Re: [ccp4bb] current location for the Superimposé web server ?
Hello, Follow up concerning my query to the bb (in case anyone is interested), reply received from Robert Preissner through Philip E. Bourne and Joel Sussman: The Superimposé server was stopped in May 2018. Fred. Vellieux On 2/28/20 7:47 AM, Fred Vellieux wrote: *Hello, I am looking for the Superimposé web server (from Charité, Berlin). * *This web server must have moved from the place where it is supposed to reside, farnsworth.charite.de , not found. Does anyone know where this web server resides now ? TIA, Fred. * To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB&A=1 To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB&A=1
Re: [ccp4bb] CCP4BB Digest - 27 Feb 2020 to 28 Feb 2020 (#2020-61)
Hi again, First, I wish to thank everyone for their responses. I hope that no one minds that I include them in my response letter to the Editor ? The idea of citing the Karplus and Diedrichs paper in Science has been essentially consumed already in our first response letter, which accompanied the submission of the revised manuscript, and did not work. (Instead of the Science citation from 2012 we used a quote from the paper suggested below by Karplus and Diederichs, Curr Opin Struct Biol. 2015 Oct; 34: 60–68. ) As Phoebe mentioned, it would be good to use the momentum. My intention of writing to the bulletin board was not only to get help with argumentation, but also to raise the issue of how to address resolution cutoffs in crystallographic data in publications to avoid such situations in general. I also think that the issue is more complicated than the last shell criterion with I/Isig > xx, Rmerge < yy, or cc1/2 > zz, or ... 1. Number of reflections in a shell effects the numbers significantly. The larger numbers of the shells there are the larger the numbers will be. The number of reflections in a shell depends also on the highest resolution and unit cell size. Hence we have a dependance of potential criteria on several parameters. 2. Refinement and data processing programs use different numbers of shells and even different ways of calculating shells. REFMAC typically uses 20 equal volume sliced shells), PHENIX is more complicated, as far as I understand (shell number may depend on the number of TEST set reflections in individual shell, shells can be defined according to equal slicing volume, some kind of log dependency or even linearly according to real space resolution), in MAIN there are 20 shells by default, but one can choose any of the mentioned slicing rules. I suggest that we use this discussion to shape up guidelines that can later proposed for consideration to the IUCr committee for macromolecules. I prefer soft as opposed to strict borders. In the end, the structures do not speak for themselves, but are a mean to support one or more biologically relevant conclusions. ??? best wishes, dusan turk > On 29 Feb 2020, at 01:00, CCP4BB automatic digest system > wrote: > > > Date:Fri, 28 Feb 2020 16:03:22 + > From:"Phoebe A. Rice" > Subject: Re: What resolution - X-ray diffraction round this time > > Can we get some momentum for the "standard table 1" including TWO numbers - > outer limit used in refinement, and nominal resolution based on some standard > such as I/sigI =2 (or 3, or whatever the community can agree on)? That would > hopefully cut down on all the reviewer complaints of overstated resolution. > > ~~~ > Phoebe A. Rice > Dept. of Biochem & Mol. Biol. and > Committee on Microbiology > https://voices.uchicago.edu/phoebericelab/ > > > On 2/28/20, 6:56 AM, "CCP4 bulletin board on behalf of Malý Martin" > wrote: > >Dear colleagues, > >I agree with all the previous responses, it is a pity to throw away >useful high-resolution data. The problem of high-resolution cutoff >estimation is also nicely summarized in another paper by Andrew Karplus >and Kay Diederichs "Assessing and maximizing data quality in >macromolecular crystallography" >https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4684713/ . It is suggested >using CC1/2 for the selection of the cutoff for data processing (not >I/sigI or R_whatever). Later on, the decision should be validated >performing the paired refinement protocol. > >Good luck with the argumentation. >Martin > > >On 2/28/20 11:08 AM, LMB wrote: >> Ask the referee - (apart from the other suggestions here) >> >> ‘How would removing data Improve my model?” >> >> Sent from my iPad >> >>> On 28 Feb 2020, at 08:22, dusan turk wrote: >>> >>> Hi, >>> >>> Browsing through the recent discussion on EM data resolution cutoff >>> it occurred to me that the X-ray diffraction community isn’t that >>> unanimous either. >>> >>> My stand: >>> >>> When the default resolution cutoff provided with the data processing >>> software in electron density map calculation and refinement delivers >>> quality maps noisier than expected and/or too high R-factors I start >>> adjusting the resolution cutoff by lowering the resolution and trying >>> alternative space group. Hence, I allow the data processing programs >>> to suggest where to draw the line (be it CC1/2, I/sigI, R merge, R >>> sym, R p.i.m. and R r.i.m, …) , unless there are problems. >>> >>> Doing so, I came into a dispute with a referee who shaped his request: >>> >>> "It is well accepted that the criteria for resolution cutoff should >>> consider both I/SigI and Rmerge for the outer most shell. For data >>> sets collected at synchrotron sources, the criteria of I/SigI > 5 and >>> Rmerge <50% can be taken as a good practical reference.” >>> >>> So where do we stand? Which are t
Re: [ccp4bb] Hydrogens in PDB File
