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UPDATE-- Difficult dataset and refinement--P422? I422?Twinning?
by Zhou, Tongqing (NIH/VRC) [E] 08 May '12

08 May '12

Hi Jürgen, Kelly and Nat, I tried XDS and MosFilm, they simply cannot index the data. I reprocessed the data yesterday with HKL2000 starting from frame #30 (splitting 1-29, 30-360), I did get it indexed to I4 and refined to very nice x/y chi^2 and covering most spots, I then used the index/refinement numbers for the whole data set. it seems that other spots were from a "nicely" positioned secondary crystal which fooled the program to pick P4 space group. I scaled the data as I4 and I422 to see which one is better. The I4 data set, ~95% complete overall, 9.2% R-sym. Xtriage suggested it should have higher symmetry I422 or twin (-k, -h, -l, almost 50%). MR sent well and best SG is I4, 2 mol/ASU, r and r-free went to about 27% and 32%, but if I used the twin-law in phenix.refine and ask phenix to generate r-free-flags with twinning in mind, the numbers went to ~22% and 27%. Shell Lower Upper Average Average Norm. Linear Square limit Angstrom I error stat. Chi**2 R-fac R-fac 50.00 7.32 440.7 8.8 5.1 1.481 0.027 0.030 7.32 5.81 167.2 4.2 3.2 1.868 0.045 0.048 5.81 5.08 196.3 5.1 3.9 2.130 0.053 0.058 5.08 4.62 243.1 6.2 4.7 2.264 0.056 0.061 4.62 4.29 236.7 6.4 4.9 2.057 0.055 0.055 4.29 4.03 167.6 5.6 4.7 1.819 0.067 0.066 4.03 3.83 139.5 5.6 5.0 1.740 0.083 0.075 3.83 3.66 110.2 5.5 5.1 1.645 0.102 0.091 3.66 3.52 92.5 5.6 5.3 1.459 0.117 0.101 3.52 3.40 76.5 5.7 5.4 1.372 0.143 0.124 3.40 3.30 59.3 5.7 5.5 1.278 0.179 0.146 3.30 3.20 47.3 5.7 5.6 1.161 0.212 0.179 3.20 3.12 40.7 5.8 5.7 1.103 0.241 0.201 3.12 3.04 31.6 5.8 5.8 1.057 0.298 0.254 3.04 2.97 28.9 5.9 5.9 1.031 0.331 0.271 2.97 2.91 22.6 5.9 5.9 0.985 0.400 0.340 2.91 2.85 19.0 6.0 6.0 0.991 0.484 0.405 2.85 2.80 17.1 6.1 6.1 0.957 0.518 0.437 2.80 2.75 14.6 6.2 6.2 0.908 0.577 0.485 2.75 2.70 13.0 6.4 6.3 0.931 0.645 0.550 All reflections 112.9 5.9 5.3 1.455 0.092 0.066 The I422 data (below), 1 mol/ASU, r and r-free were about 27% and 32%. Shell Lower Upper Average Average Norm. Linear Square limit Angstrom I error stat. Chi**2 R-fac R-fac 50.00 7.32 430.0 8.4 5.0 1.344 0.025 0.028 7.32 5.81 163.4 4.0 3.0 1.489 0.040 0.042 5.81 5.08 192.4 4.8 3.6 1.694 0.046 0.052 5.08 4.62 231.6 5.6 4.3 1.790 0.049 0.053 4.62 4.29 227.8 5.8 4.5 1.534 0.045 0.043 4.29 4.03 161.3 5.0 4.2 1.401 0.055 0.052 4.03 3.83 135.3 5.0 4.4 1.367 0.066 0.059 3.83 3.66 106.2 4.9 4.4 1.302 0.082 0.071 3.66 3.52 88.1 4.8 4.5 1.253 0.097 0.082 3.52 3.40 75.0 4.9 4.6 1.193 0.113 0.096 3.40 3.30 56.7 4.8 4.6 1.121 0.144 0.115 3.30 3.20 44.8 4.8 4.7 1.061 0.172 0.141 3.20 3.12 38.0 4.8 4.7 1.011 0.203 0.166 3.12 3.04 29.1 4.7 4.7 1.029 0.253 0.209 3.04 2.97 27.7 4.8 4.8 0.985 0.261 0.209 2.97 2.91 21.4 4.9 4.8 0.917 0.320 0.266 2.91 2.85 18.3 4.9 4.8 0.949 0.380 0.316 2.85 2.80 16.1 4.9 4.9 0.955 0.431 0.358 2.80 2.75 14.2 5.0 5.0 0.940 0.483 0.399 2.75 2.70 12.6 5.2 5.2 0.886 0.536 0.462 All reflections 110.9 5.1 4.5 1.236 0.076 0.054 So my questions are: 1. Is it really twinned since including twin-law in refinment gave much better numbers? 