- phenixbb - phenix-online.org

TLS refinement and interpretation of the B factors
by 向嵩 09 May '22

09 May '22

Dear Phenix users, I have sent this message to phenixbb before I subscribed to it and did not see it show up in the bulletin board. I am sending it again and apologize for the possible duplicated messages. I have been trying to understand the B factors generated by TLS refinements and appreciate any help in this subject. In my particular case, I was trying to analyze the dynamics of different portions of a protein molecule by looking at the B factors. The structure was refined with the isotropic B factor and TLS and the TLS groups were determined automatically. After the refinement, I tried to analyze the TLS matrices with phenix.tls_analysis. The analysis failed for many of the TLS groups, the program complains that some of the matrices are not positive semidefinite. I have several questions related to this: 1, in cases like this, can I tweak the initial values of the matrix elements or add constrains to make the TLS refinement results physically meaningful? i.e. the refined TLS matrices can be interpreted in terms of elemental motions. I noticed that Afonine et al mentioned in a recent paper (doi: 10.1107/S2059798318005764) that a large fractions of TLS matrices in the PDB databank can not be interpreted by elemental motions. I wonder if there is a way to make the refined matrices more meaningful in the current implementation of TLS refinement in phenix? 2, what is the "best" B-factor to look at in an analysis for protein dynamics? I am interested in the dynamics of portions of protein that are smaller than the TLS groups. I am guessing that if the TLS matrices are not physically meaningful, one should look at the overall B-factors? If these matrices are meaningful, is it a good practice to look at the residual B factors (overall B-factor minus the TLS contributions)? 3, how to calculate the residual B factors? I could not find a manual for phenix.tls mentioned in the phenix refinement instruction. I tried with the extract_tls=true option with my PDB and got a PDB file with some negative B factors. Is it another sign that the refined TLS matrices are not physically meaningful or am I using the phenix.tls program wrong? Your help in this subject is greatly appreciated. Song Xiang

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Diffraction Methods in Structural Biology - Gordon Research Conference - July 24-29, 2022
by James Holton 07 May '22

07 May '22

Many of you may recall approximately 1000 years ago we were looking forward to getting together for another great Diffraction Methods GRC. Now, after a 4-year break, the meeting is on! https://www.grc.org/diffraction-methods-in-structural-biology-conference/20… It will be in-person at Bates College in Lewiston, ME, USA, on July 24-29 of 2022. Strange how it is strange to be considering meeting in person, but recent GRCs have proven they can be conducted safely. We've learned a lot about viruses in recent years, both in our lives and in our labs. Artificial Intelligence has come a long way, and the role of biological structure, and indeed science in general, is impacting the everyday lives of human beings more than ever before. It is time we got together to talk about all this. Yes, we've gotten a lot of work done remotely, but some things just have to wait until you are face-to-face. Preferably over a Maine lobster dinner. GRCs are not about listening to talks, they are about the discussion that comes after. Newcomers and Veterans sharing and debating ideas until far too late at night. It is my sincere hope that fighting this virus, and looking toward a brighter future, will inspire even more visionary and collaborative ideas for the role structure will play in that future. I can't imagine a better theme of discussion for this next meeting. Some may also recall that in 2020 we were accepting tax-deductible donations to help attendees from underrepresented groups overcome the financial barriers to GRC attendance. Those funds are still available, and donations are also still possible. I ask that applicants who feel they may qualify please self-identify to me, off-list, in an email. It is my goal to bring as many diverse backgrounds and points of view as possible into this meeting, because that is what makes for the most productive discussions. -James Holton MAD Scientist -James Holton MAD Scientist and Chair of the 2020/2022 Diffraction Methods GRC

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get list of missing atoms table in validation
by Wei Wang 05 May '22

05 May '22

Dear all, I would like to get an externally readable list of the missing atoms table in “Summary" session of "Comprehensive validation (X-ray/Neutron)". Currently it seems the information is integrated and clickable only inside the gui window. I want to access the table via sed. Is there a way to extract that table out? Best regards, Wei

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2022 Cold Spring Harbor Macromolecular Crystallography Course
by Paul Adams 03 May '22

