Re: [ccp4bb] real real-space-refinement
On 31/07/2020 20:33, James Holton wrote: But, if you real really want to do real real-space, then I suppose coot is doing that? I'm actually not sure. If by "real-space refinement" you mean the same thing as described by Bob Diamond [1], then no, the sum of the squared difference between the calculated and "observed" density is not what is minimized in Coot. Paul. [1]"A real-space refinement procedure for proteins" R.Diamond (1971) Acta Cryst A27 436-452. To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1 This message was issued to members of www.jiscmail.ac.uk/CCP4BB, a mailing list hosted by www.jiscmail.ac.uk, terms & conditions are available at https://www.jiscmail.ac.uk/policyandsecurity/
[ccp4bb] Postdoc position at the University of Pittsburgh, protein design and engineering for drug development
Dear Colleagues, I post this for Dr. Yi Shi at the University of Pittsburgh. *Description:* a postdoctoral position is available in the Shi laboratory of antibody biotechnology and proteomics at the Department of Cell Biology, University of Pittsburgh School of Medicine (UPMC). The laboratory is at the forefront of developing transformative new technologies that integrate proteomics, computational biology, and structural approaches for large-scale antibody (especially camelid VHH antibody/ nanobody) discovery and characterization. The lab is also interested in applying these techniques to develop novel nanobody-based therapies for some of the most devastating diseases including COVID-19, cancer, and neurodegeneration. The lab is currently funded by several grants including the NIH, MJFF, and Alzheimer Foundation. More information about our research could be found here at www.shi-lab.org. For the successful incumbent, the initial appointment is 12 months with the possibility of renewal. Compensation will follow standard NIH Post-Doc salary rates and is based on experience. We strongly support the career development of our colleagues and trainees. A listing of Pitt employee benefits is available at https://www.hr.pitt.edu/current-employees/benefits Highly motivated individuals with a background in biochemistry, bioengineering, biophysics or structural biology (especially membrane protein structural biology) are welcome to apply. Successful candidates should have excellent communication skills and the desire to lead research projects. Interested candidates may submit their CV including names and contact information for two-three references, a short description of their research experience, and research goals to Yi Shi at (yi@pitt.edu). For more information about our research visit: https://www.shi-lab.org/ *Pittsburgh:* As the second-largest city in Pennsylvania, Pittsburgh is best known as "the city of Bridges". In 2015, Pittsburgh was listed among the "eleven most livable cities in the world" and has been considered as one of the most livable cities in the United States. *The University of Pittsburgh is an Equal Opportunity Employer**.* -- - Cheng Zhang To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1 This message was issued to members of www.jiscmail.ac.uk/CCP4BB, a mailing list hosted by www.jiscmail.ac.uk, terms & conditions are available at https://www.jiscmail.ac.uk/policyandsecurity/
Re: [ccp4bb] real real-space-refinement
Dear Thierry, - Le 31 Juil 20, à 21:58, Fischmann, Thierry <22f7fda0a875-dmarc-requ...@jiscmail.ac.uk> a écrit : > Jim wrote: > “ technically, not in Phenix either. The real-space refinement in Phenix > simply > picks peaks in the density and then pulls nearby atoms toward them. . Like a > black hole gobbling up nearby planets (snip)” > Do you have a reference to support this assertion The methodology of the "dirty but very fast" real space refinement in Phenix is described in detail in the article : Afonine, P.V.et al. (2018) "Real-space refinement in PHENIX for cryo-EM and crystallography". Acta Cryst ., D 74 , 531-544. The articel contains also arguments for such kind of procedure, its advantages (the principal goal and advantage is a very high speed) and obvious limitations (one of them mentioned by James). > Hopefully someone from the Phenix team will give some clarifications. But the > statement above indicates that Phenix is capable of performing real space > refinement by some other means than the “black hole approach” Sorry, here I cannot comment. Best regards, Sacha Urzhumtsev To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1 This message was issued to members of www.jiscmail.ac.uk/CCP4BB, a mailing list hosted by www.jiscmail.ac.uk, terms & conditions are available at https://www.jiscmail.ac.uk/policyandsecurity/
