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Re: [phenixbb] Different cell content than expected in Xtriage
by Singh, Bishal 19 Aug '15

19 Aug '15

Dear Folmer, Thank you very much for your words. Yes! I refined the model after molecular replacement. Electron density is nicely defined for all residues including their side chains. There is no extra electron density. All refinement parameters are fine. Rwork and Rfree are around 22% and 25%, respectively. The model that I used for phaser was a homology model generated from a structure with 60 % sequence identity to my protein. I prefer to use homology model than providing sequence identity in phaser because I think that the local structural errors are more precisely defined in homology models. Thanking you again! Kind regards, Bishal From: Folmer Fredslund <folmerf(a)gmail.com<mailto:[email protected]>> Date: Tue, 18 Aug 2015 15:34:23 +0200 To: noro <b.singh(a)dkfz-heidelberg.de<mailto:[email protected]>> Subject: Re: [phenixbb] Different cell content than expected in Xtriage Hi Bishal, Did you try to refine the model from phaser? Did you check the packing of the 2 molecules? Did you look at the electron density from phaser and see if some of it could look like an extra molecule? It is not impossible to have 70% solvent content, so I don't think you should be too worried, just investigate the result. You probably have another molecule there, phaser was just not able to find it, but you should be able to manually fit it easily. Btw, how similar is your model to your own protein? Hope this helps Folmer Fredslund 2015-08-18 15:02 GMT+02:00 Singh, Bishal <b.singh(a)dkfz-heidelberg.de<mailto:[email protected]>>: Dear all, I am solving crystal structure of a protein of size 33.7kDa in space group P 43 21 2 at 2.25 Å resolution. Unit cells constants are 121.51 121.51 156.6 90 90 90. The cell content analysis in Xtriage showed 1050 residues (~ 3X 33.7 kDa) per ASU with 50 % solvent content, whereas the molecular replacement in mr-phaser produced a dimer (2X 33.7 kDa). When we consider a dimer per ASU, it should have approximately 70 % solvent content. Please suggest me whether solution is right or not. I shall appreciate your opinions. Kind regards, Bishal _______________________________________________ phenixbb mailing list phenixbb(a)phenix-online.org<mailto:[email protected]> http://phenix-online.org/mailman/listinfo/phenixbb Unsubscribe: phenixbb-leave(a)phenix-online.org<mailto:[email protected]> -- Folmer Fredslund

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Coot mutation of Asp residue to isoAsp residue
by Xiao Lei 19 Aug '15

19 Aug '15

Dear Phenixbb members, I suspect one Asp residue in my model may be an isoAsp (isomerization of Asp). I am asking if there is way to mutate Asp residue to isoAsp(isoaspartic acid) residue in coot GUI (I'm using coot 0.8.1 EL in Mac OS X10.10.5)? I know there is a mutation button on coot, but the mutated aa lists are all natural amino acids. If I have to delete the Asp residue first and then build isoAsp into the density map, is there a way in coot to build an isoAsp residue in map? Thanks ahead. Xiao

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Different cell content than expected in Xtriage
by Singh, Bishal 18 Aug '15

18 Aug '15

Dear all, I am solving crystal structure of a protein of size 33.7kDa in space group P 43 21 2 at 2.25 Å resolution. Unit cells constants are 121.51 121.51 156.6 90 90 90. The cell content analysis in Xtriage showed 1050 residues (~ 3X 33.7 kDa) per ASU with 50 % solvent content, whereas the molecular replacement in mr-phaser produced a dimer (2X 33.7 kDa). When we consider a dimer per ASU, it should have approximately 70 % solvent content. Please suggest me whether solution is right or not. I shall appreciate your opinions. Kind regards, Bishal

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"Table 1" wavelength
by mohamed noor 17 Aug '15

17 Aug '15

Dear all A trivial question but just to be sure: For a structure phased and built using MR-SAD with a dataset collected at one wavelength and refined with a higher-resolution dataset at a different wavelength, how should the wavelength be reported in "Table 1"? And what if the higher-resolution dataset is not isomorphous to the anomalous dataset? Is it sensible to do MR-SAD first, redo MR with the better dataset against the MR-SAD model and then refine at the higher resolution (about 0.8 A better)? Thanks.

