July 2014 - phenixbb - phenix-online.org

B factors too high for low resolution data refinement
by 李翔 15 Jul '14

15 Jul '14

Hi everyone, I am working on my low-resolution data (~6.5 A). When I was doing refinement with PHENIX, the B factors remain pretty high (~400, not sure whether it is normal for the data with such resolution.) And B factors further increased after group B factor refinement (~500). Does it mean the group B factors refinement not working or is there a way I can somehow define a limit for B-factors? (I read one previous topic a few years ago which indicates the B factor can not be controlled. Not sure whether it is available now). Thank you very much for your help! Sincerely, Xiang -- Li Xiang Department of chemistry, Purdue University Email：lixiang1642(a)gmail.com

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So what is unique?
by Kenneth A. Satyshur 15 Jul '14

15 Jul '14

There is some disagreement on terms used to deposit data. We need a definition and an algorithm for each definition. "Unique Reflections" My definition is all the possible reflections out to the highest resolution reported not related by symmetry. Where can I find this? The .mtz contains a list of all HKL calculated to the highest resolution. Usually, we are not able to measure all these diffraction spots due to limits of the detector, mechanical limits, crystal orientation, etc. 'Total reflections' The depositions server asks for total reflections. I assume it wants only those unique reflections we were able to collect, regardless of the sigma cut off. These are called 'observed'. The total we use in refinement will be a subset of the 'unique observed' that are cut on sigma. However, some crystallographers believe that we should not cut on sigma since some of the intensities may in fact be zero. Is this a question for the Refmac and Phenix people? Please give us some guidance and maybe a reference or two that we can use. thanks -- Kenneth A. Satyshur, M.S.,Ph.D. Senior Scientist University of Wisconsin Madison, Wisconsin 53706 608-215-5207

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cifs still in pdb v2
by Guenter Fritz 14 Jul '14

14 Jul '14

Dear Pavel and Nat, can you include in future phenix versions the FAD.cif, NAD.cif and data_NAI.cif / NAI.cif in pdb v3? Currently both are still pdb v2. I usually simply copy the cifs pdb v3 into the mon_lib directory , but sometimes I forget it and stumble over it. Thanks and best wishes, Guenter

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Maximum-likelihood score calculation
by Jouko Virtanen 12 Jul '14

12 Jul '14

Hi all, I would like to calculate the maximum-likelihood score given an input structure and an mtz file. How can I do that? Thanks in advance. Best regards, Jouko

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Real space implementation of DEN refinement?
by Oliver Clarke 11 Jul '14

11 Jul '14

Hi all, Just wondering whether there is any capacity to include DEN restraints in phenix.real_space_refine, e.g. as implemented in the program DireX (see ref below), or any possibility of implementing such in a future release of PHENIX? This would be very handy for refinement of models into low resolution maps (particularly EM maps). Also, on a different but related topic, does anyone have any suggestions as to how to handle model validation (local and global) at low resolution in real-space? It seems as though there is not a solid consensus on how to handle this. Many thanks, Oliver. 1. Schröder, G. F., Brünger, A. T. & Levitt, M. Combining Efficient Conformational Sampling with a Deformable Elastic Network Model Facilitates Structure Refinement at Low Resolution. Structure 15, 1630–1641 (2007).)

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Twinning in data reduction and phenix.refine
by Chen Zhao 11 Jul '14