03 May '22

Dear Colleagues, The July 15th deadline for applications to the CSHL Macromolecular Crystallography Course to be held later this year, October 10 through October 25 2022, is rapidly approaching. This is a unique opportunity to become expert in the use of crystallography to solve challenging macromolecular structures. The official course announcement can be found here: https://meetings.cshl.edu/courses.aspx?course=C-CRYS&year=22 please pass this on to anyone who you think might be interested and would benefit, especially students and postdocs. This comprehensive course is an outstanding place to learn both the theoretical and practical aspects of macromolecular crystallography, and consists of extensive lectures and practical sessions from experts in the field. The instructor team is: Janet Newman, Tom Peat, Jim Pflugrath, Tassos Perrakis, and myself. We are assisted by world recognized experts in crystallization, data collection, data processing, structure solution, refinement, and validation (see the course link above for more details). The goal of the course is for researchers who have had some exposure to structural biology to become experts in crystallography. This is an experience in macromolecular crystallography learning that cannot be found anywhere else, and the student/teacher ratio is often 1:1. We expect participants to crystallize one or more proteins and determine their structures in a two week period. Students may also work on their own projects, but not exclusively. They will become very well-versed in the theory of diffraction and crystal structure determination, while also having lots of fun. Both synchrotron data collection and in-house diffraction data collection will be used to solve structures. The course will also include the use of computationally predicted structures to aid in solving the crystallographic phase problem. The course is limited to 16 participants due to the very hands-on nature of the experiments and the intimate seminar and laboratory settings. Please check the above web link for more details. In particular, please note the information about the various fellowships, scholarships, and stipends that are available. The course is supported with funds provided by the National Institute of General Medical Sciences, for which we are extremely grateful. Also there are stipends available from the Leona M. and Harry B. Helmsley Charitable Trust and the Howard Hughes Medical Institute to help offset the cost of tuition. Scholarship support is also provided by Regeneron Pharmaceuticals. If anyone has any questions, please send me an e-mail, we will be happy to answer all queries. -- Paul Adams (he/him/his) Associate Laboratory Director for Biosciences, LBL (https://biosciences.lbl.gov/leadership/) Principal Investigator, Computational Crystallography Initiative, LBL (https://cci.lbl.gov) Vice President for Technology, the Joint BioEnergy Institute (http://www.jbei.org) Principal Investigator, ALS-ENABLE, Advanced Light Source (https://als-enable.lbl.gov) Division Deputy for Biosciences, Advanced Light Source (https://als.lbl.gov) Laboratory Research Manager, ENIGMA Science Focus Area (http://enigma.lbl.gov) Adjunct Professor, Department of Bioengineering, UC Berkeley (http://bioeng.berkeley.edu) Member of the Graduate Group in Comparative Biochemistry, UC Berkeley (http://compbiochem.berkeley.edu) Building 33, Room 250 Building 978, Room 4126 Building 977, Room 268 Tel: 1-510-486-4225 http://cci.lbl.gov/paul ORCID: 0000-0001-9333-8219 Lawrence Berkeley Laboratory 1 Cyclotron Road BLDG 33R0345 Berkeley, CA 94720, USA. Executive Assistant: Michael Espinosa [ MEEspinosa(a)lbl.gov ] [ 1-510-333-6788 ] Phenix Consortium: Ashley Dawn [ AshleyDawn(a)lbl.gov ][ 1-510-486-5455 ] --

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Phenix/Coot issue
by Xavier Brazzolotto 30 Apr '22

30 Apr '22

Dear Phenix and Coot users and developers I've recently observed a kind of conflict between Phenix and Coot. I am using Phenix v1.20.1-4487 and Coot 0.9.8.1 from thee CCP4 8.0 suite under macOS Monterey (v12.3.1). If I launch Coot through Phenix via the « Open in Coot » of a refine job for example (the path to Coot has been defined in Phenix preferences), Coot opens correctly and displays the structure with the « Connected to Phenix tab". However if I wand to do some RSR then I get an error message stating "Refinement setup failure » : « Failed to find restraints for » and a list of the residues involved in RSR. In parallel if I use Coot by itself (launched through the terminal), RSR works well… but upon opening Coot states that there is some "Nomenclature errors » with a list of different residues. I wonder if there would be some conflict between residue libraries between Phenix and Coot ? Just to leet you know I’ve also recently tried to install/comlplie the Coot v1 pre-version to have a loot at it and I do also have libccp4 installed in my Homebrew. Maybe everything comes from that ? Xavier