2. When refining with twin-law, Are the maps by phenix.refine output already "corrected" mtz/map? 3. If I use xray_data.r_free_flags.generate=True, can I write the data mtz out with the generated r-free-flags so that each round of refinement uses the same CV set? Thnaks, Tongqing Tongqing Zhou, Ph.D. Staff Scientist Structural Biology Section Vaccine Research Center, NIAID/NIH Building 40, Room 4609B 40 Convent Drive, MSC3027 Bethesda, MD 20892 (301) 594-8710 (Tel) (301) 793-0794 (Cell) (301) 480-2658 (Fax) ****************************************************************************** The information in this e-mail and any of its attachments is confidential and may contain sensitive information. It should not be used by anyone who is not the original intended recipient. If you have received this e-mail in error please inform the sender and delete it from your mailbox or any other storage devices. National Institute of Allergy and Infectious Diseases shall not accept liability for any statements made that are sender's own and not expressly made on behalf of the NIAID by one of its representatives. ****************************************************************** From: Bosch, Juergen [mailto:[email protected]] Sent: Monday, May 07, 2012 1:34 PM To: PHENIX user mailing list Subject: Re: [phenixbb] Difficult dataset and refinement--P422? I422?Twinning? Why don't you index over the whole dataset (XDS) ? Or at least over a couple of images (Mosflm) Jürgen On May 7, 2012, at 12:50 PM, Kelly Daughtry wrote: I agree. The fact that two "crystals forms" appear, with related unit cells: P422 data: unit_cell = 191.6999969 191.6999969 311.0539856 90 90 90 space_group = "P 4 2 2" I422 data: unit_cell = 191.783 191.783 103.775 90 90 90 space_group = "I 4 2 2" indicated to me you have mis-indexed the P422 data. It is likely I422. Kelly ******************************************************* Kelly Daughtry, Ph.D. Post-Doctoral Fellow, Raetz Lab Biochemistry Department Duke University Alex H. Sands, Jr. Building 303 Research Drive RM 250 Durham, NC 27710 P: 919-684-5178 ******************************************************* On Mon, May 7, 2012 at 12:18 PM, Nathaniel Echols <nechols(a)lbl.gov<mailto:[email protected]>> wrote: On Mon, May 7, 2012 at 8:46 AM, Kelly Daughtry <kddaught(a)bu.edu<mailto:[email protected]>> wrote: Looking at the P422 data, it look like you have pseudo translational symmetry. Did you try processing this data as I422? >From the P422_xtriage.log: The full list of Patterson peaks is: x y z height p-value(height) ( 0.500, 0.500, 0.165 ) : 78.635 (6.634e-07) ( 0.000, 0.000, 0.330 ) : 56.292 (2.795e-05) ( 0.500, 0.500, 0.497 ) : 30.920 (1.207e-03) One word of caution: an exceptionally high off-origin peak can mean that the unit cell was measured too large, and you've integrated extra reflections that are really non-existent. (I'm not sure what the threshold for this is, but 80% seems pretty large.) Splitting and various indexing artifacts can sometimes lead to this. I'd recommend running labelit.index on the images and seeing what it thinks the lattice should be. -Nat _______________________________________________ phenixbb mailing list phenixbb(a)phenix-online.org<mailto:[email protected]> http://phenix-online.org/mailman/listinfo/phenixbb _______________________________________________ phenixbb mailing list phenixbb(a)phenix-online.org<mailto:[email protected]> http://phenix-online.org/mailman/listinfo/phenixbb ...................... Jürgen Bosch Johns Hopkins University Bloomberg School of Public Health Department of Biochemistry & Molecular Biology Johns Hopkins Malaria Research Institute 615 North Wolfe Street, W8708 Baltimore, MD 21205 Office: +1-410-614-4742 Lab: +1-410-614-4894 Fax: +1-410-955-2926 http://web.mac.com/bosch_lab/

