Dear Paul and Jon, Thank you for your replies and comments. I guess I can provide a bit of background: there are 8 monomers in the asymmetric unit (534 residues each) and each chain is glycosylated with Asn-NAG-NAG-BMA-MAN-MAN (assumed from the electron density map ; the protein was expressed and secreted from insect cells). So there are 40 sugars in total in the structure. Resolution is 2.4 A, space group is P1, and current R and Rfree are 17.4 and 21.4. I’m using the very last distribution of both Coot and CCP4.
I definitely used the carbohydrate module in Coot to build the sugars (this feature is just fantastic!). I then used refmac (with weight = 0.02) to refine the structure using the default monomer libraries for the sugars, but explicitly providing the correct LINKR records (NAG-ASN, BETA1-4, BETA1-4, ALPHA1-3, ALPHA1-2) . However this led to their relatively distorted geometries. I have now tried “Refine Tree” in Coot for all 8 carbohydrate trees, and send the structure straight to PDB validation (without refinement in Refmac). The number of bond length outliers went down from 4 to 0, and the number of bond angle outliers went down from 125 to 34. There is definitely an improvement there. So the problem seems to lie with the default monomer libraries for the sugars provided with the CCP4 distribution that I used for refinement. I must admit that I did not look into these and assumed they would be fine. So Jon, yes please: if you don’t mind sending these through that would be great ☺! Hopefully this will tightened things a bit and solve the issue. On a related note, is there a way to export the monomer definitions from Coot to feed into Refmac? Cheers, Cyril From: CCP4 bulletin board <CCP4BB@JISCMAIL.AC.UK> On Behalf Of Jon Agirre Sent: Thursday, 18 March 2021 10:57 pm To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Enhanced Validation of Small-Molecule Ligands and Carbohydrates Dear Cyril and Paul, COVID struggles permitting, we will _very_ soon have Acedrg dictionaries for most pyranoses in the monomer library. These contain validated conformers, updated torsion restraints and, of course, modern geometry. Cyril: would you like to test these dictionaries with your structure? Best wishes, Jon On Thu, 18 Mar 2021 at 08:56, Paul Emsley <pems...@mrc-lmb.cam.ac.uk<mailto:pems...@mrc-lmb.cam.ac.uk>> wrote: The geometry targets for protein are well established (by which I mean "agreed upon") and so the distribution On 18/03/2021 07:19, Cyril Hamiaux wrote: Dear Jasmine (and all), Many thanks for the article. I have a question regarding the Z cutoffs used to flag geometry outliers in the PDB validation reports: geometry outliers are flagged with |Z|>2 for ligands (including carbohydrates), but with |Z|>5 for proteins. What is the rational for using different criteria for ligands vs proteins? After all isn’t this just one structure? The geometry targets for protein are well established (by which I mean "agreed upon") and so we can presume that the distribution for refinement is similar to that of the validation. One cannot say as much for ligands at the moment. the wwPDB wants to flag only what they have confidence are wrong. In the CCP4 world, yes, it's all homogeneous (or that is the intent (carbohydrates in particular need some attention in the monomer library (I'd recommend that you just run Acedrg to update the dictionaries (or use Coot, where this have been done for you))), but the wwPDB doesn't score with CCP4 software (not today at least). PS. I am refining a 2.4 A resolution structure of a N-glycosylated protein and end up with a large number of geometry outliers for the sugars when I generate PDB validation reports (especially angle outliers). Are you using/did you use the N-linked glycan builder in Coot? because this really shouldn't happen if you are (it uses modern Acedrg-based dictionaries). If not, you can still use "Refine Tree" and it should (might?) do something helpful. The huge majority of these outliers are flagged with |Z|<5 (ie they would not be flagged if they were protein bonds or angles). The electron density for the sugars is quite good and I made sure that correct LINKR records are used during refinement in Refmac. I must admit that I’m a bit struggling to improve this. Refmac can report distance deviations, so you can see where the restraints are fighting the density. You can do something similar in Coot too. Paul. From: CCP4 bulletin board <CCP4BB@JISCMAIL.AC.UK><mailto:CCP4BB@JISCMAIL.AC.UK> On Behalf Of Jasmine Young Sent: Thursday, 18 March 2021 2:46 am To: CCP4BB@JISCMAIL.AC.UK<mailto:CCP4BB@JISCMAIL.AC.UK> Subject: [ccp4bb] Enhanced Validation of Small-Molecule Ligands and Carbohydrates A new article in Structure<https://doi.org/10.1016/j.str.2021.02.004> describes new features, including branched representations and 2D SNFG images for carbohydrates, identification of ligands of interest, 3D views of electron density fit, and 2D images of small molecule geometry. These enhancements and processes for validation of 3D small-molecular structures reflect recommendations from the wwPDB/CCDC/D3R Ligand Validation Workshop and the adoption of software through community collaborations. This manuscript also highlights enhancements made since the initial implementation of Validation Reports as described in Validation of the Structures in the Protein Data Bank (2017) Structure 25: 1916-1927 doi: 10.1016/j.str.2017.10.009<https://doi.org/10.1016/j.str.2017.10.009>. Enhanced Validation of Small-Molecule Ligands and Carbohydrates in the Protein Data Bank Zukang Feng, John D. Westbrook, Raul Sala, Oliver S. Smart, Gérard Bricogne, Masaaki Matsubara, Issaku Yamada, Shinichiro Tsuchiya, Kiyoko F. Aoki-Kinoshita, Jeffrey C. Hoch, Genji Kurisu, Sameer Velankar, Stephen K. Burley, and Jasmine Y. Young (2021) Structure doi: 10.1016/j.str.2021.02.004<https://doi.org/10.1016/j.str.2021.02.004> [undefined]<https://doi.org/10.1016/j.str.2021.02.004> <http://www.wwpdb.org/news/news?year=2021#top> -- Regards, Jasmine =========================================================== Jasmine Young, Ph.D. Biocuration Team Lead RCSB Protein Data Bank Research Professor Institute for Quantitative Biomedicine Rutgers, The State University of New Jersey 174 Frelinghuysen Rd Piscataway, NJ 08854-8087 Email: jasm...@rcsb.rutgers.edu<mailto:jasm...@rcsb.rutgers.edu> Phone: (848)445-0103 ext 4920 Fax: (732)445-4320 =========================================================== ________________________________ To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1 The contents of this e-mail are confidential and may be subject to legal privilege. If you are not the intended recipient you must not use, disseminate, distribute or reproduce all or any part of this e-mail or attachments. If you have received this e-mail in error, please notify the sender and delete all material pertaining to this e-mail. Any opinion or views expressed in this e-mail are those of the individual sender and may not represent those of The New Zealand Institute for Plant and Food Research Limited. ________________________________ To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1 ________________________________ To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1 -- Dr Jon Agirre Royal Society Olga Kennard Research Fellow 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 ________________________________ To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1 The contents of this e-mail are confidential and may be subject to legal privilege. If you are not the intended recipient you must not use, disseminate, distribute or reproduce all or any part of this e-mail or attachments. If you have received this e-mail in error, please notify the sender and delete all material pertaining to this e-mail. Any opinion or views expressed in this e-mail are those of the individual sender and may not represent those of The New Zealand Institute for Plant and Food Research Limited. ######################################################################## To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=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/