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3Fe4S clusters in real-space refinement
by Rhys Grinter 28 Apr '22

28 Apr '22

Hi PHENIX BB, I'm currently refining a structure with 3Fe4S clusters into some high resolution cryoEM maps (1.57 Ang). However, refinement messes up the Fe4S cluster, by moving the existing Fe and S atoms into the wrong positions in the density. Is anyone aware of this issue and/or knows of a simple way to prevent this from happening? Cheers, Rhys -- Dr Rhys Grinter Lab Head Molecular Physiology of Microbial Pathogens (MP2) Lab Monash University +61 (0)3 9902 9213 +61 (0)403 896 767

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Re: [phenixbb] NaN of AlphaFold
by Randy John Read 26 Apr '22

26 Apr '22

Did you try looking at a surface representation of the structure? The one that Pavel generated from a more simplistic random sequence looked vaguely plausible as a chain trace, but had very large cavities when viewed as accessible surface, e.g. in ChimeraX. Randy > On 24 Apr 2022, at 01:36, James Holton <jmholton(a)lbl.gov> wrote: > > I downloaded all sequences from the PDB to do a frequency analysis, which I think will enable a more sophisticated "random peptide" than giving all 20 amino acids equal likelihood with no neighbor correlations. Turns out the most common heptapeptide in the PDB is: > XXXXXXX > > because, apparently, NCBI lists gaps like this. If I eliminate "X", the most common heptapeptide is: > > SHHHHHH > > Right. His tags. If I eliminate those, the next one is: > GLVPRGS > thrombin cleavage site. Ugh! > > Cleaving off those, the next one is: > AAAAAAA > apparently also used to denote unknown amino acids. > > Ignoring those, the next one is: > YFPEPVT > this is from the human antibody heavy chain. Thousands of those in the PDB. This made me realize that there is going to be a lot of "homology bias". > > I next tried the NCBI refseq_select to try and get something non-redundant, and then I get: > GSGKSTL > lots of ABC transporters. >10k of this sequence in the db. > > On the other hand, of the ~330e6 heptapeptide sequences I'm looking at, 52% of them only appear once. Another 19% of my list of unique sequences occur twice, 9% occur 3x, etc. It is a VERY steep curve. Only 4% of my sample sequences occur more than 10 times, and 0.05% occur more than 100x. I never thought of the PDB this way, but I take this as indicative of how much repetition there is in the sampling. Perhaps I need to brush up on my statistics, but I did not expect this. > > So, I said "heck with it" and simply extracted all heptads that only occur EXACTLY ONCE in the PDB. There are 7.4e6 of them. I chose one at random as the starting 7 residues: FLACISE (from 6ekr). Rather than simply appending random heptads, I took the final ISE and extracted all one-off heptads that begin with ISE. There are 1905 of those. I chose one at random: ISENDGN (from 6ixy). I then extracted heptads beginning with DGN, etc. Final sequence of randomly-assembled yet abbuting heptads from random places in the PDB is then: > > > random1 > FLACISENDGNDGDKSNLAQDTDLALGTWVRDISIALRSGFIAEEYPAWWKESVVPYYNQLNGN > DIEHSHSALLPTGIYFLEIPLVGTRDVALDTEGETTEEAFRHLDGIVGHPIVIGRRAADGGPLVIRGL > NEVLLRLSEVYNAWKLLKVQEILKGLEAYNRAMDYETILREGTWKPAGYTPNALAGGAQCGNAD > IVNERIISDGVEASQLATLERRENRALQTALEIENGELEQKWGRLPSAXPAVERQRYRGPSTATW > EGFSTDGRFPSEVDIFDRETLDGVEGLAKDVEALKGVLMGLFAPYWKWCTHNCIGYAARFVALN > KAIRTALTFARTHSNNETIHNEVVGMIPDIDIAVSDINSQEYTKTRSERQGGVLKEHLNALNAKIEPS > VNLKVSA > > A quick BLASTP shows a surprisingly high homology match: > <o4TgiNP8vaFOcUkl.png> > > I mean... not great, but higher than I'd expect for "completely random" ? > > > The alphafold2 colab result looks like this: > > > <rHffn0Fa0WfwR4Yg.png> > > So still lots of helical content. This shape makes me want to make this sequence and see if it folds. It probably doesn't. But if this thing weren't red I'd wonder. > > Discuss? > > -James > ----- Randy J. Read Department of Haematology, University of Cambridge Cambridge Institute for Medical Research Tel: +44 1223 336500 The Keith Peters Building Fax: +44 1223 336827 Hills Road E-mail: rjr27(a)cam.ac.uk Cambridge CB2 0XY, U.K. www-structmed.cimr.cam.ac.uk