Dear Dale, your last sentence is of great importance: "leaving the (hopefully) manually inspected and curated Hydrogen atoms in the deposited PDB" I believe this hope is unrealistic. Most people do probably not think or understand what refinement programs do about hydrogen atoms. In Refmac5 it has long been an option to generate hydrogen atoms for refinement but do not put them out into the PDB file. Like Ethan, I believe this is best practice. Of course, in case hydrogen atoms have been curated, one may leave them in for deposition. It is not useful to see all the H-atoms in Coot, and chemists omit hydrogen atoms as well even for 2D drawings. @Matthew Whitley: Adding hydrogen atoms in calculated (riding) positions should be rather independent of resolution of the data, since their major role is in improving anti-bumping restraints, and since their major contribution to the diffraction data is in the low resolution data. Best, Tim On Sunday, March 1, 2020 9:26:29 AM CET Dale Tronrud wrote: > Dear Ethan, > >To move away from an abstract discussion of hydrogen atoms I'd like > to describe a concrete example. In 2008 I deposited a model of the FMO > (Bacteriochlorophyll containing) protein. The ID code is 3EOJ. The > model was refined to a data set cut off at 1.3 A resolution using the > criteria of the day. I used shelxl for the final stage of refinement > and added riding hydrogen atoms to the mix. When I deposited the model > I succumb to peer pressure and removed the hydrogen atoms. > >If you look at the map calculate by the Electron Density Server you > will see many peaks in the Fo-Fc map indicating the missing hydrogen > atoms. (I have attached a screen-shot from Coot but I recommend that > you fire up Coot and explore the map yourself.) In my picture you can > see the three peaks around a methyl group. Above and to the left is the > peak for the hydrogen of a CH bridging atom in the Bacteriochlorophyll-a > ring. To the right and in the distance is a peak for the hydrogen of a > CH2 group. Not every hydrogen is represented by a positive peak, but > they exist throughout the map. This Fo-Fc map is useless for the > purpose of assessing the quality of my model, since the true residuals > are hidden among all these hydrogen peaks. > >A critic might say that these peaks are simply the result of the > model being biased toward the presence of hydrogen atoms and therefore > an artifact. A model refined to this data set w/o hydrogen atoms would > not likely show peaks indicating that hydrogen atoms need to be built. > >I would say that the map calculated from a Hydrogen-free model is the > biased one. I am 99% confident in the location of most of the riding > hydrogen atoms and leaving them out results in a model that is > fantastically unlikely. The absence of peaks in an apo map is a flaw in > that map. I would describe it as "vacuum bias". "Biasing" a model > toward reality is not a problem. > >This example shows that the current PDB is incompatible with models > whose Hydrogen atoms have been stripped. To get proper maps and > validation reports one has to either preserve the Hydrogen atoms in the > model, or modify all the software that uses coordinate files to add the > hydrogen atoms back in. That is a major programming task, which the > authors have, apparently, been unwilling to do. > >I will go further and disagree with you that even this is a solution. > It is very difficult to add the Hydrogen atoms back into 3EOJ, and I > expect this difficulty is the reason software has not been written that > successfully does it. > >There are two major problems to be overcome in 3EOJ. shelxl has an > option to twirl the methyl groups and select the torsion angle with the > best fit to the map. The hydrogen atoms in the pictured methyl group > weren't built as staggered -- All values for the torsion angle were > tested and it happens that the best fit placed them in a staggered > conformation. That is a much more interesting result. There are other > methyl groups around the edges of the Bchl-a molecules that are crowded > and the methyl groups are observed to have torsion angles that are not > standard for riding Hydrogen atoms. The neighboring methyl groups avoid > H-H bumps by twisting and that twist can be detected by shelxl in the > 1.3 A data. > >The second problem is the matter of Histidine residues. There are > two Nitrogen atoms in the side chain. A hydrogen atom could be on > either one, and sometimes both have hydrogens. A very clever program > could work out from the hydrogen bonding pattern the most likely > placement, but I've not seen any program that is very good with hydrogen > bonding networks. Worst still, I've often seen programs place the > hydrogen atom *between* the Nitrogen and Magnesium atoms of a Histidine > ligand to a Bacteriochlorophyll a. This mistake will certainly lead to > very bad geometry! >
Re: [ccp4bb] Hydrogens in PDB File