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redundancy of merged multiple datasets
by CPMAS Chen 08 May '12

08 May '12

Dear all, I have merged multiple datasets obtained from the same crystals, different spots of course, it certainly increases the signal/noise ratio and makes the resolution go higher a little bitter. But, the con is, the redundancy is increased to ~20 after I merged 6 datasets. Whereas the individual dataset has the redundancy around 3~5. Is this acceptable? Thanks! *************************************************** CPMAS Chen Research Associate University of Pittsburgh School of Medicine Department of Anesthesiology ******************************************************

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Re: [phenixbb] UPDATE-- Difficult dataset and refinement--P422? I422?Twinning?
by Kay Diederichs 08 May '12

08 May '12

> On May 8, 2012, at 11:11 AM, Zhou, Tongqing (NIH/VRC) [E] wrote: > > Hi J?rgen, Kelly and Nat, > > I tried XDS and MosFilm, they simply cannot index the data. > Hi, "simply cannot index" is hard to believe. 90% of indexing failures are due to wrong header values, 9% to the spindle rotating in the "wrong" direction, and the last 1% has other causes. I have yet to see a single-crystal dataset that cannot be indexed. If you are willing to confidentially share (at least some of) your data, I could try to process them with XDS - at least you get another opionion. Easiest way: copy them into a Dropbox or Wuala folder and share that. best, Kay

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phenix.refine crashes
by Jianghai Zhu 08 May '12

08 May '12

Hi, I am using phenix.refine dev-1051. When I was trying flip_peptide, it crashes with the following error messages. Validate: |--------START--------| |--------FINAL--------| residue map_cc 2mFo-DFc mFo-DFc map_cc 2mFo-DFc mFo-DFc Traceback (most recent call last): File "/home/jzhu/phenix/phenix-dev-1051/build/intel-linux-2.6-x86_64/../../phenix/phenix/command_line/refine.py", line 11, in <module> command_line.run(command_name="phenix.refine", args=sys.argv[1:]) File "/home/jzhu/phenix/phenix-dev-1051/phenix/phenix/refinement/command_line.py", line 146, in run call_back_handler=call_back_handler) File "/home/jzhu/phenix/phenix-dev-1051/phenix/phenix/refinement/driver.py", line 1499, in run ncs_manager = self.ncs_manager) File "/home/jzhu/phenix/phenix-dev-1051/phenix/phenix/refinement/strategies.py", line 928, in refinement_machine params = params.flip_peptides) File "/home/jzhu/phenix/phenix-dev-1051/cctbx_project/mmtbx/refinement/flip_peptides.py", line 445, in run log=log) File "/home/jzhu/phenix/phenix-dev-1051/cctbx_project/mmtbx/refinement/fit_rotamers.py", line 749, in validate_changes sites_cart=sites_cart) File "/home/jzhu/phenix/phenix-dev-1051/cctbx_project/mmtbx/refinement/flip_peptides.py", line 152, in __call__ return (self.rama_lookup.evaluate_sites(res_type,i_seqs,sites_cart)<0.002) File "/home/jzhu/phenix/phenix-dev-1051/cctbx_project/mmtbx/rotamer/ramachandran_eval.py", line 76, in evaluate_sites "isoleucine or valine"]) AssertionError -- Jianghai

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Re: [phenixbb] phenixbb Digest, Vol 78, Issue 9
by CPMAS Chen 08 May '12

