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Heme geometry restraint
by Yangqi Gu 21 Jul '20

21 Jul '20

Dear all, I have a question regarding the heme c geometry restraint. I am aware that heme c could undergo distortion when bound to protein, but I am not sure at what resolution I could claim such distortion (such as ruffling/bending/doming). I have a map at 3.6 A resolution and whenever I did the refinement in phenix with real space refinement, some of the hemes are distorted and I am wondering if it is justified to leave it that way or do I have to impose the flat plane geometry. If so, how should I proceed to impose such geometry? Thank you in advance! Best, Yangqi

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mr_rosetta question
by Wei Wang 18 Jul '20

18 Jul '20

Dear all, I would like to get help on a mr_rosetta run. I have a case that 5 copies of searching model was found by Phaser, and I would like to find the sixth one. In addition I would like to improve the building of the MR solution. In such case I’m considering a mr_rosetta run, with some parameters like this: phenix.mr_rosetta seq_file=seq.dat search_models=placed_5_copies.pdb already_placed=true ncs_copies=1 … My question is, should I use the already placed 5 copies model as search_models, with ncs_copies=1, or should I use the original searching model with ncs_copies=6 (in this case I guess I should get rid of the other four copies from the result of Phaser)? For seq.dat, should I use the sequence from one copy of searching model, or paste 5 copies into the seq.dat? Thanks, Wei

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ligand possibly bound to active site cysteine
by Jorge Iulek 18 Jul '20

18 Jul '20

Dear all, I am refining a structure of a Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (converts glyceraldehyde 3-phosphate into D-glycerate 1,3-bisphosphate) , https://www.brenda-enzymes.org/enzyme.php?ecno=1.2.1.12 . <https://en.wikipedia.org/wiki/Glycerate_1,3-bisphosphate> It turns out that its active center cysteine presents bound ligands , covalently or not to be determined if possible (data resolution 2.51 A). I would like to get help on two issues, (1) what the ligand might be and (2) how to treat it (correct me) in phenix.refine. 1) The protein was expressed in E. coli; it had much contact with glycerol and crystallization conditions include the "ethylene-glycols-mix" ("a mixture of diethylene glycol, triethylene glycol, tetraethylene glycol, and pentaethylene glycol"). Nevertheless, no NAD cofactor was added, and there is no electron density for it. Otherwise, phosphate was also present in crystallization condition. In a previous study, I learned that glycerol might also contain minor amounts of ethylene glycol. I wonder, nevertheless, about glyceraldehyde (and note resemblance with the substrate). Catalytic mechanism includes a hemithioacetal intermediate (https://febs.onlinelibrary.wiley.com/doi/abs/10.1046/j.1432-1327.1998.25204… ) such that cysteine SD is bound covalently to a carbon. I wonder also how much this might attack an ethylene glycol and their likes. Pictures for the density are shown at for the 4 monomers of the a. u., first 4 photos: https://photos.app.goo.gl/Y7MyugqwRFD4sjgDA (blue 1 sig for e. d. maps, green 3 sig for Fourier difference maps) . Density is different among them to different degrees. The nearby threonine, in some cases, seems to interact with a blob (and it is helped by other threonine and a serine) which + - might accommodate a phosphate. I have tried to fit a number of molecules, e.g., the substrate (but not really good in all monomers for the phosphate moiety), glycerol, ethylene glycol and its di and tri (found also in other places in the structure) and now I went for glyceraldehyde (though, I have doubts that there is other - apart from the one eventually bound to S - tertiary carbon). Apart from the difficulties on searching for the best fitting molecule (and consider their intrinsic flexibility) I do not manage to establish distance between them and Cys SD (and there goes the second question). 2) I could not devise how to set a proper distance between any of the ligands and the Cysteine, be it to check for a covalent bond or to establish a van der Waals restriction. I tried: bond { action = *add delete change atom_selection_1 = chain A and resid 153 and name SG atom_selection_2 = chain N and resid 5 and name C3 symmetry_operation = None distance_ideal = 1.803 sigma = 0.1 slack = None limit = -1.0 top_out = False } Results are also show for my Glyceraldehyde trial, last 4 photos, https://photos.google.com/album/AF1QipO71L7GJYKv_MmjTc_0GzsH2xtFR_V-2ICBirPb . Note clashes. Curiously , for some of the bonds to be added, I receive the message: " Atom "HETATM 9835 O2 3GR N 5 .*. O " rejected from bonding due to valence issues." which seems to point to oxygen atoms, though I declare carbon atoms. Helps welcome, thank you. Jorge

