- phenixbb - phenix-online.org

Solving MR solution without sequence information
by Rosenberg, Eric (NIH/NCI) [F] 06 Feb '23

06 Feb '23

Hi all, I'm in a bit of bind here and am seeking some advice. For context, a former graduate student in our lab set crystal trays of an MBP fusion protein, the fused part after MBP being ~400 amino acids long. This region is also predicted to be mostly unstructured, but has a C-terminal SH3 domain. Our graduate student then graduated and before throwing out some of her trays a year or two later, we found some hits of the MBP fusion protein that actually diffracted to 2.9 Angstrom. I spent some time working on it after we collected the data (June 2021), but because I didn't know what crystallized specifically, it was impossible to phase, and replicating seemed next to impossible, too. The Matthew's coefficient was predicting ~130 amino acids in the ASU, space group C222 or C2221. Since whatever crystallized was clearly a degradation product of the MBP fusion, I tried phasing with SH3 domains and a lot of other things to no avail. As a final last ditch effort I eventually submitted the .mtz file to SBGrid to perform a Wide Search MR job, and low and behold it actually found MR solutions that had TFZ scores ~17 in space group C2221! So here's my current situation-I have been able to phase the data set using the MR search model, but again, I don't know what specifically it is that I've crystallized. I'm currently able to get the Rfree to ~0.4, but can't seem to improve it. I am really at a loss of what to do, since there are obvious backbone issues with the protein (as seen from iterative build composite omit maps), but every time I try to manually correct them it seems to make the Rfree worse. The MR solution does not align very well at all to the MBP fusion, only ~20 identity, and again, I don't know to which ~130 amino acids I crystallized out of the ~400 of the MBP fusion. Is it one continuous stretch, two copies of a shorter stretch, etc.? I tried phasing with a polyalanine model of the MR search model and then tried autobuilding just a polyalanine sequence to get the backbone right, but that doesn't seem to work. Autobuild also fails when trying to various fragments of the MBP fusion sequence. Other than opening coot and manually building the entire polypeptide chain, is there an easier method? I think that once the backbone is totally right the phases will improve so I can start putting in side chains, but I'm not sure. My latest effort is to just use Sculptor prior to Phaser in order to force the sequences to match, but again, I don't know precisely what sequence was crystallized. I have tried both the Phenix and CCP4 software suites, for reference. Any and all help would be much appreciated (and yield an acknowledgement on a paper, if this ever works). Best, Eric Rosenberg CRTA Postdoctoral Fellow Randazzo Lab Laboratory of Cellular and Molecular Biology National Cancer Institute, US

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Re: [phenixbb] Solving MR solution without sequence information
by Dom Bellini 04 Feb '23

04 Feb '23

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Re: [phenixbb] Solving MR solution without sequence information
by Kay Diederichs 03 Feb '23

03 Feb '23

Hi Eric, I'd try the following: 1) use SIMBAD to potentially find other/better models 2) use ISOLDE for model building - maybe ask Tristan Croll for help 3) submit your best model to the DALI server to retrieve closer models HTH, Kay -- Kay Diederichs http://strucbio.biologie.uni-konstanz.de email: Kay.Diederichs(a)uni-konstanz.de Tel +49 7531 88 4049 Fachbereich Biologie, Universität Konstanz, Box M647, D-78457 Konstanz This e-mail is digitally signed. If your e-mail client does not have the necessary capabilities, just ignore the attached signature "smime.p7s".

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Postdoctoral position in YSBL, University of York
by Jon Agirre 31 Jan '23