11 Jul '14

Dear all, I am now dealing with a pseudo-merohedral twin (3.2 A, P1 twinned into C2), and I have a couple of questions concerning the data reduction and refinement: 1) R-free flag in refinement a) For twin refinement, I know the reflections related by the twin law should have the same R-free flag. If I process my data in P1, and generate the R-free flag by phenix.refine using the key word xray_data.r_free_flags.use_lattice_symmetry=True, can phenix generate the R-free flags considering the 2-fold point group symmetry and deal with the twinning properly? b) I read that the R-factor could go down to around 40% in twin refinement in the presence of twinning even if the model is completely wrong. For my understanding, this could be due to the fact that the reflections are correlated by the twin law. If the R-free flags are chosen in the highest lattice symmetry, will the R-free still suffer from this problem? 2) Twin fraction In phenix.xtriage, the predicted twin fraction is about 30%. However, in phenix.refine, the twin_fraction is always between 46% to 50%. Why could this happen? 3) Data reduction I am not very clear about this point, since I just started dealing twinning. But what I am thinking is whether it is a good idea to merge the Friedel pair in the presence of twinning. Since the two twin domains have different hkl index if considering one as the reference, is the Friedel law still valid? This might be a very simple mathematical problem. I really appreciate your answers! Sincerely, Chen

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Anomalous or not?
by CPMAS Chen 10 Jul '14

10 Jul '14

Hi, Phenix Users, I recently collected data at SSRL. I used autoxds and multiscale to merge couple of datasets and hoped to see some anomalous signals. Now comes the questions. 1. if I use the default sigma(=2), then autoxds will report weak anomalous signals and leave the friedel flag off. If I lower sigma to 1.5, it will report strong anomalous signals. 2. when I used Xtriage under Phenix to check the data quality, it reported weak anomalous signal or at above 10A. 3. I used aimless (in CCP4) to merge data, it also reported weak anomalous signal. Which result should I trust? By the way, how can I view/display the ***.anamplot file, which is apparently xmgr format file, but I can not display in CCP4i. Thanks! -- *************************************************** Charles Chen Research Associate University of Pittsburgh School of Medicine Department of Anesthesiology ******************************************************

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Re: [phenixbb] Anomalous or not?
by Ryan Spencer 10 Jul '14

10 Jul '14

The question then becomes how do you know the bromine is in the crystal? Is it covalent on the protein? Is it covalent on the ligand? How do you know if the ligand is in the structure? We have seen rapid crystal degredation with Br at its edge. Do you see the resolution significantly degrade after a few frames? I don't think it's a processing issue with xds or with phenix. Ryan -------- Original message -------- From: CPMAS Chen <cpmasmit(a)gmail.com> Date: 07/10/2014 9:52 AM (GMT-08:00) To: Ryan Spencer <rspencer(a)uci.edu> Cc: Francis Reyes <Francis.Reyes(a)colorado.edu>,phenixbb(a)phenix-online.org Subject: Re: [phenixbb] Anomalous or not? Well, let me make this clear. 1. I am using Br anomalous signal to identify the potentially bound ligand. 2. I do shoot the crystals at 0.92A. 3. the different crystals have different resolution, but anomalous signal was weak as reported by autoxds. Charles On Thu, Jul 10, 2014 at 12:25 PM, Ryan Spencer <rspencer(a)uci.edu> wrote: So I assume you were not able to shoot the crystal at ~0.92 A at SSRL or are the crystals sensitive? If you were limited to the single wavelength beam which is around 1A you’re dealing with a really low f” of 0.5322. There have been crystals solved with Sulfur using an in-house Cu sources (takes a lot of merged sets and anomalous is stronger at certain crystal angles) which gives about the same anomalous scattering as Br. Is the bromine covalent or is it soaked ion? Alternatives – soak with potassium iodide and get a dataset from an in-house source (iodine f” 6.6 at 1.54), or at least the anomalous locations. You may get a partial model to use for replacement at that point. Did each processed dataset have the same level of anomalous signal when processed individually? Ryan From: phenixbb-bounces(a)phenix-online.org [mailto:[email protected]] On Behalf Of CPMAS Chen Sent: Thursday, July 10, 2014 8:55 AM To: Francis Reyes Cc: phenixbb(a)phenix-online.org Subject: Re: [phenixbb] Anomalous or not? Francis, My anomalous plot for Br is more like your Cobalt 1 site less redundancy. Ryan, As my Br anomalous signals are weak, I merged signals from multiple crystals. Meanwhile I am looking into the detail about the autoxds processing as suggested by Tim. Thanks, On Thu, Jul 10, 2014 at 10:02 AM, Francis Reyes <Francis.Reyes(a)colorado.edu> wrote: In addition to Tim's comments above, using loggraph to see the anomalous CC plot from scala is a good qualitative indicator of whether you have anomalous signal.. Some data points for what reasonable plots (and their corresponding XDS SigANO's) look like: https://dl.dropboxusercontent.com/u/19558536/AnomalousCC.pdf F On Jul 10, 2014, at 9:03 AM, CPMAS Chen <cpmasmit(a)gmail.com> wrote: > > Which result should I trust? By the way, how can I view/display the ***.anamplot file, which is apparently xmgr format file, but I can not display in CCP4i. > -- *************************************************** Charles Chen Research Associate University of Pittsburgh School of Medicine Department of Anesthesiology ****************************************************** -- *************************************************** Charles Chen Research Associate University of Pittsburgh School of Medicine Department of Anesthesiology ******************************************************