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mr_rosetta build test
by Wei Wang 18 Jul '20

18 Jul '20

Dear all, I would like to ask a question about the testing of mr_rosetta build. Is the “phenix_regression.wizards.test_command_line_rosetta_quick" still available? I checked my early build on Mac it is there, but that build was ~3 years ago. However on a recent build on Ubuntu 18.04 the option is not available (I ran the rosetta.run_tests and it’s ok though). Regards, Wei

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occupancy_sum_constraints
by Petros Giastas 15 Jul '20

15 Jul '20

Dear all, I have a metal compound interacting with a protein and I am facing the following problem. Initially, the metal is equatorially complexed with chlorides, which are progressively exchanged by water molecules. This hydrolysis is clear from our spectroscopic analysis, but also from the anomalous maps, which show weaker peaks for the most exposed chlorides. The question regarding phenix.refine is: How can I refine the occupancies of the alternative species (chlorides and water molecules) and to constrain their sum into the occupancy of the metal, which is also under refinement? I cannot find the proper selection syntax to deal with this situation. Can you help me on that? Thank you in advance. Petros P.S. I remember I could treat this kind of problem with shelxl but it has been a lot of time since I last used shelxl for high resolution protein refinement and I would not like to go back.

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PhD position (Denmark), Structural biology with a focus on sterol uptake mechanisms
by Bjørn Panyella Pedersen 13 Jul '20

13 Jul '20

Dear colleagues, We have a open PhD position my my group. Please pass along to anyone who might be interested. Thanks! Deadline is July 31st. /Bjørn STRUCTURAL AND FUNCTIONAL STUDIES of eukaryotic membrane proteins involved in sterol transport (http://pedersenlab.dk/source/phd_position_2020.pdf) A PhD position is available in membrane protein biochemistry and structural biology in the research group of Dr. Bjorn P Pedersen at the Department of Molecular Biology and Genetics, Aarhus University, Denmark. The group studies the structure-function relationship of membrane proteins involved in metabolite uptake, primarily sterol and sugar uptake, as well as their regulation from a structural perspective. The PhD project will involve expression, purification and structural/functional characterization of membrane proteins involved in sterol transport and homeostasis (Winkler et al. Cell 179 (2019)). Methods learned and used will be cryo-electron microscopy and X-ray crystallography, as well as various biochemical methods to elucidate function. The project is fully funded by the Danish Council for Independent Research, and will be based in the laboratory of Dr. Bjorn P. Pedersen (http://pedersenlab.dk). It would ideally suit an outstanding biochemistry graduate with a strong interest in structural biology. The candidate should have molecular biology, protein production and purification experience. Experience with membrane proteins or protein structure determination will be an advantage. Full training will be provided in all necessary areas of protein biochemistry and structural biology. Good English language skills are essential. Salary will be according to the collective agreement between the Danish Confederation of Professional Associations and the Danish State. The position will be filled as soon as a suitable candidate is identified. Deadline is July 31st. Applicant should send a CV, cover letter and names of two referees to Dr. Bjorn P Pedersen (bpp(a)mbg.au.dk).