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Post-doctoral position in Structural Biology at the Goethe-University Frankfurt, Germany
by Maike Bublitz 14 Aug '15

14 Aug '15

Dear all, please find below a job announcement posted on behalf of my colleague Jun. Prof. Inga Hänelt at the Goethe-University Frankfurt: The Structural Biology of Membrane Transporters group of Inga Hänelt, Institute of Biochemistry at the Goethe University in Frankfurt am Main invites a post-doctoral researcher to join the Emmy Noether project “Determinants of ion channel versus transporter mechanism in the K(+) transporter superfamily“. The project is in need of a candidate with strong biochemical skills for membrane protein purification and/or experience in protein crystallization. If you are interested in further information please follow the link below. http://biochem.uni-frankfurt.de/fileadmin/user_upload/MechanismsMembraneTra… <http://biochem.uni-frankfurt.de/fileadmin/user_upload/MechanismsMembraneTra…> Contact Information for this post: Dr. Inga Hänelt Juniorprofessor for Biochemistry Institute of Biochemistry, Biocenter N220/1.03 Goethe-University Frankfurt Max-von-Laue-Str. 9 60438 Frankfurt Phone: 0049-69-798-29261 Fax: 0049-69-798-29495 Email: Haenelt(a)biochem.uni-frankfurt.de <mailto:[email protected]> -- Dr. Maike Bublitz Department of Biochemistry, Biocenter Goethe University Frankfurt Max-von-Laue-Str. 9 60438 Frankfurt am Main Germany phone: +49 (0)69 798 29274 bublitz(a)em.uni-frankfurt.de <mailto:[email protected]>

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on b factor refine for phenix.real_space_refine
by Smith Lee 14 Aug '15

14 Aug '15

Dear All, I first run a default phenix.real_space_refine with the map and initial model, with the model got, I run phenix.real_space_refine with run=adp, I find for the second phenix.real_space_refine step with run=adp, it can gives a reasonable value of b-factors for each residues in the model, and it cannot modify the coordinates of any atoms in the model (in comparison with the model got by the default phenix.real_space_refine in the first step). I just read a Nature paper from David Baker group "Atomic-accuracy models from 4.5-Å cryo-electron microscopy data with density-guided iterative local refinement", it says "Because cryo-EM maps are frequently better in resolution in some regions than others, atomic B factors are fit against cryo-EM density data to maximize the real-space correlation between model and map". For Real-space B-factor refinement, the math functions used by that Nature paper include, ( f is a scattering factor fit to each element) My question is, does b-factor refine only give a reasonable b-factor to each atom or residue in the model (so that we know the displacement of each atom or residue), or besides giving reasonable b-factor, it can also change and optimize the x y z coordinates of each atom in the model, and the phenomenon I observed in phenix.real_space_refine on that it only gives reasonable b-factor without modification of the x y z coordinates was only limited to the case I see in phenix.real_space_refine? Best regards. Smith

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Strong anomalous signal but AutoSol fails
by mohamed noor 11 Aug '15

11 Aug '15

Dear developers I have a low resolution anomalous dataset which Aimless suggests has an effective resolution to 3.3 A and anomalous signal to 3.5 A. However, SAD phasing with AutoSol is not successful with the final R factor around 50 %. I also have another dataset collected at a remote wavelength without anomalous signal to 3 A but they are not isomorphous (> 2 A difference in c axis). The anomalous signal comes from the ligand heme c, which is bound covalently to the protein, so its occupancy should be 1. The protein is quite small with about 120 residues. Xtriage suggests an NCS of 6 to 20 with most likely number to be 13. Is there any reason why a reasonable solution cannot be found? There is no twinning. I am using the latest nightly 1.10 pre2124. Thanks.

