Re: [ccp4bb] unknown density

can confirm jon´s comment. I find these in virtually every high-res structure. some of them wobble a bit given the AA close by, such as Arg. Dr. Matthias Barone AG Kuehne, Rational Drug Design Leibniz-Forschungsinstitut für Molekulare Pharmakologie (FMP) Robert-Rössle-Strasse 10 13125 Berlin

Re: [ccp4bb] Characterising potential drug-binding pockets

Hi Sergei I assume that you used the term "drug-like fragments" because you would like to sort out candidates? The paper referenced by Bernhard is a good way to go, but it reflects exactly the problem of PPIs, namely that proteins with well defined binding pockets for substrates (such as

Re: [ccp4bb] B-factors very high

4 Jan 2021 at 15:03, Barone, Matthias mailto:bar...@fmp-berlin.de>> wrote: dear silvia, apart from the resolution, it would be good to know your current refinement strategy and if you fed the structure to pdbredo yet. How did that turn out? the polygon values are picked from structure

Re: [ccp4bb] B-factors very high

dear silvia, apart from the resolution, it would be good to know your current refinement strategy and if you fed the structure to pdbredo yet. How did that turn out? the polygon values are picked from structures at similar resolution afaik. In any case, a better list of B-factors is the one

Re: [ccp4bb] Micro/Macro crystal seeding experience

Hi Raphael I can second Tom and Matthews comments. We got crystals only after including an ion exchange column (even though we work with a GST construct cleaved off with thrombin). Well diffracting crystals then only grow by seeding in reduced precipitant concentration. Additionally, the seeds

Re: [ccp4bb] R free rising

Hi Nika You said you refine with phenix, correct? And that refining the phaser solution yielded in Rwork of 22% and a gap of 7%. For refining the rebuilt model, you had to open a new phenix.refine job, did you provide the native mtz there? Or did you maybe refine against the output mtz of the

Re: [ccp4bb] Cysteine oxidation product

Hi Dhiraj how did you measure the affinities? What are the two affinities, incl standard error (or even amount of data points you used in the titration exp)? Im just asking because Im curious as what you mean by "increase". If you add DDT to an ITC for example, this will create a lot of

Re: [ccp4bb] ligand bound to only one chain in the crystal

Hi Christian One of our proteins crystallizes always as non-crystallographic dimer. We occasionally find inhibitors bound to a second non-canonical site. Usually, the inhibitors bound to the second binding sites are sufficiently resolved only on one of the protein dimer. In these cases I often

Re: [ccp4bb] AW: [ccp4bb] over-fitting? over-refinement?

Eleanor rises a very important practical point here..."sidechains at the solvent interface have multiple conformations, and that as a result the water networks should also have partial occupancies". I was fighting with such a model for half a year and also tested XSHEL (there was a thread in

Re: [ccp4bb] How to save two superposed protein structures in PDB format

Hi Abhik Additional to Phils suggestions, you can use chimera to superpose and render the images right away. For this, load the pdb codes right into chimera (file -> fetch ID), then tools -> structure comparison -> match maker Change the Chain pairing to specific chains in ref and match

Re: [ccp4bb] Regarding difference in ITC and structure data

Hi Monika. Can you please give us some more information about the ITC experiments? Does the affinity change between maltose, maltotriose, maltotetraose and maltopentaose or do they all bind equally strong? And, does N stay at roughly equimolar ratios or is there a trend from 1 towards 0.2 for

Re: [ccp4bb] Help With Setting up a Stereo view system

Hi Marian I used the quadro 4000 myself and had two Acer monitors connected to it, one with display port, one with VGA connector. Only the VGA showed 3D, the other would not. Have you tried going analog with this card? best, matthias Dr. Matthias Barone AG Kuehne, Rational Drug Design

Re: [ccp4bb] protein oligomer

You could try SERp? Gave some good suggestions for our non-cristallizing paralog. However, some of the mutants I created did not express well/were rather insoluble. So that might be a bad idea in your case.. anyway here's the link: https://services.mbi.ucla.edu/SER/ Best, Matthias

Re: [ccp4bb] Looking for suggestions with protein expression

Hi Umar Artem is right, we should have some more infos about how you proceed after disrupting the cells. The part I find curious is your usage of "usually". Usually soluble when? Before you centrifugate the sample? Before you go into the äkta? That would be a first hint to know what you mean

Re: [ccp4bb] Ligand building

Hi Hari If I understood you correctly, you want to modify and subsequently dock it onto a complex structure, right? For me, the following workflow turned out to be most straightfwd: - build it in MOLOC (http://www.moloc.ch/). It lets you very easily modify an existing molecule without the

Re: [ccp4bb] Ligand Refinement in coot

do you mean earlier whne coot was connected to phenix during the refinement? If you load the pdb and the mtz directly into coot, you have to load the cif file beofre starting the real space refinement in coot. Dr. Matthias Barone AG Kuehne, Rational Drug Design Leibniz-Forschungsinstitut für