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phenix writing files with wrong job number
by Green, Todd Jason 09 Jul '20

09 Jul '20

Hello All- Phenix has started to write files from each job with incorrect job number notation. It puts them into the correct directory (ie. with correct job number, ex. Refine_23) but with file names having wrong job number (ie. where they are job number - 1, ex. protein_refine_22.pdb). This causes issues with the gui. I can edit the names post run, but is there a permanent fix. Just start a new project? Probably upgrade to the new version is the first choice. FYI I'm running 1.17.1-3660-000 on a macbook. Seems to have just started spontaneously. Thanks for the help- Todd

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The 5'end guanosine triphosphate is not linked to the rest of RNA model by Phenix refine
by Zixian Li 08 Jul '20

08 Jul '20

Dear Phenix Community, I'm at beginner level of using Phenix. I have difficulty to refine a protein-RNA complex in Phenix. I refined the protein model without RNA first, then I built the RNA model into the difference density map in Coot through Calculate>Other Modelling Tools>Ideal DNA/RNA, where I input the sequence and fit nucleotides into the density. At the 5' end it's guanosine triphosphate (GTP) but the modeler tool in Coot only generates nucleoside monophosphate. So I built the RNA model from the second nucleotide at 5' end downstream until where RNA density was too poor to model. I did the real space refine zone in Coot to fit this main part of the RNA into its density the best possibly. For the GTP at 5' end, I realized there is GTP.cif restraint file in Phenix monomer library. So I obtained an arbitrary pdb file for GTP using eLBOW and corrected atom names matching the definition in GTP.cif file. Then I can also fit GTP into the RNA density at 5' end using real space refine zone in Coot. Now I have a broken RNA model with GTP unlinked to the ribose-phosphate backbone. For Phenix refinement, I supplied a cif_link_GTP-G.params file to define the linkage for the two RNA chains in the complex: refinement.pdb_interpretation { apply_cif_link { data_link = p residue_selection_1 = chain C and resname GTP and resid 1 residue_selection_2 = chain C and resname G and resid 2 } apply_cif_link { data_link = p residue_selection_1 = chain D and resname GTP and resid 1 residue_selection_2 = chain D and resname G and resid 2 } } After refinement, I noticed O3' of GTP appears connected to P of the following nucleotide G in the refined model in Coot. However, if I do the real space refine zone in Coot, the linkage becomes broken, i.e., I cannot refine the RNA model as a whole: Coot only allows separate real space refine for GTP and the rest. It seems Coot still cannot recognize GTP and the rest as one body. In the refinement settings there is a button for Automatic linking options. Interestingly, I noticed if I leave the automatic covalent linking as default, Phenix writes Link records in the output pdb after refinement. But it also links one of the protein residues to the GTP in chain D. In order to avoid the unwanted intermolecular linkage, I clicked Link none for Automatic linking options, then there is no any link records written in the output pdb despite of the defined linkage in cif_link_GTP-G.params file. So it seems for me Phenix ignores the input cif_link_GTP-G.params file, it writes Link records only if the Automatic linking option is activated. I worried whether it could be due to the syntax of the cif_link_GTP-G.params file. So I tried the following syntax in two separate files, the same issue was observed. cif_link_GTP-G_chC.params refinement.pdb_interpretation.apply_cif_link { data_link = p residue_selection_1 = chain C and resname GTP and resid 1 residue_selection_2 = chain C and resname G and resid 2 } cif_link_GTP-G_chD.params refinement.pdb_interpretation.apply_cif_link { data_link = p residue_selection_1 = chain D and resname GTP and resid 1 residue_selection_2 = chain D and resname G and resid 2 } I also need to point out that p linkage is pre-defined in the list of links (mon_lib_list.cif) in Phenix monomer library. I apologize for this long explanation of the problem. I hope it is clear. I really appreciate your advice in advance. Sincerely, Zixian Li

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Heteroatoms addition to crystal structures
by Shirin Akbar 06 Jul '20

06 Jul '20

Dear all, I am using the phenix.real_space_refine program for refinement of crystal structure in cryo-EM map (ribosome map, 3.4 A resolution). Prior to real_space_refinement I had removed all the heteroatoms. What is the correct way to add the cations (eg. Mg2+ ions) to the structure and how do I do it? Can anyone kindly suggest.. Thanks & Regards Shirin -- *Shirin Akbar*

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real-space refinement resolution
by Alexei Gorelik 30 Jun '20

30 Jun '20

Hi, When refining a model against a density-modified cryo-EM map, should one use the resolution of the original or modified map? Thanks

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