of negative density upon ligand fitting as proof of correct inhibtor modeling? I would appreciate if you could comment on this issue. Or tell me that I'm dying to see the inhibitor and hence imagining things! Kind Regards, Naveed Nadvi.
Re: [ccp4bb] Criteria for Ligand fitting
Naveed, From your description, it appears as if some covalent changes have occurred in the 2 weeks of soaking. Perhaps your inhibitor is getting turned over by the enzyme ? Also, if you don't have your inhibitor in the cryo, you could be soaking it out prior to data collection, which may explain the weak density. Good luck, Jay -Original Message- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Naveed A Nadvi Sent: Tuesday, April 24, 2012 12:02 AM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Criteria for Ligand fitting Dear Crystallographers, We have obtained a 1.7 A dataset for a crystal harvested from crystallization drop after 2 weeks of soaking with inhibitor. The inhibitor has an aromatic ring and also an acidic tail derived from other known inhibitors. The active site hydrophobic crown had been reported to re-orient and a charged residue is known to position for forming a salt-bridge with similar ligands. When compared to apo strucutres, we can clearly see the re-orientation of these protein residues. However, there are no clear density visible for the ligand in the Fo-Fc map. Some density is visible in the 2Fo-Fc map with default settings in COOT. We were expecting co-valent modifcations between the inhbitor, co-factor and protein residues. In fact, the Fo-Fc map suggested the protein residue is no longer bonded to the co-factor (red negative density) and a green positive density is observed nearby for the protein residue. These observations, along with the extended soaking and the pre-determined potency convince us that the inhibitor is present in the complex. When I lower the threshold of the blue 2Fo-Fc map (0.0779 e/A^3; 0.2 rmsd) we can see the densities for the aromatic ring and the overall structural features. These densities were observed without the cofactor and the inhibtor in the initial MR search model. The R/Rfree for this dataset without inhibitor was 0.20/0.24 (overall Bfactor 17.4 A^2). At 50% occupancy, modeling the inhibtor showed no negative desities upon subsequent refinement. With the inhibtor, the R/Rfree was 0.18/0.22 (overall Bfactor 18.8 A^2). The temp factors of the inhibitor atoms (50% occ) were 15-26 A^2. My understanding is phase from the MR search model may influence Fo-Fc maps, and the 2Fo-Fc map minimizes phase bias. Since the inhibitor was absent from the MR search model, can these observations be used to justify the fitting of the ligand in the map? Given the low map-level used to 'see' the ligand, would this be considered noise? Can I justfiy the subsequent fall in R/Rfree and the absence of negative density upon ligand fitting as proof of correct inhibtor modeling? I would appreciate if you could comment on this issue. Or tell me that I'm dying to see the inhibitor and hence imagining things! Kind Regards, Naveed Nadvi.
Re: [ccp4bb] Criteria for Ligand fitting
Interesting. One of the most helpful ways of ascertaining whether you do have a bound ligand (or, as Jay suggests, bound but modified ligand/protein) is, of course, to compare the electron density you see in the complex with the corresponding unliganded (let’s say apo) complex (in your case, the crystals before soaking). One can calculate m|Fo(soak)|-m|Fo(apo)| maps (i.e. in reciprocal space). Separate maps can be generated using phases from both the soaked and apo structure, i.e. (m|Fo(soak)| -m|Fo(apo)|) exp–iphi(soak) and (m|Fo(soak)| -m|Fo(apo|)) exp –iphi(apo). These can be compared to check the level of phase bias in the maps. Of course omitting the ligand or active site waters may further reduce phase bias and there are other approaches to reducing phase bias that can be incorporated. To go a step further, you can even calculate a difference map, in real space, where you subtract the electron density of your (m|Fo(soak)| -m|Fo(apo)|) exp –iphi(apo) map from your (m|Fo(soak)| -m|Fo(apo)|)exp –iphi(soak) map; this will help show any phase bias. This exercises the mind when it comes to remembering what a positive peak and what a negative means (and exercised my mind when trying to describe these maps correctly in ascii - which I may well have got wrong!) Should the cells of your soaked and apo structures differ my more than a percent or so in dimensions or more than a degree or so in angles, you may be restricted to working solely in real space (a high Rmerge for Fo(apo) to Fo(soak) will tell you whether the structures are too isomorphous fro reciprocal space comparison – perhaps this is the case as you mention you used MR to solve the structure?). In this case, you can still generate an (m|Fo(apo)|) exp -iphi(apo) map and subtract it, in real space, from an (m|Fo(soak)|) exp i-phi(soak) map, effectively giving you a rho o(soak) - rho o(apo) map. It’s important to look at all these maps to get a clear picture of the differences your processed data show, as opposed to the differences which result from your two sets of phases. It’s worth noting, you should not expect to see strong peaks in these maps in positions where water molecules are displaced by your bound ligand. 