31 Jan '23

Dear all, Applications are invited for a Postdoctoral Research Associate position, funded by the Royal Society, for up to 30 months to work with Dr Jon Agirre in the Department of Chemistry. The successful applicant will join a vibrant team of researchers at the heart of the world-leading York Structural Biology Laboratory (YSBL). The work of the laboratory over the years has included the delivery of world-leading methods and experimental discoveries, training and nurturing hundreds of outstanding scientists in the process. Role In collaboration with Dr. Elisa Fadda (Maynooth University, Ireland), we have developed proof-of-concept software that models protein glycosylation into AlphaFold models. This task has been automated and integrated into the new Python interface of the carbohydrate-specific Privateer software. In parallel, we have developed infrastructure and analyses that allow us to get a clear picture of glycans and their interactions. We expect that this technology will enable the successful applicant to, based on existing glycobiological knowledge, extend the Privateer software to predict, build and adapt models of glycans onto AlphaFold models. Main duties and responsibilities: - To develop and extend the Privateer software to predict, build and adapt models of glycans onto AlphaFold models. - To further our existing collaborations with CCP4, CCP-EM, PDB-REDO, the Protein Data Bank and the AlphaFold Protein Structure Database. For informal enquiries: please contact Dr Jon Agirre, jon.agirre(a)york.ac.uk To apply: https://jobs.york.ac.uk/vacancy/research-associate-513495.html Department The Department of Chemistry is one of the largest and most successful departments at York and we are renowned internationally for our research. As a department, we strive to provide a working environment that allows all staff and students to contribute fully, to flourish, and to excel. We are proud of our Athena Swan Gold Award. The Department of Chemistry values all employees for the qualities and skills they bring to the workplace and aims to be a diverse and egalitarian community in which all can thrive. Many thanks for making it this far in the advert! With best wishes, Jon -- Dr Jon Agirre [image: A button with "Hear my name" text for name playback in email signature] <https://www.name-coach.com/jon-agirre> Royal Society Olga Kennard Research Fellow CCP4 ED&I champion | instruct-ERIC representative @ 3D-Bioinfo (Elixir) York Structural Biology Laboratory, Department of Chemistry University of York, Heslington, YO10 5DD, York, UK http://www.york.ac.uk/chemistry/research/ysbl/people/staff/jagirre/ Office: /B/K/065 Phone: +44 (0) 1904 32 8252 Twitter: @glycojones

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Feb 9 Free Webinar: New Dimensions - How Computation Is Accelerating Structure Based Drug Design
by Mitchell D. Miller 31 Jan '23

31 Jan '23

Dear all, On Thursday, February 9, 2023 from 12:00 p.m. to 1:00 p.m. ET (17:00-18:00 UTC) Karen Akinsanya, President of Research & Development - Therapeutics, Schrödinger, Inc will present a zoom webinar titled New Dimensions - How Computation Is Accelerating Structure Based Drug Design This zoom webinar is free, but registration is required. Please see the registration link on the National Academies of Sciences, Engineering, and Medicine event page along with Karen Akinsanya's abstract and bio. https://www.nationalacademies.org/event/02-09-2023/new-dimensions-how-compu… A flyer for the webinar is also available https://www.nationalacademies.org/documents/embed/link/LF2255DA3DD1C41C0A42… This is the second presentation in the series -- Advancing Drug Discovery: A Webinar Series of the National Academies of Sciences, Engineering, and Medicine The series is being hosted jointly by the U.S. National Committee for Crystallography (USNC/Cr), the U.S. National Committee for the International Union of Pure and Applied Chemistry (USNC/IUPAC) and the U.S. National Committee for CODATA. More information about the series is on the NASEM website and it will be updated as additional webinars in the series are confirmed. The website also has links to recordings of "past events". https://www.nationalacademies.org/our-work/advancing-drug-discovery-a-webin… Please forward this to any you think would be interested. Sincerely, Mitch P.S. for those who missed the January 10, 2023 webinar presented by Ken Duncan, Deputy Director, The Bill & Melinda Gates Foundation on Pandemic Preparedness: Accelerating the Discovery of New Therapeutics The recording is available on the National Academies of Sciences, Engineering, and Medicine event page along with Ken Duncan's abstract and bio. https://www.nationalacademies.org/event/01-10-2023/pandemic-preparedness-ac…

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RapiData at SSRL 2022 - application deadline extended by two weeks!
by Smith, Clyde 12 Jan '23

12 Jan '23

We have extended the deadline for applications for a place at the RapiData 2022 @ SSRL workshop to next Friday, February 4th 2022! There are still a few spots left, so get your application in! The workshop will be held virtually from May 4 - 14 2022. The course will comprise lectures on the following topics: Specimen preparation, data collection strategies, X-ray light sources, X-ray detectors, data reduction, structure solution by MAD, SAD and molecular replacement, complementary methods (spectroscopy, small angle scattering and cryoEM). There will also be several days of software tutorials (HKL3000,XDS, CCP4/DIALS, Phenix, AutoSharp/AutoProc, Shelx, Coot) and you will all get to connect remotely to the SSRL beamlines to collect both monochromatic and MAD/SAD data. There is also an opportunity to ship your own samples to SSRL prior to the workshop, and collect your own data. Please visit https://www-ssrl.slac.stanford.edu/rapidata/rapidata-2022/index.html and use the links on the "registration" page to apply for the course. Successful applicants will be notified later in February 2022 and invited to register at that time (there will be no fee for registration). Regards, Clyde Smith (csmith(a)slac.stanford.edu) and Silvia Russi (srussi(a)slac.stanford.edu) Workshop Organizers

