Thanks, I obviously didn't read your description of your problem carefully enough! Best wishes, Randy

On 4 Mar 2019, at 01:38, Julian Esselborn wrote:

Dear Randy, thanks for your answer Randy, but unfortunately we are not just having a trimer. Rather it is a structure that in the end comes together as a dodecameric cage, but from a tri-fold crystallographic symmetry, which means a tetramer in the ASU. However, thinking in the symmetry of the cage, each monomer participates in two different 3-fold axes, such that there is no way to assemble a tetramer such that all 3-fold axes will fall either on the crystallographic 3fold or will be within the ASU.

For the moment I worked with the different alternative conformations, each close to one binding ligand at different ends of the ASU and the density is good enough to not let them wander too far apart. I will probably in a last run fix them to one position in between and we should be good. However, not exactly the ideal solution, I guess.

yours

Julian

Am 02.03.2019 um 01:01 schrieb Randy Read:

...

Dear Julian Perhaps I’m missing something, but I’m not seeing why it should ever be particularly difficult to reduce case 3 to case 2. If the chains forming a trimer are really crystallographically unique, then you just have to shift them one at a time to get them to form a trimer within the same asymmetric unit. You also have to make sure that the associated metal atoms are all on the local 3-fold and that there’s only one copy of each (with occupancy one) and you should be fine. In case you haven’t found it, probably the easiest way to do this is in coot. Open your PDB, view it as CAs + Ligands, turn on symmetry (Draw -> Cell and symmetry -> Symmetry by Molecule -> Display as CAs, check box to show symmetry atoms) and find the trimer you want. Center on an atom in one of the chains from a symmetry copy, which you would like to have as the master copy, then Extensions -> Modelling -> Symm Shift Reference Chain Here. Do that for the other chain, then check and if necessary fix the metal ions (which may or may not have followed the chain when you were moving things around). Chaniging symmetry copies for one chain at a time should sort out your problem, if I’ve understood it correctly. Best wishes, Randy Read ----- Randy J. Read Department of Haematology, University of Cambridge Cambridge Institute for Medical Research Tel: +44 1223 336500 Wellcome Trust/MRC Building Fax: +44 1223 336827 Hills Road E-mail: [email protected] Cambridge CB2 0XY, U.K. www-structmed.cimr.cam.ac.uk

...

On 2 Mar 2019, at 01:22, Julian Esselborn wrote:

Dear community, we have a somewhat complicated problem to which I don't seem to find a solution.

We have a structure, which has a number of 3-fold and 2-fold symmetry axes in the final assembly structure. The 3-fold axes are hold together by metal atoms on the axis. However, we have three cases of these axes: 1. Symmetry axis falls onto the crystallographic symmetry axis. We can deal with this; setting metal to 0.33 occupancy and setting metal-protein distance constraints. This is a proper symmetry axis. 2. Symmetry axis doesn't fall onto a crystallographic symmetry axis, but all three monomers coming together are within the same symmetry copy of the ASU. Even easier, metal stays at occ=1 and we just set constraints to chain A, chain B, chain C. 3. The really challenging case, where the axis doesn't fall onto the symmetry axis, but the three monomers coming together are in different symmetry copies of the ASU.

Cases (2) and (3) are pseudo-symmetric in the crystallographic sense.

Usually a bit of intelligent moving around of the monomers to their crystallographic symmetry positions should push all monomers of case (3) into neighboring positions within the same ASU ending up as case (2); problem solved.

BUT: In our structure we have too many 3fold axes, such that there will always be one of them ending up as case (3). E.g. the three monomers are chains A, B, C, but they do not end up neighboring in the ASU. Rather the axis is formed by A, B' and C'' (with ' denoting different symmetry copies of the ASU). We could assign the metal to chain A with occ=1 and no metal in B and C. However, we would need to set a distance constraint from the metal to it's ligands in protein monomer B and protein monomer C. But it is only the ligand in B' and C'', which are actually close to the metal in A. The ligands in B and C are at the other end of the ASU. Is there a way to set a distance constraint such that it measures the distance to a crystallographic symmetry copy?

A different idea was to just assign an alternative position alt A, alt B and alt C to the metal, with A being close to the ligand in monomer A, B close to monomer B and C close to monomer C. That way we could make constraints. But we would need to cross fingers that the three metal atoms actually end up in the same spot once applying the crystallographic symmetry (and remember, that 3-fold axis is not constructed by applying a 3fold rotational symmetry around that axis; rather an actual crystallographic symmetry somwhere else brings them together; means the ligands with their metal atoms can actually move independently).