1.7A should be plenty high enough resolution for this and all can be done easily in CCP4. Don't be put off by what seems a tedious set of maps to calculate, these can be quite instructional and generating a master mtz file containing h, k, l, fo(soak), fo(apo), fc(soak), fc(apo), sigf(soak), sigf(apo), phi(soak), phi(apo) will allow you to pick and choose maps as you see fit. Let us know what you find, Colin -Original Message- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Pandit, Jayvardhan Sent: 24 April 2012 14:56 To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Criteria for Ligand fitting Naveed, From your description, it appears as if some covalent changes have occurred in the 2 weeks of soaking. Perhaps your inhibitor is getting turned over by the enzyme ? Also, if you don't have your inhibitor in the cryo, you could be soaking it out prior to data collection, which may explain the weak density. Good luck, Jay -Original Message- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Naveed A Nadvi Sent: Tuesday, April 24, 2012 12:02 AM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Criteria for Ligand fitting Dear Crystallographers, We have obtained a 1.7 A dataset for a crystal harvested from crystallization drop after 2 weeks of soaking with inhibitor. The inhibitor has an aromatic ring and also an acidic tail derived from other known inhibitors. The active site hydrophobic crown had been reported to re-orient and a charged residue is known to position for forming a salt-bridge with similar ligands. When compared to apo strucutres, we can clearly see the re-orientation of these protein residues. However, there are no clear density visible for the ligand in the Fo-Fc map. Some density is visible in the 2Fo-Fc map with default settings in COOT. We were expecting co-valent modifcations between the inhbitor, co-factor and protein residues. In fact, the Fo-Fc map suggested the protein residue is no longer bonded to the co-factor (red negative density) and a green positive density is observed nearby for the protein residue. These observations, along with the extended soaking and the pre-determined potency convince us that the inhibitor is present in the complex. When I lower the threshold of the blue 2Fo-Fc map (0.0779 e/A^3; 0.2 rmsd) we can see the densities for the aromatic ring and the overall structural features. These densities were observed without the cofactor and the inhibtor in the initial MR search model. The R/Rfree for this dataset without inhibitor was 0.20/0.24 (overall Bfactor 17.4 A^2). At 50% occupancy, modeling the inhibtor showed no negative desities upon subsequent refinement
Re: [ccp4bb] Criteria for Ligand fitting
Naveed, You mention: The active site hydrophobic crown had been reported to re-orient and a charged residue is known to position for forming a salt-bridge with similar ligands. When you induce structural changes on ligand binding, lattice forces that stabilize one particular conformation may be able to fight against ligand binding. The result is not always absence of ligand. You will get a statistical sampling of various viable solutions but the answer in not satisfactory. You should try co-crystallization instead of soaking! For hydrophobic ligands the problem is different. (I assume that the acidic tail gives your ligand excellent water solubility so I will not mention what to do in that case). Enrico. -Original Message- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Naveed A Nadvi Sent: Tuesday, April 24, 2012 12:02 AM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Criteria for Ligand fitting Dear Crystallographers, We have obtained a 1.7 A dataset for a crystal harvested from crystallization drop after 2 weeks of soaking with inhibitor. The inhibitor has an aromatic ring and also an acidic tail derived from other known inhibitors. The active site hydrophobic crown had been reported to re-orient and a charged residue is known to position for forming a salt-bridge with similar ligands. When compared to apo strucutres, we can clearly see the re-orientation of these protein residues. However, there are no clear density visible for the ligand in the Fo-Fc map. Some density is visible in the 2Fo-Fc map with default settings in COOT. We were expecting co-valent modifcations between the inhbitor, co-factor and protein residues. In fact, the Fo-Fc map suggested the protein residue is no longer bonded to the co-factor (red negative density) and a green positive density is observed nearby for the protein residue. These observations, along with the extended soaking and the pre-determined potency convince us that the inhibitor is present in the complex. When I lower the threshold of the blue 2Fo-Fc map (0.0779 e/A^3; 0.2 rmsd) we can see the densities for the aromatic ring and the overall structural features. These densities were observed without the cofactor and the inhibtor in the initial MR search model. The R/Rfree for this dataset without inhibitor was 0.20/0.24 (overall Bfactor 17.4 A^2). At 50% occupancy, modeling the inhibtor showed no negative desities upon subsequent refinement. With the inhibtor, the R/Rfree was 0.18/0.22 (overall Bfactor 18.8 A^2). The temp factors of the inhibitor atoms (50% occ) were 15-26 A^2. My understanding is phase from the MR search model may influence Fo-Fc maps, and the 2Fo-Fc map minimizes phase bias. Since the inhibitor was absent from the MR search model, can these observations be used to justify the fitting of the ligand in the map? Given the low map-level used to 'see' the ligand, would this be considered noise? Can I justfiy the subsequent fall in R/Rfree and the absence of negative density upon ligand fitting as proof of correct inhibtor modeling? I would appreciate if you could comment on this issue. Or tell me that I'm dying to see the inhibitor and hence imagining things! Kind Regards, Naveed Nadvi. -- Enrico A. Stura D.Phil. (Oxon) ,Tel: 33 (0)1 69 08 4302 Office Room 19, Bat.152, Tel: 33 (0)1 69 08 9449Lab LTMB, SIMOPRO, IBiTec-S, CE Saclay, 91191 Gif-sur-Yvette, FRANCE http://www-dsv.cea.fr/en/institutes/institute-of-biology-and-technology-saclay-ibitec-s/unites-de-recherche/department-of-molecular-engineering-of-proteins-simopro/molecular-toxinology-and-biotechnology-laboratory-ltmb/crystallogenesis-e.-stura http://www.chem.gla.ac.uk/protein/mirror/stura/index2.html e-mail: est...@cea.fr Fax: 33 (0)1 69 08 90 71
