Re: [ccp4bb] MR on low resolution soaking data.
The easy Q first: Wilson plot B values are very unreliable for 4A data - b=20 is almost certainly wrong, but until you have a model to refine it is hard to get a proper estimate. Q2: With a decent model the ligand should show up as a blob, even at this resolution. You might have trouble fitting it, but at least you would know whether it had bound or not. Q3: Why the MR doesnt work - I cant answer this.. Check the data for any serious problems - missing strong intensities can mislead. Is the spce group certain? Is there any relationship between the P212121 and F222 cells? Eleanor w it ang li wrote: Dear colleagues, We are now trying to soak some ligands into a protein, which is about 60kd in size and the structure has been solved before. But the molecular replacement cannot give a right solution. Below is some contrast of the data: Native 2A P212121 monomer Soaked4A F222 monomer (more than 70% solvent) or dimer(more possible) I wonder if it is possible to find the ligand in the case of such low resolution, provided the ligand is not so small. What facts could probably lead to the failure of MR? Molrep gave a model of monomer but the rfree is as high as 0.7, while phaser could get no result. I tried phenix.explore_metric_symmetry to find the two spacegroups are not compatible, and the Rmerge of the data seems reasonable. One more question is: the wilson B of the data is lower than 20 from ccp4. Is it common for a 4A data? Since I donnot have the experience of handling this low resolution data yet. By the way, any suggestions about refinement methods in low resolution will be appreciated! Best wishes Yang
Re: [ccp4bb] MR on low resolution soaking data.
Dear All, Thank you for your help! There do have something needed to be checked carefully, as you suggested. Peter Zwart showed me the right way of explore_symmetry_metric, which indicated there is some relationship between the two unit cells. I hope it can explain the strange wilsonB and failure of MR. I will try to reprocess the data later to make sure. Best wishes Yang On Tue, Jun 8, 2010 at 1:57 AM, Eleanor Dodson c...@ysbl.york.ac.uk wrote: The easy Q first: Wilson plot B values are very unreliable for 4A data - b=20 is almost certainly wrong, but until you have a model to refine it is hard to get a proper estimate. Q2: With a decent model the ligand should show up as a blob, even at this resolution. You might have trouble fitting it, but at least you would know whether it had bound or not. Q3: Why the MR doesnt work - I cant answer this.. Check the data for any serious problems - missing strong intensities can mislead. Is the spce group certain? Is there any relationship between the P212121 and F222 cells? Eleanor w it ang li wrote: Dear colleagues, We are now trying to soak some ligands into a protein, which is about 60kd in size and the structure has been solved before. But the molecular replacement cannot give a right solution. Below is some contrast of the data: Native 2A P212121 monomer Soaked4A F222 monomer (more than 70% solvent) or dimer(more possible) I wonder if it is possible to find the ligand in the case of such low resolution, provided the ligand is not so small. What facts could probably lead to the failure of MR? Molrep gave a model of monomer but the rfree is as high as 0.7, while phaser could get no result. I tried phenix.explore_metric_symmetry to find the two spacegroups are not compatible, and the Rmerge of the data seems reasonable. One more question is: the wilson B of the data is lower than 20 from ccp4. Is it common for a 4A data? Since I donnot have the experience of handling this low resolution data yet. By the way, any suggestions about refinement methods in low resolution will be appreciated! Best wishes Yang
Re: [ccp4bb] MR on low resolution soaking data.
since the ligands bare being soaked into a known solved structure, why is MR necessary? Why not just start out with some rigid body refinement of the native structure to account for possible slight differences in the cell dimensions? -- === All Things Serve the Beam === David J. Schuller modern man in a post-modern world MacCHESS, Cornell University schul...@cornell.edu On 06/07/10 15:17, yang li wrote: Dear colleagues, We are now trying to soak some ligands into a protein, which is about 60kd in size and the structure has been solved before. But the molecular replacement cannot give a right solution. Below is some contrast of the data: Native 2A P212121 monomer Soaked4A F222 monomer (more than 70% solvent) or dimer(more possible) I wonder if it is possible to find the ligand in the case of such low resolution, provided the ligand is not so small. What facts could probably lead to the failure of MR? Molrep gave a model of monomer but the rfree is as high as 0.7, while phaser could get no result. I tried phenix.explore_metric_symmetry to find the two spacegroups are not compatible, and the Rmerge of the data seems reasonable. One more question is: the wilson B of the data is lower than 20 from ccp4. Is it common for a 4A data? Since I donnot have the experience of handling this low resolution data yet. By the way, any suggestions about refinement methods in low resolution will be appreciated! Best wishes Yang
Re: [ccp4bb] MR on low resolution soaking data.
You haven't given much detail to work with so I can only guess about your problem. A Wilson B of 20 for a 4 A data set is ridiculous, but the uncertainty in the Wilson B calculation at 4 A is enormous, so what might be a more reasonable statement would be to say your Wilson B calculates to 20 +_ 300 A^2 and the true value would be in that range. I don't think a precise Wilson B is important for MR so I wouldn't worry about it. An R value of .7 after MR is very large. Its size implies a systematic problem with your model - I would be looking for a second monomer. You haven't said anything about the structure of your monomer. Often a ligand will bind in the cleft between two domains, and the domains move relative to each other upon binding. You may have to perform separate searches for each domain or construct a range of trial models with different angles between the domains. Don't worry about the ligand until you solve the protein structure. Whether you see it in the end will depend on how big it is and how good your 4 A data are. Of course, it's possible that it doesn't bind at all. Dale Tronrud On 06/07/10 12:17, yang li wrote: Dear colleagues, We are now trying to soak some ligands into a protein, which is about 60kd in size and the structure has been solved before. But the molecular replacement cannot give a right solution. Below is some contrast of the data: Native 2A P212121 monomer Soaked4A F222 monomer (more than 70% solvent) or dimer(more possible) I wonder if it is possible to find the ligand in the case of such low resolution, provided the ligand is not so small. What facts could probably lead to the failure of MR? Molrep gave a model of monomer but the rfree is as high as 0.7, while phaser could get no result. I tried phenix.explore_metric_symmetry to find the two spacegroups are not compatible, and the Rmerge of the data seems reasonable. One more question is: the wilson B of the data is lower than 20 from ccp4. Is it common for a 4A data? Since I donnot have the experience of handling this low resolution data yet. By the way, any suggestions about refinement methods in low resolution will be appreciated! Best wishes Yang
