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TaBr SAD Autosol and Phaser
by Ian S 09 Apr '18

09 Apr '18

Hi Phenixbb, I trying to phase a structure soaked in TaBr and am running into some technical problems. My anomalous signal goes out to about 4.5 and I have a 1.9A native data set. SIR hasn't worked so far. Autosol SAD has given me several convincing solutions when searching for Tantalum (not TX), finding 11 sites (not clustered) at 6 angstroms: FOM =0.478, map=0.1.3, and corr of 0.82 and a final score of 37+-15.71. By only playing around with the sites I can get an overall score of 73 +- 4.58, map skew to 0.5, and a RMS density to 0.79, thought the R-factor increases to .43 and the FOM . Perhaps not suprisingly at this resolution, the map is uninterpretable (maybe some helices) so I'm trying to improve the phases by model building and by using Phaser and giving it TX sites. If I put the Ta sites in Phaser with the same dataset as Autosol, I get a FOM of .342. If I use the TX label it drops to 0.143. What is happening here? I know that typically TaBr has one or two major sites (my protein isnt massive, only about 1200 aa predicted in the ASU) , so I use only the top two major sites and have the same problem. Does this indicate that the Ta sites Autosol found are not in fact the sites I'm looking for? Or maybe that there really are 10 TaBr sites all with low occupancy? Any suggestions would be appreciated. Thank you. Best, Ian

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linking only one conformer to a thiol
by Krzysztof Bzymek 07 Apr '18

07 Apr '18

Hi, I'm trying to refine a structure with a disulfide bond between a cysteine and a thiol-containing peptide. There are two conformations for the thiopeptide, in one of them the thiol is facing away from SG of the cysteine (~7 A away). I defined the link as: refinement.pdb_interpretation { apply_cif_link { data_link = DRG-CYS residue_selection_1 = chain A and resname DRG and resid 10 residue_selection_2 = chain B and resname CYS and resid 20 } } I also tried: refinement.pdb_interpretation { apply_cif_link { data_link = DRG-CYS residue_selection_1 = chain A and resname DRG and resid 10 and altloc A residue_selection_2 = chain B and resname CYS and resid 20 } } After refinement (with a corresponding data_link.eff file defining the bonds and angles), the thiol of the other conformer that was facing away from the cysteine moves out of the electron density and now faces the cysteine (still about ~5.7 A away). Is there a way to define a link between only one conformer and have the other refine independently? I did try refinement without the above link definition (relying on the proximity of the two cysteines and automatic linking in phenix) but some of the bond and dihedral angles showed higher deviation from ideal values expected for disulfide bonds. Thanks, Chris

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23rd Annual SCSB Symposium, April 28th, Galveston Texas
by Mark A. White 05 Apr '18

05 Apr '18

You and your colleagues are cordially invited to join us for the 23rd Annual Structural Biology Symposium to be held at Levin Hall on the University of Texas Medical Branch Campus on beautiful Galveston Island on Saturday, April 28, 2018. The meeting is organized by the Sealy Center for Structural Biology & Molecular Biophysics and co-sponsored by the Keck Center for Computational & Structural Biology. This year’s speakers include: Grant Jensen, Ph.D., California Institute of Technology “Progress and Challenges in Electron Cryotomography” Ayyalusamy Ramamoorthy, Ph.D., University of Michigan “Membrane-Assisted Dynamic Protein-Protein Interactions by NMR" Andrew Routh, Ph.D., University of Texas Medical Branch “Parallel ClickSeq and Nanopore Deep-Sequencing Characterizes the Emergence and Evolution of Defective Viral Genomes” Joan-Emma Shea, Ph.D., University of California Santa Barbara “Binding and Assembly of Intrinsically Disordered Peptide” Yizhi Tao, Ph.D., Rice University “The Tail of a Nematode Virus” Adam Zlotnick, Ph.D., Indiana University “Multiple Mechanisms for Assembly-Directed HBV Antivirals” To register or for more information, visit http://scsb.utmb.edu/symposium -- Yours sincerely, Mark A. White, Ph.D. Associate Professor of Biochemistry and Molecular Biology, Manager, Sealy Center for Structural Biology and Molecular Biophysics Macromolecular X-ray Laboratory, Basic Science Building, Room 6.658A University of Texas Medical Branch Galveston, TX 77555-0647 Tel. (409) 747-4747 mailto://[email protected] http://xray.utmb.edu QQ: "For a man to conquer himself is the first and noblest of all victories." - Plato

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Rfree and a low resolution data set
by Toon Van Thillo 05 Apr '18