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Postdoctoral Position in Membrane Protein Structural Biology
by Noinaj, Nicholas (NIH/NIDDK) [E] 07 Jul '14

07 Jul '14

Two postdoctoral positions in structural biology are available starting in August in my new laboratory within the Biological Sciences department at Purdue University. As part of the Markey Center for Structural Biology, my lab will integrate electron microscopy and X-ray crystallography with biochemical methods to study the biogenesis of beta-barrel membrane proteins in Gram-negative bacteria with a focus on eventual therapeutic development against pathogenic strains of bacteria (See Noinaj et al., Structure (in press, 2014), Nature, 501:385-390 (2013), and Nature, 483:53-58 (2012)). The Markey Center for Structural Biology at Purdue is recognized worldwide for its leadership in structural biology of viruses, membrane proteins, receptors, signaling proteins and enzymes in addition to methods development in crystallography, NMR and electron microscopy. My lab is located within the Hockmeyer Hall of Structural Biology which boasts state-of-the-art shared resources including a Titan Krios cryo-TEM, Bruker Avance-III 800 MHz NMR, Rigaku X-ray generators and detectors, and other advanced instrumentation available at the Bindley Bioscience Center, the Birck Nanotechnology Center, and the new Center for Drug Discovery. In addition, Purdue is in close proximity to the Advanced Photon Source at Argonne National Lab for convenient access to synchrotron data collection. The successful candidate(s) should be highly motivated, have strong written and communication skills, have (or expect) a recent PhD in structural biology, biophysics, biochemistry, or other related field of study and have previous experience in working with membrane proteins and/or X-ray crystallography or electron microscopy. Applicants should send a CV, names of three referees and a statement describing your interests and relevant work experience to Nicholas Noinaj at nicholas.noinaj(a)gmail.com<mailto:[email protected]>. Nicholas Noinaj Purdue University Department of Biological Sciences 333 Hockmeyer Hall 915 W State St West Lafayette, IN 47907

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Autobuiold from polyalanine model
by Chieh Yin 07 Jul '14

07 Jul '14

Dear Phenix Users, I am doing the molecular replacement to solve a membrane protein structure(transmembrane and soluble domain). The structure of the soluble domain is known, 200AAs. And there is a homology structure of transmembrane domain, 350AAs. I used the polyalanine model of the homologous model of transmembrane domain to do the first search. I got a solution with TFZ 16. Then used the partial solution to do the second search with the soluble domain as the model.I also got a solution with TFZ about 15. The density map showed I got the right solution. And the simple rigid body refinement yielded a Rfactor/Rfree 0.29/0.32. Then I used the polyalanine model and the soluble domain as the starting model to do the phenix autobuild. But it seemed that Autobuild didn't replace the alanine with the real sequence AAs. I tried to set the rebuild in place to false but I still got the same result. The Autobuild even didn't change any amino aside reside from the starting model. I hope you could figure out the problem for me. Thank you in advance! Best regards, Jie

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