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 the wt protein (assuming your protein has one site to bind one titrand)? I have attached a pdf to make it a bit easier to show. It could either be that maltose binds much weaker than the pentamer or that the pentamer binds with 1:5 stoichiometry. In both cases you are able to get an affinity for the pentamer but not for the monomer, whose saturation curve would be too flat to fit. Do you see any dH for the maltose and maltotriose with the mutant, or is it just linear and not fittable like the red curve in the pdf? The reason that you see them however in the structure could be because of higher protein concentration and excess. Saturation depends on both variables. You use 100x molar excess in the crystallization droplet, and maybe a higher protein concentration to easily saturate a weak interaction of, say 5-8mM. In a crystal you got even higher protein concentration which would allow the mutant to bind maltose and maltotriose even better. So, if you see indeed some dH released by the maltose binding to your mutant, I would first try to push the syringe and cell concentrations to the limit. If you see no dH change, follow Philippe's suggestion and changing the temperature would be a good idea. best, matthias Dr. Matthias Barone AG Kuehne, Rational Drug Design Leibniz-Forschungsinstitut für Molekulare Pharmakologie (FMP) Robert-Rössle-Strasse 10 13125 Berlin Germany Phone: +49 (0)30 94793-284 From: CCP4 bulletin board on behalf of DUMAS Philippe (IGBMC) Sent: Saturday, August 1, 2020 10:42:16 AM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Regarding difference in ITC and structure data Monika Did you try ITC experiments at different temperatures ? Delta H may be null, or close to zero, at some temperature without implying that there is no binding ! Philippe Dumas De: "monika chandravanshi" À: "CCP4BB" Envoyé: Samedi 1 Août 2020 09:57:24 Objet: [ccp4bb] Regarding difference in ITC and structure data Dear All, I am working on a carbohydrate-binding protein, which co-crystallizes with maltose, maltotriose, maltotetraose and maltopentaose and the same can be supported by ITC experiments as well. Also, the mutant protein (X2Y) co-crystallizes with maltose, maltotriose, maltotetraose and maltopentaose, however, the binding of only maltotriose and maltotetraose could be observed through ITC. For your information, the ITC conditions are the same for all the ligands and the ligand concentration used in ITC is same as used in crystallization (100x of protein concentrations). Moreover, from structural analysis, we have observed that the binding mode of all ligands is the same. I request your suggestion on why maltose and maltopentaose do not show any binding to the mutant protein in ITC experiments. Looking forward to suggestions. Best Regards, Monika To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1 To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1 To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1 This message was issued to members of www.jiscmail.ac.uk/CCP4BB, a mailing list hosted by www.jiscmail.ac.uk, terms & conditions are available at https://www.jiscmail.ac.uk/policyandsecurity/ ITC_vs_crystal.pdf Description: ITC_vs_crystal.pdf
Re: [ccp4bb] real real-space-refinement
The isolde suggestion already made is an excellent one. The hardest part of that is getting the right version of chimeraX working. But, once you've done that its pretty straightforward. Hopefully not for too much longer. All going well, ISOLDE 1.0 (to work in ChimeraX 1.0) should be out this week. On 2020-07-31 20:33, James Holton wrote: Not in CCP4, no. And, technically, not in Phenix either. The real-space refinement in Phenix simply picks peaks in the density and then pulls nearby atoms toward them. Like a black hole gobbling up nearby planets. It took me a while to realize that! If you manage to turn off geometry restraints (as I eventually did) all the atoms end up on top of each other. Might seem like a horrible idea, but for poor resolution data and reasonably good geometry restraints it has a high radius of convergence and is incredibly fast when compared to "real real-space refinement". Refining against map voxels directly is a very very slow process. But, if you real really want to do real real-space, then I suppose coot is doing that? I'm actually not sure. The isolde suggestion already made is an excellent one. The hardest part of that is getting the right version of chimeraX working. But, once you've done that its pretty straightforward. One program that has not been mentioned, but does "real real" space refinement is: "rsref" https://chapman.missouri.edu/wp-content/uploads/sites/2/software/rsref/html/rsref_doc.html It is not too hard to install and use. I can't say I've gotten results appreciably different from reciprocal-space refinement, and that led me to ask myself why exactly I thought it would be different. The Fourier transform is symmetric after all. But I do expect that if you have unmodeled regions, such as big, spiky metals, or large tracts of disordered, ropy stuff, then localizing the refinement could be beneficial. Now, of course, you can also do localized refinement in reciprocal space by just smoothing out parts of the map that are "uninteresting". The vast area of noise around the protein in a cryoEM