08 May '12

Thank you all. Actually, as Pavel said there is a easy way to do so, I changed the cif file of the ligand, where I had restraints on the dihedral modified. Message: 3 > Date: Sat, 05 May 2012 13:48:06 -0700 > From: Pavel Afonine <pafonine(a)lbl.gov> > To: phenixbb(a)phenix-online.org > Subject: Re: [phenixbb] how to define a molecule in two rigid_body > Message-ID: <4FA59206.7090207(a)lbl.gov> > Content-Type: text/plain; charset=ISO-8859-1; format=flowed > > Hi Charles, > > I think you are trying to achieve two goals, "keep conformation you > like" and "make sure it fits given density", while one of the two > contradicts to the other. That is either the density you are trying to > fit in your molecule or your expectations about the geometry of the > molecule (or both) are not quite right. > > I think this is how much one can tell given the information you provided. > > I don't think you need to do that sophisticated rigid-body kind of > refinement that you described in your original post. The solution must > be easier. May be if you send me the model and data and tell which part > of the model you think is in trouble then probably I could help more. > > Pavel > > On 5/5/12 10:22 AM, Nathaniel Echols wrote: > > On Sat, May 5, 2012 at 6:39 AM, CPMAS Chen<cpmasmit(a)gmail.com> wrote: > >> I am almost at final stage. but the refinement always shows error > density at > >> the single bond connecting the two rings. I have tried manually change > the > >> orientation of these two rings, but there are two concerns that I > thought > >> why I did not get the perfect fitting. > >> 1. I might not choose the perfect the orientation of these rings, and > these > >> give me a non-perfect fitting, and high b-factor. > >> 2. since phenix always tries to change the conformation a little bit to > fit > >> the density, so this also might cause some errors, or not the > conformation I > >> like. > >> > >> Then, my sense is that, if I can somehow define the two parts of the > >> molecule as two rigid bodies, then phenix only can has free rotation of > >> these two rings, and the fitting might come out good. > > You can define custom planarity restraints for the rings - there isn't > > good documentation for this, but here is what to do: > > > > 1. If you're using the command line, look here in the .eff file: > > > > refinement { > > geometry_restraints { > > edits { > > planarity { > > atom_selection = resseq 1001 and (name C1 or name C2 or name > > C3 or name C4 or name C5 or name C6) > > sigma = 0.1 > > } > > } > > } > > } > > > > (Other parameters omitted for clarify, of course. I'm not sure what > > an appropriate sigma would be - you'll probably have to experiment.) > > > > 2. If you're using the GUI, make sure you have the latest nightly > > build, and on the middle configuration tab, click "Custom geometry > > restraints", which will open a window that includes a tab for creating > > planarity restraints. > > > > Technically, the two planes won't actually be "rigid bodies" (i.e. > > "constrained"), but if you make the planarity restraint sufficiently > > tight there will be very little deviation from ideality. (Just don't > > make it too tight, or you'll confuse the minimizer.) > > > > -Nat > > _______________________________________________ > > phenixbb mailing list > > phenixbb(a)phenix-online.org > > http://phenix-online.org/mailman/listinfo/phenixbb > > > > ------------------------------ > > -- *************************************************** CPMAS Chen Research Associate University of Pittsburgh School of Medicine Department of Anesthesiology ******************************************************

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Difficult dataset and refinement--P422? I422?Twinning?
by Zhou, Tongqing (NIH/VRC) [E] 07 May '12

