Dear Shramana, I don’t know why MR is not working well with individual monomers, if you have a refined model of the same thing. Is the resolution particularly low? However, the point you make here that perhaps wasn’t in the previous email is that the space group and cell dimensions are the same. Have you just tried rigid-body refinement with the solved structure? Perhaps this is a space group (e.g. trigonal) where there are alternative ways to index the data. If it is, then you should see if there’s a way to make this data set agree with the solved one using some alternative indexing, and then do rigid-body refinement. Of course, it is in principle possible that it’s just an accident that the space group and cell dimensions are the same, but the crystal forms are actually different, but this is less likely to be the case. Randy Read ----- Randy J. Read Department of Haematology, University of Cambridge Cambridge Institute for Medical Research Tel: +44 1223 336500 The Keith Peters Building Fax: +44 1223 336827 Hills Road E-mail: [email protected] Cambridge CB2 0XY, U.K. www-structmed.cimr.cam.ac.uk

On 9 Apr 2020, at 16:35, Shramana Chatterjee wrote:

Dear Randy,

Thank you for your suggestion. I should mention that I have tried with single copy, best fitted dimer as well with the whole structure (on different set of runs) but in al the cases PhaserMR is unable to produce identical number of copies comparable with the solved structure although the cell dimension and space group is same with the solved one. What can be the possible reason and how to get rid of that?

Thanks again for your reply.

On Thu, Apr 9, 2020 at 4:30 AM Randy Read wrote: Dear Shramana,

I may be reading this incorrectly, but it sounds like you’re providing the entire PDB file from the solved model to Phaser to solve the other structure by MR. You need to edit the PDB file before using it as a model so that what you have is a sensible model for what is in the other crystal form, because Phaser will just take the whole thing as a single rigid body. Given the 100% sequence identity, I would use a single copy as a model and search for the appropriate number of copies (perhaps 4, from what you say). If the model was more distant, then it might be worth looking at it to see if it could plausibly be a dimer or tetramer in solution, and then you could use a higher-order structure. But MR with identical models can usually place a reasonable number of copies independently, and then you don’t have to make any assumptions about quaternary structure.

Best wishes,

Randy Read

----- Randy J. Read Department of Haematology, University of Cambridge Cambridge Institute for Medical Research Tel: +44 1223 336500 The Keith Peters Building Fax: +44 1223 336827 Hills Road E-mail: [email protected] Cambridge CB2 0XY, U.K. www-structmed.cimr.cam.ac.uk

...

On 9 Apr 2020, at 04:43, Shramana Chatterjee wrote:

Hi,

I am trying to solve a structure using a data solved by SAD as an reference (ensemble in phaser). The structure I am trying to solve has 100% sequence identity with the solved one. The problem that I am facing during Phaser MR is that, in the solved structure there are 6 molecules in the ASU although I am getting maximum 4 molecules in the ASU and also Rfree is around 0.49 just after the phaser. Phaser is showing a good values of LLG (>500) and TFZ (>8).

It would be very helpful if I get any suggestion about the above-mentioned problem.

Thank you in advance.

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Using only rigid body refinement worked well with Rfree ~0.29. Can it produce a bias map meaning is it possible to miss some important part (like solvent or ligand) due to bias-ness of the map? On Thu, Apr 9, 2020 at 3:07 PM Shramana Chatterjee wrote:

Thanks a lot. The resolution is ~3ang. for both the cases and the space group is tetragonal. I haven't tried yet just the rigid body refinement.I will start from only rigid body refinement, it may produce an identical one.

Thanks and regards, Shramana.

On 9 Apr 2020 2:29 pm, "Randy Read" wrote:

Dear Shramana,

I don’t know why MR is not working well with individual monomers, if you have a refined model of the same thing. Is the resolution particularly low?

However, the point you make here that perhaps wasn’t in the previous email is that the space group and cell dimensions are the same. Have you just tried rigid-body refinement with the solved structure? Perhaps this is a space group (e.g. trigonal) where there are alternative ways to index the data. If it is, then you should see if there’s a way to make this data set agree with the solved one using some alternative indexing, and then do rigid-body refinement.

Of course, it is in principle possible that it’s just an accident that the space group and cell dimensions are the same, but the crystal forms are actually different, but this is less likely to be the case.

Randy Read

----- Randy J. Read Department of Haematology, University of Cambridge Cambridge Institute for Medical Research Tel: +44 1223 336500 The Keith Peters Building Fax: +44 1223 336827 Hills Road E-mail: [email protected] Cambridge CB2 0XY, U.K. www-structmed.cimr.cam.ac.uk

...

On 9 Apr 2020, at 16:35, Shramana Chatterjee wrote:

Dear Randy,

Thank you for your suggestion. I should mention that I have tried with single copy, best fitted dimer as well with the whole structure (on different set of runs) but in al the cases PhaserMR is unable to produce identical number of copies comparable with the solved structure although the cell dimension and space group is same with the solved one. What can be the possible reason and how to get rid of that?

Thanks again for your reply.

On Thu, Apr 9, 2020 at 4:30 AM Randy Read wrote: Dear Shramana,

I may be reading this incorrectly, but it sounds like you’re providing the entire PDB file from the solved model to Phaser to solve the other structure by MR. You need to edit the PDB file before using it as a model so that what you have is a sensible model for what is in the other crystal form, because Phaser will just take the whole thing as a single rigid body. Given the 100% sequence identity, I would use a single copy as a model and search for the appropriate number of copies (perhaps 4, from what you say). If the model was more distant, then it might be worth looking at it to see if it could plausibly be a dimer or tetramer in solution, and then you could use a higher-order structure. But MR with identical models can usually place a reasonable number of copies independently, and then you don’t have to make any assumptions about quaternary structure.

Best wishes,

Randy Read

----- Randy J. Read Department of Haematology, University of Cambridge Cambridge Institute for Medical Research Tel: +44 1223 336500 The Keith Peters Building Fax: +44 1223 336827 Hills Road E-mail: [email protected] Cambridge CB2 0XY, U.K. www-structmed.cimr.cam.ac.uk

...

On 9 Apr 2020, at 04:43, Shramana Chatterjee wrote:

Hi,

I am trying to solve a structure using a data solved by SAD as an reference (ensemble in phaser). The structure I am trying to solve has 100% sequence identity with the solved one. The problem that I am facing during Phaser MR is that, in the solved structure there are 6 molecules in the ASU although I am getting maximum 4 molecules in the ASU and also Rfree is around 0.49 just after the phaser. Phaser is showing a good values of LLG (>500) and TFZ (>8).

It would be very helpful if I get any suggestion about the above-mentioned problem.

Thank you in advance.

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