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Refinement Strategy vs. R gap
by Yuri 17 Mar '11

17 Mar '11

Pavel, It seemed like I had to close the GUI window and reopen it for the default (automatic aniso/iso) to take effect. I ran a TLS with 5 groups and ended up with better statistics. Rwork 0.18 Rfree 0.239 -- Yuri Pompeu

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Refinement Strategy vs. R gap
by Yuri 17 Mar '11

17 Mar '11

Hello phenixers, I am refining a liganded structure at 2.1 and I am pretty far into my modeling. Rfree = .265 rwork= .20 My asu has 399 residues one prosthetic group (3178 atoms) and 236 HOH. I just want to do one last refinement round before submission and I am not sure about the strategy I should select as I am running into the following: I- If I refine individuals sites and real space (XYZ) and individual ADP with aniso/iso selection automatic my Rfree goes up slightly and Rwork drops to about .196. I also select optimize x-ray/ADp weight I should say. II- When I choose no XYZ refinement with anisotropic ADP for protein chain only with weight optimization Rwork plummets to 0.15 and Rfree also drops to 0.25. But my gap is now .10 !! I checked my log file and it looks like in default automatic individual ADP phenix.refine is picking all the residues to be anisotropic even though I would not try that at 2.1 A. Could someone point me in the right direction, there is probably something I am overlooking. Thank you -- Yuri Pompeu

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Peptides bonds in cyclic peptides
by Krzysztof Bzymek 16 Mar '11

16 Mar '11

Hi, I've been trying to refine a structure of a protein with a cyclic peptide bound to it. Adding LINK/CONECT to the pdb file works in coot but the bonds are not present after refinement in phenix. I tried creating a param file (I followed recommendations for making ASN/NAG bonds posted here a while ago, but changed the names of connected residues) and included it in refinement, but the bond was not formed and the amino and carboxy groups of respective amino acids were far apart. I also got a message after refinement that a cif file was not specified. What's the solution? Thanks, Chris

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Only one solution in AutoMR with very negative LLG
by Zhang yu 16 Mar '11

16 Mar '11

Hi, I am working on a DNA-protein complex, the protein apo structure is already known. Recently I got a dataset of the DNA-protein complex, I tried to find a solution by autoMR in Phenix with the known protein coordinate as template. I got only one solution with very negative LLG (around -4000), and after rigid body refinement, the both Rwork and Rfree is around 0.55. Was there anyone in the same situation as me? What does that mean if there is just one solution while with very high negative LLG? Thanks

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Need help in refining my twinned P6322(apparent) dataset
by John Pak 15 Mar '11

15 Mar '11

Hey Phenix community, Here are the details of my current situation - any assistance in the matter would be greatly appreciated as I've been struggling mightily for the past month trying to get this structure refined (to no avail). Details: -3.0 angstrom dataset from a membrane protein crystal -reduced in a P3 Bravis lattice using HKL2000 -Pointless and Xtriage suggest P622 symmetry -Solved structure via MR - ran Phaser in all P622 based space groups, found 1 convincing solution in P6322 (LLG ~110) which makes biological sense (1 monomer in ASU that lies on the screw axis to recapitulate the biological trimer) -31% sequence identical search molecule -no pseudotranslation (via Xtriage) Xtriage suggests that the P6322 dataset is nearly perfectly twinned (twin fraction = 0.49). Mindful of the fact that there are no twin operators for P6322, I'm thinking that the true space group is P321, P312 or P63 and I've tried a number of things listed below: 1. Refined in P6322 (simulated annealing+individual B). Missing side chains and incorrectly modelled loops apparent in maps. Start R=0.49, Rfree=0.5. Refinement stalls at R=0.41, Rfree=0.45 after building. 2. Ran Phaser in P312 (using unrefined model) - no solution. 3. Ran Phaser in P321 (using unrefined model). One nice solution (LLG ~220) - lattice looks very similar to P6322 crystal packing. Twin refinement in phenix using operator suggested by Xtriage (simulated annealing + individual B) results in R=0.38, Rfree=0.41 (no rebuilding performed). 4. Ran Phaser in P63 (using unrefined model). Three solutions (LLGs all ~210) - solution #3 looks very similar to P6322 crystal packing. Twin refinement in phenix using operator suggested by Xtriage of solution #3 gives R=0.37, Rfree=0.40 (no rebuilding performed). Solutions #1 and #2 do not give packing arrangements that look like the P6322 solution. Twin refinement of Sol 1 and 2 give higher R factors. So I'm at a loss as to which space group my crystal truly belongs to, and where I should be focusing my refinement. The maps all look similar before and after DM. Thanks for any assistance or recommendations that you might have! -- John E. Pak, Ph.D. Postdoctoral Associate, Stroud Lab Department of Biochemistry& Biophysics UCSF MC2240 Genentech Hall Room S414 600-16th St. San Francisco, CA 94158-2517 Lab #: 415-476-3937 Fax #: 415-476-1902 Cell #: 415-215-0048