05 Apr '18

Hi all, Currently I am refining a data set which showed anisotropic diffraction. Aimless suggested cutoffs at 2.3, 2.6 and 3.6 angstrom for the h,k and l axis. I chose a general 3.6 cutoff to obtain satisfactory statistics for Rmeas, I/sd(I) and CC1/2. At this resolution the data set consists of approximately 2800 reflections. Generally 5% of the set is set aside as the Rfree test set and I found that a minimum of 500 reflections in total is used to produce a reliable Rfree. However, 5% only amounts to 140 reflections in this case. I am hesitant to include more reflections as I would have to go up to 20% of the reflections to obtain more than 500 reflections for the test set. In a discussion on the CCP4 message boards some time ago it was suggested to do multiple refinements with different test sets: https://www.jiscmail.ac.uk/cgi-bin/webadmin?A2=ind1411&L=ccp4bb&F=&S=&P=125… In the thread it was also discussed that a least squares approach is prefered when using a small test set. However, when using a LS target, the resulting Rfree is very high (10% higher than when using the automatic option) and phenix.refine? produces awful geometry (24% ramachandran outliers, 105 clashcore...). It seems that the refinement is performed without restraints? Optimize X-ray/stereochemistry weight does not result in improved stereochemistry. My question is if the LS approach is still relevant and if so, is there an explanation (and solution) for the bad statistics? Kind regards, Toon Van Thillo

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A new capability on the STARANISO server: "PDBpeep"
by Gerard Bricogne 29 Mar '18

29 Mar '18

Dear all, Ever since the WebGL viewer became available on the STARANISO server, we have found ourselves using it with increasing frequency to take a quick look (a "peep") at the diffraction data deposited with various PDB entries - for example, to try and identify a root cause for some sub-optimal refinement results, or, quite often, just out of sheer curiosity! This involved a totally straightforward procedure whereby the diffraction data file associated with a given PDB entry was downloaded from the PDB and subsequently uploaded to the STARANISO server. Gradually, however, this operation became so popular among some of us that we thought it would be useful to implement this simple procedure as an autonomous capability - and thus was born "PDBpeep" ! You can access this new feature by connecting to http://staraniso.globalphasing.org/cgi-bin/PDBpeep.cgi and enter a PDB code into the box. As indicated on that page, this provides only a cursory look at the overall quality of each dataset, and any further analysis or output can only be obtained by submitting the datafile to the STARANISO server. Better results would clearly be obtainable if the raw images for these datasets had been deposited and could be reprocessed, with the untruncated output of that processing then being submitted to the STARANISO server (reprocessing the images with autoPROC would combine those two steps into a single one). Most of the deposited datasets have been isotropically truncated, and their 3D view in WebGL often suggests that this truncation was too drastic. A number of entries will show infelicities - such as cusps and/or missing angular ranges, or even stripes caused by gaps between the modules of pixel detectors if the beam centre is at a position symmetric relative to those gaps - all marked up in blue. Our purpose in sharing this capability with the community is to bring a further contribution to the process of making everyone more "data quality aware" and keen to scrutinise more closely the protocols by which they collect diffraction data, or have such data collected on their behalf. We will be grateful to receive feedback about PDBpeep, just as we have been about the STARANISO server itself. With best wishes, The STARANISO developers.

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MR Rosetta fails
by Aaron Oakley 29 Mar '18

29 Mar '18

Salvete! I am trying (unsuccessfully) to run MR Rosetta. Im using a MacBookPro running OS X 10.13.3. The job terminates with Sorry: mr_rosetta failed... Sorry routine failed Please see log file for full error message The relevant log file rebuild.log terminates with protocols.jd2.JobDistributor: [ WARNING ] The following options have been set, but have not yet been used: -in:file:extended_pose 1 I get the same error with phenix-1.13-2998 and phenix-dev-3092. I'm using rosetta_bin_mac_2018.09.60072_bundle. Advice accepted with thanks. a++ Aaron Oakley Associate Professor School of Chemistry | Science, Medicine and Health University of Wollongong NSW 2522 Australia T +61 2 4221 4347 | F +61 2 4221 4287

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non-consecutive residue selection in phenix.polder
by Schulz, Eike-Christian 28 Mar '18

28 Mar '18

Dear all, To generate unambiguous ligand density in a number of structures we would like to omit the surrounding active site residues in the polder-map calculation. However, so far we were unable to figure out how non-consecutive residues can be selected for phenix.polder. https://www.phenix-online.org/documentation/reference/atom_selections.html The documentation only explains how to select ranges of residues but not how to select say 5 individual residues. Is there a syntax to achieve this ? Thanks for your advice, Eike

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iNEXT EM and X-ray bundles!
by a.perrakis＠nki.nl 26 Mar '18