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Jan 10 Free Webinar: Pandemic Preparedness: Accelerating the Discovery of New Therapeutics
by Mitchell D. Miller 03 Jan '23

03 Jan '23

Dear all, On Tuesday, January 10, 2023 from 12:00 p.m. to 1:00 p.m. ET (17:00-18:00 UTC) Ken Duncan, Deputy Director, The Bill & Melinda Gates Foundation will present a zoom webinar titled Pandemic Preparedness: Accelerating the Discovery of New Therapeutics This zoom webinar is free, but registration is required. Please see the registration link on the National Academies of Sciences, Engineering, and Medicine event page along with Ken Duncan's abstract and bio. https://www.nationalacademies.org/event/01-10-2023/pandemic-preparedness-ac… A flyer for the webinar is also available https://www.nationalacademies.org/documents/embed/link/LF2255DA3DD1C41C0A42… This is the first presentation in a new series -- Advancing Drug Discovery: A Webinar Series of the National Academies of Sciences, Engineering, and Medicine The series is being hosted jointly by the U.S. National Committee for Crystallography (USNC/Cr), the U.S. National Committee for the International Union of Pure and Applied Chemistry (USNC/IUPAC) and the U.S. National Committee for CODATA. More information about the series is on the NASEM website and it will be updated as additional webinars in the series are confirmed. https://www.nationalacademies.org/our-work/advancing-drug-discovery-a-webin… Please forward this to any you think would be interested. Sincerely, Mitch

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staraniso/phenix.refine
by Andrea Piserchio 22 Dec '22

22 Dec '22

Dear all, I am trying to validate and then (hopefully) deposit a structure generated using the autoproc/staraniso staraniso_alldata-unique.mtz file as input for phenix.refine. Autoproc also produces a cif file ( Data_1_autoPROC_STARANISO_all.cif) specifically for deposition. Long story short, the PDB validation server complains about the lack of a freeR set for both files. I realized that, at least for the cif file, the r_free_flag is missing (but why does the .mtz for the isotropic dataset work??),so I then tried to use for validation the *.reflections.cif file that can be generated by phenix.refine. This file can actually produce a validation report, but I still have some questions: 1) Is it proper to use the .reflections.cif file for this purpose? During the upload I do see some errors (see pics); also, the final results show various RSRZ outliers in regions of the structure that look reasonably good by looking at the maps on coot, which seems odd ... 2) In case the *.reflections.cif is not adequate/sufficient for deposition (I sent an inquiry to the PDB, but they did not respond yet), can I just add a _refln.status column to the autoproc cif file (within the loop containing the r_free_flag) where I insert “f” for r_free_flag = 0 and “o” everywhere else? Thank you in advance, Andrea

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Labeling centric and acentric reflections
by Andre LB Ambrosio 22 Dec '22

22 Dec '22

Hi, Is there a way to label centric and acentric reflections in the .mtz file and output an additional column, let's say, with values 0 (acentric) and 1 (centric), similar to R_free flags? Many thanks for your attention. -- Andre LB Ambrosio Associate Professor, IFSC/USP - Brazil www.ifsc.usp.br/alba

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Re: [phenixbb] staraniso/phenix.refine
by Gerard Bricogne 21 Dec '22