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fwd: Re: Need help in refining my twinned P6322(apparent) dataset
by Nathaniel Echols 15 Mar '11

15 Mar '11

---------- Forwarded message ---------- From: "Van Den Berg, Bert" <Lambertus.VandenBerg(a)umassmed.edu> To: "PHENIX user mailing list" <phenixbb(a)phenix-online.org> Date: Tue, 15 Mar 2011 08:01:42 -0400 Subject: Hi John, How do the maps look for cases 3 and 4? Are they great or are there obvious things to correct? Does your model have the correct sequence or is it that of the search model? I’m asking because your R’s seem reasonable to me after a first round of refinement and using a 30% identical search model for MR. So maybe there is no problem? Bert On 3/15/11 2:55 AM, "John Pak" <john.pak(a)msg.ucsf.edu> wrote: Hey Phenix community, Here are the details of my current situation - any assistance in the matter would be greatly appreciated as I've been struggling mightily for the past month trying to get this structure refined (to no avail). Details: -3.0 angstrom dataset from a membrane protein crystal -reduced in a P3 Bravis lattice using HKL2000 -Pointless and Xtriage suggest P622 symmetry -Solved structure via MR - ran Phaser in all P622 based space groups, found 1 convincing solution in P6322 (LLG ~110) which makes biological sense (1 monomer in ASU that lies on the screw axis to recapitulate the biological trimer) -31% sequence identical search molecule -no pseudotranslation (via Xtriage) Xtriage suggests that the P6322 dataset is nearly perfectly twinned (twin fraction = 0.49). Mindful of the fact that there are no twin operators for P6322, I'm thinking that the true space group is P321, P312 or P63 and I've tried a number of things listed below: 1. Refined in P6322 (simulated annealing+individual B). Missing side chains and incorrectly modelled loops apparent in maps. Start R=0.49, Rfree=0.5. Refinement stalls at R=0.41, Rfree=0.45 after building. 2. Ran Phaser in P312 (using unrefined model) - no solution. 3. Ran Phaser in P321 (using unrefined model). One nice solution (LLG ~220) - lattice looks very similar to P6322 crystal packing. Twin refinement in phenix using operator suggested by Xtriage (simulated annealing + individual B) results in R=0.38, Rfree=0.41 (no rebuilding performed). 4. Ran Phaser in P63 (using unrefined model). Three solutions (LLGs all ~210) - solution #3 looks very similar to P6322 crystal packing. Twin refinement in phenix using operator suggested by Xtriage of solution #3 gives R=0.37, Rfree=0.40 (no rebuilding performed). Solutions #1 and #2 do not give packing arrangements that look like the P6322 solution. Twin refinement of Sol 1 and 2 give higher R factors. So I'm at a loss as to which space group my crystal truly belongs to, and where I should be focusing my refinement. The maps all look similar before and after DM. Thanks for any assistance or recommendations that you might have! -- John E. Pak, Ph.D. Postdoctoral Associate, Stroud Lab Department of Biochemistry& Biophysics UCSF MC2240 Genentech Hall Room S414 600-16th St. San Francisco, CA 94158-2517 Lab #: 415-476-3937 Fax #: 415-476-1902 Cell #: 415-215-0048

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Mar 15, 2011 deadline- User proposal submission for Collaborative Crystallography at BCSB
by Banumathi Sankaran 15 Mar '11