I'm a bit at a loss how to deal with this and would appreciate input.

Thanks a lot in advance!

all the best

Julian

---- Julian Esselborn Postdoctoral Researcher Tezcan group University of California, San Diego

_______________________________________________ phenixbb mailing list [email protected] http://phenix-online.org/mailman/listinfo/phenixbb Unsubscribe: [email protected]

-- Dr. Julian Esselborn AG Photobiotechnologie Ruhr-Universität Bochum ND 2/170 0032-27049 Universitätsstr. 150 44801 Bochum _______________________________________________ phenixbb mailing list [email protected] http://phenix-online.org/mailman/listinfo/phenixbb Unsubscribe: [email protected]

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

Thanks a lot Pavel! This was exactly what I was looking for and now I realized I should have seen that myself. Worked for one of my problems, where I just needed a distance restraint. Unfortunately I also had one, where I would have needed a planarity restraint and there is no symmetry option for those I think. So I ended up using the alternative position workaround for that one. Am 04.03.2019 um 17:53 schrieb Pavel Afonine:

Hi Julian,

perhaps you can approach this by defining custom bonds between symmetry copies, like this:

refinement.geometry_restraints.edits {   bond {     atom_selection_1 = chain A and resseq 123 and name N     atom_selection_2 = chain B and resseq 321 and name OD1     symmetry_operation = -x-1/2,y-1/2,-z+1/2     distance_ideal = 2.1     sigma = 0.02   } }

Pavel

On 3/1/19 17:22, Julian Esselborn wrote:

...

Dear community, we have a somewhat complicated problem to which I don't seem to find a solution.

We have a structure, which has a number of 3-fold and 2-fold symmetry axes in the final assembly structure. The 3-fold axes are hold together by metal atoms on the axis. However, we have three cases of these axes: 1. Symmetry axis falls onto the crystallographic symmetry axis. We can deal with this; setting metal to 0.33 occupancy and setting metal-protein distance constraints. This is a proper symmetry axis. 2. Symmetry axis doesn't fall onto a crystallographic symmetry axis, but all three monomers coming together are within the same symmetry copy of the ASU. Even easier, metal stays at occ=1 and we just set constraints to chain A, chain B, chain C. 3. The really challenging case, where the axis doesn't fall onto the symmetry axis, but the three monomers coming together are in different symmetry copies of the ASU.

Cases (2) and (3) are pseudo-symmetric in the crystallographic sense.

Usually a bit of intelligent moving around of the monomers to their crystallographic symmetry positions should push all monomers of case (3) into neighboring positions within the same ASU ending up as case (2); problem solved.

BUT: In our structure we have too many 3fold axes, such that there will always be one of them ending up as case (3). E.g. the three monomers are chains A, B, C, but they do not end up neighboring in the ASU. Rather the axis is formed by A, B' and C'' (with ' denoting different symmetry copies of the ASU). We could assign the metal to chain A with occ=1 and no metal in B and C. However, we would need to set a distance constraint from the metal to it's ligands in protein monomer B and protein monomer C. But it is only the ligand in B' and C'', which are actually close to the metal in A. The ligands in B and C are at the other end of the ASU. Is there a way to set a distance constraint such that it measures the distance to a crystallographic symmetry copy?

A different idea was to just assign an alternative position alt A, alt B and alt C to the metal, with A being close to the ligand in monomer A, B close to monomer B and C close to monomer C. That way we could make constraints. But we would need to cross fingers that the three metal atoms actually end up in the same spot once applying the crystallographic symmetry (and remember, that 3-fold axis is not constructed by applying a 3fold rotational symmetry around that axis; rather an actual crystallographic symmetry somwhere else brings them together; means the ligands with their metal atoms can actually move independently).

I'm a bit at a loss how to deal with this and would appreciate input.

Thanks a lot in advance!

all the best

Julian

---- Julian Esselborn Postdoctoral Researcher Tezcan group University of California, San Diego

-- Dr. Julian Esselborn AG Photobiotechnologie Ruhr-Universität Bochum ND 2/170 0032-27049 Universitätsstr. 150 44801 Bochum