Hi Dale, You make very valid points and there are good reasons to keep the refined hydrogen positions (methyl twists an protonation of HIS are good examples). There is a way of distinguishing refined a modelled hydrogens in mmCIF and we should start using that. About protonation of hustidines: WHAT IF does quite a decent job although there is room for improvement around ligands. About maps from EDS: These were (and perhaps are) made by running 0 cycles of unrestrained refinement in Refmac. Unrestrained refinement takes away the need to sort out restraints which was an absolute nightmare at the time. A side-effect of unrestrained refinement is that Refmac cannot (could not) add hydrogens. PDB-REDO needs restraints anyway so this does 0 cycles restrained refinement to generate the first maps and adds hydrogens in the process. This addition is not that sophisticated, but it should make the maps better. Have a look https://pdb-redo.eu/db/3eoj.zip Cheers, Robbie On 1 Mar 2020 09:26, Dale Tronrud wrote: Dear Ethan, To move away from an abstract discussion of hydrogen atoms I'd like to describe a concrete example. In 2008 I deposited a model of the FMO (Bacteriochlorophyll containing) protein. The ID code is 3EOJ. The model was refined to a data set cut off at 1.3 A resolution using the criteria of the day. I used shelxl for the final stage of refinement and added riding hydrogen atoms to the mix. When I deposited the model I succumb to peer pressure and removed the hydrogen atoms. If you look at the map calculate by the Electron Density Server you will see many peaks in the Fo-Fc map indicating the missing hydrogen atoms. (I have attached a screen-shot from Coot but I recommend that you fire up Coot and explore the map yourself.) In my picture you can see the three peaks around a methyl group. Above and to the left is the peak for the hydrogen of a CH bridging atom in the Bacteriochlorophyll-a ring. To the right and in the distance is a peak for the hydrogen of a CH2 group. Not every hydrogen is represented by a positive peak, but they exist throughout the map. This Fo-Fc map is useless for the purpose of assessing the quality of my model, since the true residuals are hidden among all these hydrogen peaks. A critic might say that these peaks are simply the result of the model being biased toward the presence of hydrogen atoms and therefore an artifact. A model refined to this data set w/o hydrogen atoms would not likely show peaks indicating that hydrogen atoms need to be built. I would say that the map calculated from a Hydrogen-free model is the biased one. I am 99% confident in the location of most of the riding hydrogen atoms and leaving them out results in a model that is fantastically unlikely. The absence of peaks in an apo map is a flaw in that map. I would describe it as "vacuum bias". "Biasing" a model toward reality is not a problem. This example shows that the current PDB is incompatible with models whose Hydrogen atoms have been stripped. To get proper maps and validation reports one has to either preserve the Hydrogen atoms in the model, or modify all the software that uses coordinate files to add the hydrogen atoms back in. That is a major programming task, which the authors have, apparently, been unwilling to do. I will go further and disagree with you that even this is a solution. It is very difficult to add the Hydrogen atoms back into 3EOJ, and I expect this difficulty is the reason software has not been written that successfully does it. There are two major problems to be overcome in 3EOJ. shelxl has an option to twirl the methyl groups and select the torsion angle with the best fit to the map. The hydrogen atoms in the pictured methyl group weren't built as staggered -- All values for the torsion angle were tested and it happens that the best fit placed them in a staggered conformation. That is a much more interesting result. There are other methyl groups around the edges of the Bchl-a molecules that are crowded and the methyl groups are observed to have torsion angles that are not standard for riding Hydrogen atoms. The neighboring methyl groups avoid H-H bumps by twisting and that twist can be detected by shelxl in the 1.3 A data. The second problem is the matter of Histidine residues. There are two Nitrogen atoms in the side chain. A hydrogen atom could be on either one, and sometimes both have hydrogens. A very clever program could work out from the hydrogen bonding pattern the most likely placement, but I've not seen any program that is very good with hydrogen bonding networks. Worst still, I've often seen programs place the hydrogen atom *between* the Nitrogen and Magnesium atoms of a Histidine ligand to a Bacteriochlorophyll a. This mistake will certainly lead to very bad geometry! Until an hydrogenation program is written that can handle all ligands, all hydrogen bonding networks (even overlapping partial
Re: [ccp4bb] Postdoctoral positions(2) in cryo-EM
Dear all, Just a reminder of the Postoctoral positions currently open in our laboratory. The positions are for 15 months initially, but extendable to up to 7 years! Harry up applying shortlisting of candidates started already! Best wishes, Ale PS. Get in touch with me directly if you require further info > On 10 Feb 2020, at 19:55, Alessandro Vannini wrote: > > Dear all, > I would like to draw your attention to two postdoctoral positions available > immediately in my laboratory to work on cryo-EM determination of complexes > operating at the interface of SMC complexes and the transcription apparatus. > The projects are well suited for individual with good experience in modern > Cryo-EM pipelines. For some projects datasets have been already collected and > await further processing while others samples are “cryo” ready. The > postdoctoral fellows will be based at the ICR in South Kensington (Glacios, > F20 in house + consortium Krios + eBIC) but is expected that will also > benefit from the top notch EM facility currently being set up at the Human > Technopole, Milan Italy (Krios, Glacios, Talos, Aquilos, CLEM and room > temperature tomography 300 Kv scope). > If interested please apply here: > > https://icr.tal.net/vx/appcentre-ext/brand-0/candidate/so/pm/1/pl/1/opp/995-Postdoctoral-Training-Fellow-in-Cryo-EM/en-G > > For general enquiries please write me directly. > > Cheers, > > Alessandro > > To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB&A=1