15 Mar '11

Dear Users, The deadline for May/June 2011 Collaborative Crystallography proposals will be *Mar 15, 2011.* ---------------------------------------------------------------------------------------- Through the Collaborative Crystallography Program (CC) at the Advanced Light Source (ALS), scientists can send protein crystals to Berkeley Center for Structural Biology (BCSB) staff researchers for data collection and analysis. The CC Program can provide a number of benefits to researchers: * Obtain high quality data and analysis through collaborating with expert beamline researchers; * Rapid turn around on projects; and * Reduced travel costs. To apply, please submit a proposal through the ALS General User proposal review process for beamtime allocation. Proposals are reviewed and ranked by the Proposal Study Panel, and beamtime is allocated accordingly. BCSB staff schedule the CC projects on Beamlines 5.0.1 and 5.0.2 to fit into the available resources. Only non-proprietary projects will be accepted. As a condition of participation, BCSB staff researchers who participate in data collection and/or analysis must be appropriately acknowledged - typically being included as authors on publications and in PDB depositions. Please consult the website for additional information at: http://bcsb.als.lbl.gov/wiki/index.php/Collaborative_Crystallography ----------------------------------------------------------------------------------------- How To Apply: To learn more, go to: http://www.als.lbl.gov/als/quickguide/becomealsuser.html To submit a proposal, go to: http://alsusweb.lbl.gov/. Scroll down to "*Structural Biology beamlines (includes protein SAXS)*" and click on "New Proposal." Enter your proposal information, with attention to the following details: * For question 3/First choice, select "5.0.1 (Monochromatic);" for question 3/Second choice, select "5.0.2 (MAD)." * Check box (yes) in response to question (7) "Do you want collaborative crystallography (beamline 5.0.1 or 5.0.2 only) * In question 4, please describe a specific research project with a clear end point. In order to request CC time for May/June 2011 allocation period, proposals must be submitted by *Mar 15, 2011.* The deadline for CC proposals for the time period July/Aug 2011 will be May 15, 2011. Regards, Banumathi Sankaran

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Jan 15, 2011 deadline- User proposal submission for Collaborative Crystallography at BCSB
by Banumathi Sankaran 15 Mar '11

15 Mar '11

Dear Users, The deadline for March/April 2011 Collaborative Crystallography proposals will be *Jan 15, 2011.* ---------------------------------------------------------------------------------------- Through the Collaborative Crystallography Program (CC) at the Advanced Light Source (ALS), scientists can send protein crystals to Berkeley Center for Structural Biology (BCSB) staff researchers for data collection and analysis. The CC Program can provide a number of benefits to researchers: * Obtain high quality data and analysis through collaborating with expert beamline researchers; * Rapid turn around on projects; and * Reduced travel costs. To apply, please submit a proposal through the ALS General User proposal review process for beamtime allocation. Proposals are reviewed and ranked by the Proposal Study Panel, and beamtime is allocated accordingly. BCSB staff schedule the CC projects on Beamlines 5.0.1 and 5.0.2 to fit into the available resources. Only non-proprietary projects will be accepted. As a condition of participation, BCSB staff researchers who participate in data collection and/or analysis must be appropriately acknowledged - typically being included as authors on publications and in PDB depositions. Please consult the website for additional information at: http://bcsb.als.lbl.gov/wiki/index.php/Collaborative_Crystallography ----------------------------------------------------------------------------------------- How To Apply: To learn more, go to: http://www.als.lbl.gov/als/quickguide/becomealsuser.html To submit a proposal, go to: http://alsusweb.lbl.gov/. Scroll down to "*Structural Biology beamlines (includes protein SAXS)*" and click on "New Proposal." Enter your proposal information, with attention to the following details: * For question 3/First choice, select "5.0.1 (Monochromatic);" for question 3/Second choice, select "5.0.2 (MAD)." * Check box (yes) in response to question (7) "Do you want collaborative crystallography (beamline 5.0.1 or 5.0.2 only) * In question 4, please describe a specific research project with a clear end point. In order to request CC time for March/April 2011 allocation period, proposals must be submitted by *Jan 15, 2011.* The deadline for CC proposals for the time period May/June 2011 will be March 15, 2011. Regards, Banumathi Sankaran

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B-factor discrepancy
by Yuri 14 Mar '11

14 Mar '11

I am refining a single-mutant structure at 2.1 A. The native was solved years ago by heavy atom phasing. The reported average B-factors for alpha carbons especially of the original structure were significantly lower than the ones I am seeing. around 15 vs. around 25. Has anyone any idea if this is perfectly fine and its simply something intrinsic to my crystals? Also at this resolution, should I try to refine any residues at all anisotropically? Thanks for the guidance

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XYZ refinement vs individual sites refinement
by Yuri 13 Mar '11

13 Mar '11

I am new to phenix and I am trying to probe two residues that are putative H-bond partners, after refinement they seem to be put in orientations that would make it impossible for them to be interacting with a ligand. My SA composite omit map may suggest that one of the Asn could be in a different position. My question: If I manually change its conformation is it wise to keep its position fixed during refinement to see what the new maps would look like? If so where would I do that under the real space or individual sites tab on GUI phenix? I am not sure what these 2 tasks accomplish exactly. Any guidance would be appreciated. Thanks Yuri Pompeu

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