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ligand refinement
by xinghuaqin＠126.com 11 Aug '15

11 Aug '15

Hi everyone, I am refining a structure of protein/ligand complex (2.75 Å) almost to the last stage. However, I found that some atoms of the ligand is too close to the protein (1.2 or 1.3 Å) after refinement. How should I avoid this? Should I use the "nonbonded_distance_cutoff" parameter? What the value should be set to? Another question is that at this resolution is it possible for me to refine the occupancy? Any suggestion is appreciated. Best Xinghua Xinghua Qin Ph.D Department of Physiology The Fourth Military Medical University 169# Changlexi Road Xi'an 710032, China Tel. 86-29-84772779 E-mail: xinghuaqin(a)126.com

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Re: [phenixbb] anomalous difference map calculation
by Ashok Nayak 11 Aug '15

11 Aug '15

Alexandra Marques <at.marques(a)fct.unl.pt> wrote: >_______________________________________________ >phenixbb mailing list >phenixbb(a)phenix-online.org >http://phenix-online.org/mailman/listinfo/phenixbb >Unsubscribe: phenixbb-leave(a)phenix-online.org

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Failed ADP refinement with active TLS and weight optimization
by Schubert, Carsten [JRDUS] 10 Aug '15

10 Aug '15

Hi, I am running into some strange issues with regards to ADP refinement especially when I am putting final touches on a structure for PDB submission. The problem manifests itself most prominent in the B-factor refinement of solvent, but also maps are affected and are becoming noisier, so I assume this problem affects the protein as well. I checked this against the current release 1.9 and the latest pre1.10 dev version, both versions show the issue. Refinement protocol includes individual_xyz, individual_adp, TLS and weight optimization. The problem I am running into is that the ADP of waters only changes by a global offset. The problem is tricky to spot but more evident when looking at solvent placed in coot with the default ADP (30). The ADP changes only by a global offset, no individual refinement takes place. Since the protein is refined by TLS the problem is not as evident, but as mentioned the fofc maps get noisier from cycle to cycle even though no manual change to the geometry was performed. To me an indicator that the B-factors are not refined properly. The problem can be circumvented by reverting back to isotropic B-factors and then refine with weight refinement for 5-10 cycles. After that TLS can be switched back on for a final polishing cycle. In stubborn cases I had to randomly assign B-factors close to Wilson-B and refine for more cycles to get out of the hole I refined myself into. Another indicator that something went awry is the weight refinement for the B-factors: ----------Individual ADP refinement---------- R-FACTORS <Bi-Bj> <B> WEIGHT TARGETS work free delta data restr 20.66 26.39 5.73 5.712 67.871 2.160 5.225 20.66 26.38 5.73 5.712 67.871 0.030 5.225 20.66 26.38 5.73 5.712 67.871 0.125 5.225 20.66 26.38 5.73 5.712 67.871 0.500 5.225 20.66 26.38 5.73 5.712 67.871 1.000 5.225 20.66 26.38 5.73 5.712 67.871 1.500 5.225 20.66 26.38 5.73 5.712 67.871 2.000 5.225 20.66 26.38 5.73 5.712 67.871 2.500 5.225 20.66 26.38 5.73 5.712 67.871 3.000 5.225 20.66 26.38 5.73 5.712 67.871 3.500 5.225 20.66 26.38 5.73 5.712 67.871 4.000 5.225 20.66 26.38 5.73 5.712 67.871 4.500 5.225 20.66 26.38 5.73 5.712 67.871 5.000 5.225 Essentially there is no change in R/Rfree with regards to different weights. I have not figured out the exact time point when this happens during the course of the refinement but it seems to be coupled to using TLS in combination with ADP refinement and during the final stages of the refinement. I can provide data offline to reproduce the issue Cheers, Carsten

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