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Refining cyclosporin A
by Armando Albert 22 Apr '22

22 Apr '22

Dear All, We are trying to refine a protein complex with cyclosporin which is a non conventional cyclic peptide. I wonder what is the best strategy to build the cif file. Shall we define the cif file for individual non-conventional amino acids and then link them or shall we define the whole structure as an individual ligand? Does any one have the corresponding cif file? Armando

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Validation of structure with modified residue
by Xavier Brazzolotto 21 Apr '22

21 Apr '22

Dear Phenix users, I don’t know if my problem is related to Phenix but for information I’m running Phenix 1.20.1-4487 under MacOS 12.3.1. I’ve finalized a structure where a ligand covalently modified the protein. I’ve generated the modified residue (named SLG for serine modified by ligand). For this I’ve generated the molecules in SMILES and used eLBOW to generate the restraints. Then I’ve modified the cif file defining the molecule as a L-peptide and replacing the atom names of the Serine part (CA, CB, OG, C, O, N, and OXT) In coot (from CCP4 : 0.9.6 EL), I’ve used the modified cif file and it allowed merging of the modified residue into the polypeptide chain as expected and further refinements went without any issue in Phenix (providing the modified cif file of course). Everything seems well interpreted. So far so good. However, now I would like to validate the structure and both Phenix validation tool and the PDB web server do not accept the final cif file. Checking this file I’ve noticed that the protein seems split into 3 pieces (chain A, first residue up to the one before the modified residue; chain B the modified residue by itself described as HETATM and chain C the rest of the polypeptide up to the C-ter). The PDB file presents only one chain A for the whole protein with the modified residue... I don’t know if this is an issue with Phenix generating this final cif file in this specific case or if I need to modify this final file by hand ? Any help is welcome. Thanks Xavier

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ligand at two-fold
by Patrick Loll 14 Apr '22

14 Apr '22

Hello all, I have a dimer in which the two protomers are related by a crystallographic 2-fold. There is a peptide ligand that sits in the interface between the two protomers. This means that the ligand will be present in two orientations, and I’ve set the occupancies to 0.5. However, the ligand is pushed out of density during refinement, presumably because orientation #1 is repelling orientation #2 (to be clear, orientations #1 and #2 are symmetry mates). To deal with this, I include a parameter file in the GUI that contains the following instruction: custom_nonbonded_symmetry_exclusions = "chain X” [where chain X is the peptide ligand] However, the refinement fails, with the following error message: "Unused parameter definitions: Please check the input file(s) for spelling errors and obsolete parameter definitions." This would seem to imply that the custom_nonbonded_symmetry_exclusions command is obsolete, but it’s still found in the list of all available keywords (?). Any help would be welcome. Thanks, Pat --------------------------------------------------------------------------------------- Patrick J. Loll, Ph. D. (he, him, his) Professor of Biochemistry & Molecular Biology Drexel University College of Medicine Room 10-102 New College Building 245 N. 15th St., Mailstop 497 Philadelphia, PA 19102 USA pjl28(a)drexel.edu pjloll(a)gmail.com

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