Re: [ccp4bb] superimposition of 3D structures with the DNA part only

Hi Fred Chimera let's you choose to selectively superpose chains of one entry onto a selective chain of the second. The easiest way is to assign the dsDNA into a separate chain before loading the pdb (if this is not already the case). If you want to selectively superpose only certain atoms of

Re: [ccp4bb] A question of density

hey Jessica a tip that might come up later on anyway: once you put every reasonable bit into the desity, what I like to to when facing such blobbs: I take a well defined water out to create a diff density at a position where I know it is real. Having a feeling of how much you have to contour

Re: [ccp4bb] refinement of 0.73A data in shelxl

10 13125 Berlin Germany Phone: +49 (0)30 94793-284 From: bogba...@yahoo.co.uk Sent: Thursday, February 6, 2020 5:01:14 PM To: Barone, Matthias Cc: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] refinement of 0.73A data in shelxl Hello, hope I can help. OK, so

Re: [ccp4bb] refinement of 0.73A data in shelxl

7:14:25 PM To: Barone, Matthias Cc: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] refinement of 0.73A data in shelxl Hi Matthias, did you use correct model parameterization and optimal refinement strategy for the resolution? Such as: - Add H atoms; - Refine all but H atoms with anisotropic ADPs

Re: [ccp4bb] nVidia 3D Vision2 glasses

Berlin Germany Phone: +49 (0)30 94793-284 From: Dirk Kostrewa Sent: Thursday, January 9, 2020 11:33:45 AM To: Barone, Matthias; CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] nVidia 3D Vision2 glasses Hi Matthias, although, Nvidia announced that the final driver w

Re: [ccp4bb] nVidia 3D Vision2 glasses

Hi Patricia All modern graphics cards still support 3D vision, but you are correct that nvidia is addressing critical issues in release 418 only till April. So if you got a new card, you should not run into problems anytime soon. Just make sure the card is supported with the new kernel

Re: [ccp4bb] Potential weak binding ligand in the active site

Hi Katherine I'm regularly struggling with inhibitors that bound at non-canonical sites and bind there with different conformations. So the situation in these complex-structures could be a bit similar to your situation. In our case, the inhibitors bind on the non-canonical site with roughly Kd

Re: [ccp4bb] why does Coot ignore CONECT?

Hi Pavel I work with peptide-scaffold chimeras on a daily basis. Initially, I tried to include the scaffolds as HETATM and use JLigand to create the links between the scaffolds and the natural amino acids. This procedure however is rather fussy and by now I build the chimera in silico, using

Re: [ccp4bb] ITC question -dimer vs monomer

No, the law of mass action does not depend on whether A is titrated into B or vice versa. The heat measured is proportional to the complex formed, relative to one partner being kept at constant concentration (the referece in the cell). The law of mass action can be re-written to describe the

Re: [ccp4bb] ITC question -dimer vs monomer

As Reza already pointed out, ITC cannot tell you anything about the sub-processes that underlay an equilibrium, be it complicated like 2A+B2 <-> AB2 + A <-> A2B2, or with (virtually) no intermediate 2A+B2 <-> A2B2 due to a fast second forward reaction , or a simple one-to-one model A+B2 <->

Re: [ccp4bb] challenges in structural biology

Something that pops up here routinely, connected to 5) what does "resolution" really mean?: While new methods are being implemented to improve the accuracy of models or to deal with weak data, how to convince editors to accept the new methods? cheers, matthias Dr. Matthias Barone AG

Re: [ccp4bb] AKTA FPLC fractionation peak by peak

Hi sorin The problem in your elution profile is that you cannot define a global threshold as the baseline is heavily bent (relative to the peak heights) and starting to fractionate at 6' is not the problem, telling the script to stop once you reach baseline at 13' is. I would try what you

Re: [ccp4bb] Issue with high Rfree (0.25) for a high-resolution dataset (1.05 Ang)

I agree with Pavel... if you got so many observations you dont need TLS but use anisotropic adps. What I want to add is the dangerous comment to "cutting back data ... to improve Rfact" Of course, if you lower the amount of observations while keeping the parameters to fit constant will lower

Re: [ccp4bb] Creating map volume of custom size

I use CCP4 mapmask to exted the map some Å beyond the asymmetric unit in order to see the density at crystal contacts Dr. Matthias Barone AG Kuehne, Rational Drug Design Leibniz-Forschungsinstitut für Molekulare Pharmakologie (FMP) Robert-Rössle-Strasse 10 13125 Berlin Germany Phone: +49

Re: [ccp4bb] Compound with flexible conformation but nM Kd

we also do structure-activity relationship and rational drug design. And I agree with Christian: never rely on one single method and try to include a homogenous assay, such as ITC or FT. you mention a tyrosine involved in the binding pocket. Did you try to track the Tyr in FT? best, matthias