21 Dec '22

Dear Andrea, As Clemens Vonrhein is not not subscribed to the phenixbb, I am sending the message below on his behalf. With best wishes, Gerard. ---------------------------------------------------------------------------- Dear Andrea, Besides the paragraph in the Release Notes of our latest BUSTER release that Luca directed you to, you may find it useful to consult the following Wiki page: https://www.globalphasing.com/buster/wiki/index.cgi?DepositionMmCif Although it has not yet been updated so as to mention explicitly the cases where refinement was done with REFMAC or phenix-refine, the procedure and commands given there will work in these two cases as well. Our intention was to get feedback from our users first, then to announce the extended capability of aB_deposition_combine more broadly - but your question clearly shows that we should make that announcement sooner rather than later. Getting back to your message: although uploading either the phenix-generated mtz file or the mmCIF file generated by aB_deposition_combine would be acceptable to the OneDep system, we would highly recommend using those from aB_deposition_combine for several reasons. First of all, these files should already contain the data from Phenix but also provide additional data blocks with richer reflection data (and metadata!), going right back to the scaled and unmerged data without any cut-off applied. Furthermore, it should ensure that the correct data quality metrics (i.e. merging statistics) are included into the mmCIF files uploaded during deposition. Of course, you don't need to use aB_deposition_combine to generate such a set of mmCIF files (model and reflection data) - these are after all just text files - but the devil is often in the details. It may be useful to add some fairly general background information in order to avoid misunderstandings and false impressions - so here are some relevant points you may wish to consider. (a) The deposition of model and associated data into the PDB should allow for two things: (1) the validation of the current model and of its parametrisation on their own, as well as a check against the data used during refinement, and (2) the provision of additional data to allow further analysis of, and improvements to, the data handling as well as the model itself. For the first task one needs to deposit the data exactly as used by the depositor to arrive at the current model, i.e. the input reflection data (as used as input to the refinement program) plus all available Fourier coefficients (as output by that refinement program) needed to compute the maps used in modeling. The second task requires deposition of less and less "processed" versions of the experimental data - ultimately going back to the raw diffraction images. This might involve several sets of reflection data, e.g. (i) scaled, merged and corrected reflection data, (ii) scaled and merged data before correction and/or cutoff, and (iii) data scaled and unmerged before outlier rejection - plus additional variants. (b) When going from the raw diffraction images towards the final model, a lot of selection and modification of the initial integrated intensities takes place - from profile fitting, partiality scaling, polarization correction, outlier rejection, empirical corrections or analytical scale determination and error model adjustments, all the way the application of truncation thresholds (isotropically, elliptically or anisotropically), conversion to amplitudes (and special treatment of weak intensities) and anisotropy correction. There are often good reasons for doing all or some of these (with sound scientific reasons underpinning them - not just waving some "magic stick"), even if under special circumstances a deviation from standard protocols is advisable. What is important from a developer's viewpoint is to provide users with choices to influence these various steps and to ensure that as many intermediate versions of reflection data as possible are available for downstream processes and deposition. (c) At deposition time, the use of a single reflection datablock is often not adequate to provide all that information (e.g. refinement programs might output not the original Fobs going in, but those anisotropically rescaled against the current model - so a second block might be needed to hold the original Fobs data, free from that rescaling). If different types of map coefficients are to be provided, they too need to come in different mmCIF datablocks (2mFo-DFc and mFo-DFc for observed data only; 2mFo-DFc filled in with DFc in a sphere going out to the highest diffraction limit; 2mFo-DFc filled in with DFc for only the reflections deemed "observable" by STARANISO; F(early)-F(late) map coefficients for radiation damage analysis; coefficients for anomalous Fourier maps etc). So ultimately we need to combine (going backwards): the refinement output data, the refinement input data and all intermediate versions of reflection data (see above) ... ideally right back to the scaled+unmerged intensities with a full description of context (image numbers, detector positions etc). This is what the "Data Processing Subgroup" of the PDBx/mmCIF Working Group has been looking at extensively over the last months, and about which a paper has just been submitted. (d) autoPROC (running e.g. XDS, AIMLESS and STARANISO) and the STARANISO server provide multi-datablock mmCIF files to simplify the submission of a rich set of reflection data. autoPROC provides two versions here: one with traditional isotropic analysis, and the another for the anisotropic analysis done in STARANISO. It is up to the user to decide which one to use for which downstream steps. To help in