26 Mar '18

Dear all, iNEXT, a European program to support access to research infrastructures for Structural Biology, is now welcoming applications for the “EM and X-ray bundles", to help people carry a project all the way from the starting sample to the initial structure determination, by EM or X-rays. To apply for either bundle you need exciting translational research projects, for which you have the macromolecule or complex you want to study in purified soluble form, and you want to engage in structural studies. In a short application form (less than one thousand words overall), you will need to describe the background of the project in your lab and current results, the general scientific background, and the value for translational research (short term but also long term value in any short of biomedical or biotechnological application). No prior experience on either method is required. For the EM bundle, projects will start with an initial characterisation for optimal stability conditions, molecular weight in solution, and negative stain EM (if you send enough sample, we can also try to crystallise it if you wish - just in case!). At this stage you will only need to send the sample to us. Projects that look most promising after that first step, will proceed to optimising the cryo-conditions in one of the iNEXT EM facilities, with your participation. Finally, projects that do best in cryo-optimisation, will proceed to collect data in one of the high-end microscopes available to iNEXT. For the X-ray bundle, projects will start with an initial characterisation for optimal stability conditions, molecular weight in solution, and crystallisation trials. Projects that either yield crystals, or promising precipitates, will proceed to extensive crystallisation trials. For all projects that will yield crystals diffraction data will be collected in one of the fully automated X-ray facilities that collaborate in iNEXT. You will only need to send the sample to us and in most cases physical presence will not be required. It should be emphasised, that no re-application will be needed at any step and iNEXT will cover all experimental expenses, and contribute to travel costs, when needed. There is a single deadline on Friday 20th April 2018. You can Submit a Proposal by clicking here<https://www.structuralbiology.eu/submit-proposal/?t=inext> or go to the iNEXT web site: http://www.inext-eu.org and follow the “Submit Proposal” link. Please note, that given the iNEXT resources availability, this is likely the only call for these “bundle” access. These will serve as pilots for likely requesting future funding from the EU, if such an opportunity becomes available. We note that researchers from all EU and associated countries can apply, but also international labs are eligible, albeit in a more limited capacity. Please forward this email beyond this bulletin list, especially to colleagues that might be less familiar with structural biology. Consider asking this colleague of yous that wanted to do a structural biology project and you never had time or the resources to try hers or his exciting idea! Preliminary results from this bundle access, or a structure, can be the basis for your next grant application! Please also keep in mind that iNEXT remains open to all kinds of Structural Audit, Enhanced Support, and High-End data collection, for projects that involve X-ray Crystallography, SAXS, solution and solid state NMR, EM single particles, EM tomography or CLEM, biophysics for looking at macromolecular interactions, advanced light imaging, and ligand and fragment screening! All these access modalities are listed on our web site! Best regards and looking forward receiving your project, The iNEXT coordination and management team and all iNEXT partners

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Phaser.find_anomalous_substructure error
by Janine Gray 25 Mar '18

25 Mar '18

Hi, I am trying to run phaser.find_anomalous_substructure to identify the anomalous peaks of some phosphorus atoms and get the following error: AttributeError: 'NoneType' object has no attribute 'as_pdb_string' Looking in the log file), it states a file opening error with my PDB file: ****************************************************************************************** *** Phaser Module: AUTOMATED EXPERIMENTAL PHASING 2.8.1 *** ****************************************************************************************** ** Steps: ** Anisotropy correction ** Experimental Phasing ** SAD Phasing CPU Time: 0 days 0 hrs 0 mins 0.02 secs ( 0.02 secs) Finished: Fri Mar 23 15:23:15 2018 ****************************************************************************************** *** Phaser Module: SINGLE-WAVELENGTH ANOMALOUS DISPERSION 2.8.1 *** ****************************************************************************************** Keyword input too long to echo to output (>10000 characters) ------------------------------------------------------------------------------------------ FILE OPENING ERROR: ATOM 1 N MET A 1 5.825 -12.222 -3.663 1.00 89.47 N Has anyone seen this before and/or knows how to fix this? Cheers, Janine

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nucleic acid secondary structure restraint sigmas
by Dyda 24 Mar '18

24 Mar '18

Hi, Does anyone one whether is it possible (and if so how) to change the target sigmas of the H-bonds restraining base pairs? It seems that base pair restraints can do several things: base_pair { enabled = True base1 = chain 'C' and resid 21 base2 = chain 'D' and resid 5 saenger_class = 19 restrain_planarity = False planarity_sigma = 0.176 restrain_hbonds = True restrain_hb_angles = True restrain_parallelity = True parallelity_target = 0 parallelity_sigma = 0.0335 What I'd like to know whether the restrain_hbonds and restrain_hb_angles sigmas can be adjusted. Or is it the case that parallelity_sigma is doing what I'm looking for? Thanks! Fred [32m******************************************************************************* Fred Dyda, Ph.D. Phone:301-402-4496 Laboratory of Molecular Biology Fax: 301-496-0201 DHHS/NIH/NIDDK e-mail:[email protected] Bldg. 5. Room 303 Bethesda, MD 20892-0560 URGENT message e-mail: 2022476710(a)mms.att.net Google maps coords: 39.000597, -77.102102 http://www2.niddk.nih.gov/NIDDKLabs/IntramuralFaculty/DydaFred *******************************************************************************[m

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