map, for example, is perhaps a candidate for noise suppression. The only trick is how to suppress noise without creating systematic error. For example, if your model does not have "bulk solvent" then this area will be modeled as vacuum, but if you simply set the map voxel values to 0.00, you will have effectively created more bulk solvent, not eliminated it. This is because 0.00 is usually the average voxel value, not the "vacuum level". Then there is the "edge" between the modified and unmodified areas. Unless you smooth it in some way this edge will be very sharp and therefore have significant Fourier coefficients at a wide range of resolutions. So, if you are not careful, your "noise suppression" can create a lot more error than it eliminates. As for what to do? The scale factor given to the "bulk solvent" model is perhaps the best value to use to replace the "bulk" solvent region. The bulk solvent mask itself, ranging from 0 to 1, might also be a reasonable weighting function for combining your original map with a single-valued map. That is, don't change the protein, but flatten the solvent. You can get this map out of refmac using the MSKOUT feature. You then smooth it in reciprocal space by applying refmac's best-fit solvent B factor using sfall and fft, then finally scale it with mapmask. I should admit, however, that I have not tried this in a while. Let me know if it works! HTH -James Holton MAD Scientist On 7/29/2020 8:20 AM, Schreuder, Herman /DE wrote: Dear BB, I would like to do a real real-space-refinement of a protein against a cryo-EM map; not the mtz-based Refmac approach. A quick internet search produced a lot of Phenix hits, but little ccp4 hits. Does somebody know how to do this using ccp4 programs, or has someone a Coot script to do this? Thank you for your help! Herman - To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1 - To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1 To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1 This message was issued to members of www.jiscmail.ac.uk/CCP4BB, a mailing list hosted by www.jiscmail.ac.uk, terms & conditions are available at https://www.jiscmail.ac.uk/policyandsecurity/
Re: [ccp4bb] Regarding difference in ITC and structure data
Monika Did you try ITC experiments at different temperatures ? Delta H may be null, or close to zero, at some temperature without implying that there is no binding ! Philippe Dumas De: "monika chandravanshi" À: "CCP4BB" Envoyé: Samedi 1 Août 2020 09:57:24 Objet: [ccp4bb] Regarding difference in ITC and structure data Dear All, I am working on a carbohydrate-binding protein, which co-crystallizes with maltose, maltotriose, maltotetraose and maltopentaose and the same can be supported by ITC experiments as well. Also, the mutant protein (X2Y) co-crystallizes with maltose, maltotriose, maltotetraose and maltopentaose, however, the binding of only maltotriose and maltotetraose could be observed through ITC. For your information, the ITC conditions are the same for all the ligands and the ligand concentration used in ITC is same as used in crystallization (100x of protein concentrations). Moreover, from structural analysis, we have observed that the binding mode of all ligands is the same. I request your suggestion on why maltose and maltopentaose do not show any binding to the mutant protein in ITC experiments. Looking forward to suggestions. Best Regards, Monika To unsubscribe from the CCP4BB list, click the following link: [ https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1 | https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1 ] To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1 This message was issued to members of www.jiscmail.ac.uk/CCP4BB, a mailing list hosted by www.jiscmail.ac.uk, terms & conditions are available at https://www.jiscmail.ac.uk/policyandsecurity/
[ccp4bb] Regarding difference in ITC and structure data
Dear All, I am working on a carbohydrate-binding protein, which co-crystallizes with maltose, maltotriose, maltotetraose and maltopentaose and the same can be supported by ITC experiments as well. Also, the mutant protein (X2Y) co-crystallizes with maltose, maltotriose, maltotetraose and maltopentaose, however, the binding of only maltotriose and maltotetraose could be observed through ITC. For your information, the ITC conditions are the same for all the ligands and the ligand concentration used in ITC is same as used in crystallization (100x of protein concentrations). Moreover, from structural analysis, we have observed that the binding mode of all ligands is the same. I request your suggestion on why maltose and maltopentaose do not show any binding to the mutant protein in ITC experiments. Looking forward to suggestions. Best Regards, Monika To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1 This message was issued to members of www.jiscmail.ac.uk/CCP4BB, a mailing list hosted by www.jiscmail.ac.uk, terms & conditions are available at https://www.jiscmail.ac.uk/policyandsecurity/