combining the reflection data from data processing with that from refinement, and in transferring all relevant meta data (data quality metrics) into the model mmCIF file, we provide a tool called aB_deposition_combine: it should hopefully work for autoPROC (with or without STARANISO) data in conjunction with either BUSTER, REFMAC or Phenix refinement results. At the end the user is provided with two mmCIF files for deposition: (1) a model file with adequate data quality metrics, and (2) a reflection mmCIF file with multiple datablocks all the way back to the scaled+unmerged reflection data. (e) It is important at the time of deposition to not just select whatever reflection data file happens to be the first to make it through the OneDep system, as this can often lead to picking up the simplest version of an MTZ or mmCIF file, but to choose, if at all possible, the most complete reflection data file containing the richest metadata. Otherwise we simply increase the number of those rather unsatisfying PDB entries whose reflection data files contain only the very minimum of information about what they actually represent and what data quality metrics (such as multiplicity, internal consistency criteria etc) would have been attached to them in a richer deposition. *** Please note that the current OneDep system seems to complain about the fact that unmerged data blocks come without a test-set flag column: this looks to us like an oversight, since test-set flags are attributes belonging to the refinement process, so that it makes no logical sense to require them for unmerged data. This will probably requires some rethinking/clarification/changes on the OneDep side. A final remark: Instead of trying to give the impression that there is only one right way of doing things, and therefore only a single set of reflection data that should (or needs to) be deposited, it would seem more helpful and constructive to try and provide a clear description of the various "views" of the same raw diffraction data that can be provided by the various approaches and data analysis methods we have at our disposal. Together with more developments regarding the PDBx/mmCIF dictionary, and coordinated developments in the OneDep deposition infrastructure, this will enable better and richer depositions to be made, helping future (re)users as well as software developers. Cheers, Clemens ---------------------------------------------------------------------------- -- On Mon, Dec 19, 2022 at 05:42:24PM -0500, Andrea Piserchio wrote: > So, > > Both the phenix-generated mtz file (silly me for not checking this first) > and the cif file generated by aB_deposition_combine can be uploaded on the > PDB server. > > Thank you all for your help!! > > Andrea > > On Sat, Dec 17, 2022 at 5:06 PM Pavel Afonine <pafonine(a)lbl.gov> wrote: > > > Hi, > > > > two hopefully relevant points: > > > > - phenix.refine always produces an MTZ file that contains the copy of > > all inputs plus all is needed to run refinement (free-r flags, for > > example). So if you use that file for deposition you have all you need. > > > > - Unless there are strongly advocated reasons to do otherwise in your > > particular case, you better use in refinement and deposit the original > > data and NOT the one touched by any of available these days magic sticks > > (that "correct" for anisotropy, sharpen or else!). > > > > Other comments: > > > > > - However, CCP41/Refmac5 does not (yet) read .cif reflection files. As > > > far as I know, Phenix Refine does not (yet) neither. > > > > Phenix supports complete input / outputs of mmcif/cif format. For > > example, phenix.refine can read/write model and reflection data in cif > > format. It's been this way for a long time now. > > > > Pavel > > > > > > On 12/16/22 17:32, Andrea Piserchio wrote: > > > Dear all, > > > > > > > > > I am trying to validate and then (hopefully) deposit a structure > > > generated using the autoproc/staraniso staraniso_alldata-unique.mtz > > > file as input for phenix.refine. > > > > > > Autoproc also produces a cif file ( Data_1_autoPROC_STARANISO_all.cif) > > > specifically for deposition. > > > > > > Long story short, the PDB validation server complains about the lack > > > of a freeR set for both files. I realized that, at least for the cif > > > file, the r_free_flag is missing (but why does the .mtz for the > > > isotropic dataset work??),so I then tried to use for validation the > > > *.reflections.cif file that can be generated by phenix.refine. This > > > file can actually produce a validation report, but I still have some > > > questions: > > > > > > 1) Is it proper to use the .reflections.cif file for this purpose? > > > During the upload I do see some errors (see pics); also, the final > > > results show various RSRZ outliers in regions of the structure that > > > look reasonably good by looking at the maps on coot, which seems odd ... > > > > > > 2) In case the *.reflections.cif is not adequate/sufficient for > > > deposition (I sent an inquiry to the PDB, but they did not respond > > > yet), can I just add a _refln.status column to the autoproc cif file > > > (within the loop containing the r_free_flag) where I insert “f” for > > > r_free_flag = 0 and “o” everywhere else? > > > > > > > > > Thank you in advance, > > > > > > > > > Andrea > > > > > > _______________________________________________ > > > phenixbb mailing list > > > phenixbb(a)phenix-online.org > > > http://phenix-online.org/mailman/listinfo/phenixbb > > > Unsubscribe: phenixbb-leave(a)phenix-online.org > > > _______________________________________________ > 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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