07 May '12

Dear All, I have collected several dataset for an antibody:antigen complex. It apparently indexed to P4 space group with large cell dimensions of 191.7000/191.7000/311.0540. When I processed as P1 and ran Pointless, it suggested P422 as space group. P4212 gives the best MR solution with 6 mol/ASU (18 chains total ). However, ridigbody and subsequent refinement with TLS could not reduce the r and r-free which stayed at ~48%. Tried to go P4, P222...(12 mol/ASU) with twinning, but the refinement failed due to insufficient computer memory (12 GB), it was also impossible to refine in P1 due to the same reason. But for some data sets, HKL2000 also gave I4 space group with c axis reduced to 1/3 of that of P4. With the I4 data (~2.7 A resolution), I422 was the best MR space group (1 mol/ASU), the refinement went down to r=29% and r-free=34%, but won't go down further. Please advise if it's possible to use the data Tongqing Tongqing Zhou, Ph.D. Staff Scientist Structural Biology Section Vaccine Research Center, NIAID/NIH Building 40, Room 4609B 40 Convent Drive, MSC3027 Bethesda, MD 20892 (301) 594-8710 (Tel) (301) 793-0794 (Cell) (301) 480-2658 (Fax) ****************************************************************************** The information in this e-mail and any of its attachments is confidential and may contain sensitive information. It should not be used by anyone who is not the original intended recipient. If you have received this e-mail in error please inform the sender and delete it from your mailbox or any other storage devices. National Institute of Allergy and Infectious Diseases shall not accept liability for any statements made that are sender's own and not expressly made on behalf of the NIAID by one of its representatives. ******************************************************************

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how to define a molecule in two rigid_body
by CPMAS Chen 05 May '12

05 May '12

Thanks, Ralf. I am almost at final stage. but the refinement always shows error density at the single bond connecting the two rings. I have tried manually change the orientation of these two rings, but there are two concerns that I thought why I did not get the perfect fitting. 1. I might not choose the perfect the orientation of these rings, and these give me a non-perfect fitting, and high b-factor. 2. since phenix always tries to change the conformation a little bit to fit the density, so this also might cause some errors, or not the conformation I like. Then, my sense is that, if I can somehow define the two parts of the molecule as two rigid bodies, then phenix only can has free rotation of these two rings, and the fitting might come out good. Charles %%%%%%%%%%%%%%%%%%%%% University of Pittsburgh School of Medicine Department of Aneshesiology %%%%%%%%%%%%%%%%%%%%%

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how to define a molecule in two rigid_body
by CPMAS Chen 05 May '12

05 May '12

Dear Colleagues, First, I would say sorry to bother your guys at Friday evening. :) I have a molecule which has two rings connected by a single bond. I would like this two rings freely rotate around the single bond, but keep each ring conformation fixed (as there are sidechains connected to each ring). Could you help me do this rigid_body definition, or suggest a better idea to do so in phenix? Regards and Thanks! Charles Chen. %%%%%%%%%%%%%%%%%%%%% University of Pittsburgh School of Medicine Department of Aneshesiology %%%%%%%%%%%%%%%%%%%%%

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geometry weight optimization with mlhl target
by Mooers, Blaine H.M. (HSC) 04 May '12

04 May '12

Dear Phenixbb, How does one optimize geometry when using the mlhl target in refinement with Henderson-Lattman coefficients in the input reflection file? The wxc_scale keyword is not recognized when I used the mlhl target, but it is recognized when I use the ml target by setting main.use_experimental_phases=false. Blaine Blaine Mooers Assistant Professor Department of Biochemistry and Molecular Biology University of Oklahoma Health Sciences Center S.L. Young Biomedical Research Center Rm. 466 Letter address: Shipping address: P.O. Box 26901, BRC 466 975 NE 10th Street, BRC 466 Oklahoma City, OK 73190 Oklahoma City, OK 73104-5419 office: (405) 271-8300 lab: (405) 271-8313 fax: (405) 271-3910 e-mail: blaine-mooers(a)ouhsc.edu<mailto:[email protected]> webpage: http://www.oumedicine.com/department-of-biochemistry-and-molecular-biology/…

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secondary structure restraints helices/helixes proline
by Morten Groftehauge 04 May '12

04 May '12

Hi guys, How does phenix.refine handle prolines in helices? When I look in high resolution structures it seems that the carbonyl of the residue four positions before the proline (also in the helix) should be more perpendicular than parallel to the helix. This makes sense since there isn't any nitrogen for it to hydrogen bond to. But when I refine my structure it seems as if phenix.refine is trying to push the carbonyl into parallel position. Am I imagining things? It's not the best data and this is a troublesome region. Cheers, Morten -- Morten K Grøftehauge, PhD Pohl Group Durham University

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