Re: [ccp4bb] Criteria for Ligand fitting
Dear Crystallographers, Thank you all for responding! I will try to respond to the suggestions collectively. I did however have some questions for some of these suggestions... @Herman Schreuder: I have performed refinement with alt conform as you suggested. I am not sure how to do group-occupancy of the inhibitor. Is that possible in Refmac? @Colin Groom: Your suggestion for using maps from different dataset is very interesting and I am excited to chase this up! But I am not very competent with the exact terms and this procedure in general. Should I do this using FFT? I have the usual master MTZ from SCALA but not the other labels yet. How can I create them? I would appreciate if you could suggest to me which CCP4 MODULES (and their documentation) I should look at! @David Mueller: apologies I wasn’t sure what details of the hydrophobic crown you asked from me... There were about 7 hydrophobic residues with Trp, Tyr and Phe. Regarding the covalent modification, apo crystals are present with a protein residue chemically bonded to the co-factor. Our inhibitor (NOT derived from substrate) was expected to displace the protein residue and chemically bind itself to the co-factor. We had observed this somewhat in the density map as a proof of inhibitor binding. There was a significant red density (COOT) where the residue and co-factor forms the bond. However, when trying to bond the co-factor and the inhibitor, that too was unfavourable. So we have left the three components un-bonded at the moment. I have tried alternate conformations for all the three components (protein residue, co-factor, inhibitor). At the moment I am playing with 50% occupancy to keep things simple. I defined the 'A' conformers when inhibitor is present and 'B' for the apo structure. (Question1: Can Refmac refine separately based on these two possibilities?). After incorporating the inhibitor in the structure and refinement of all components at 50% occupancy, we have not seen any negative density. The 2Fo-Fc (blue) map fits extremely well after refinement. But I am a little paranoid if it is due to phase bias introduced from the current model with inhibitor. Q2: How can I deduce the percentage occupancy of alt conformers? Should I look at the bfactors as a guide somehow? I am ultimately concerned whether it is acceptable to publish this enzyme-inhibitor complex given the lower occupancy and poor initial density. We have tried a large number of experiments to optimize co-crystallization by varying all common strategies: seeding, soaking, co-crystallizing and crystallization of inhibited protein. The complex crystals were finally obtained from co-crystallization drops that were seeded with apo crystal nuclei. So these crystals were more like co-crystallization rather than pure soaking. The final inhibitor concentration in the co-crystallization drop was 500-fold more concentrated than the IC50. The cryo buffer also contained same concentration of inh as mother liquor. Thank you again for your comments (I love CCP4BB)! Naveed
[ccp4bb] Criteria for Ligand fitting
Dear Crystallographers, We have obtained a 1.7 A dataset for a crystal harvested from crystallization drop after 2 weeks of soaking with inhibitor. The inhibitor has an aromatic ring and also an acidic tail derived from other known inhibitors. The active site hydrophobic crown had been reported to re-orient and a charged residue is known to position for forming a salt-bridge with similar ligands. When compared to apo strucutres, we can clearly see the re-orientation of these protein residues. However, there are no clear density visible for the ligand in the Fo-Fc map. Some density is visible in the 2Fo-Fc map with default settings in COOT. We were expecting co-valent modifcations between the inhbitor, co-factor and protein residues. In fact, the Fo-Fc map suggested the protein residue is no longer bonded to the co-factor (red negative density) and a green positive density is observed nearby for the protein residue. These observations, along with the extended soaking and the pre-determined potency convince us that the inhibitor is present in the complex. When I lower the threshold of the blue 2Fo-Fc map (0.0779 e/A^3; 0.2 rmsd) we can see the densities for the aromatic ring and the overall structural features. These densities were observed without the cofactor and the inhibtor in the initial MR search model. The R/Rfree for this dataset without inhibitor was 0.20/0.24 (overall Bfactor 17.4 A^2). At 50% occupancy, modeling the inhibtor showed no negative desities upon subsequent refinement. With the inhibtor, the R/Rfree was 0.18/0.22 (overall Bfactor 18.8 A^2). The temp factors of the inhibitor atoms (50% occ) were 15-26 A^2. My understanding is phase from the MR search model may influence Fo-Fc maps, and the 2Fo-Fc map minimizes phase bias. Since the inhibitor was absent from the MR search model, can these observations be used to justify the fitting of the ligand in the map? Given the low map-level used to 'see' the ligand, would this be considered noise? Can I justfiy the subsequent fall in R/Rfree and the absence of negative density upon ligand fitting as proof of correct inhibtor modeling? I would appreciate if you could comment on this issue. Or tell me that I'm dying to see the inhibitor and hence imagining things! Kind Regards, Naveed Nadvi.
