Thank you, yes, threading style tools to assess the likelihood of having a given amino acid in a certain position in the fold would be a good approach. I have tried one but wasn't hugely informative, in my hands anyway. All suggestions very welcome but big database science is a bit outside my skill set. Of course, sequence conservation in the family helps a lot with the assignment but there are bigger ambiguities in less conserved surface regions e.g. a disordered Lys can refine well as a Ser, Ala or Gly even, but e.g. nearby conserved acidic groups might suggest the presence of the basic amino acid which could salt-bridge with them, but why then would it be so disordered? Tangles we weave...
Sent from ProtonMail mobile -------- Original Message -------- On 29 Jul 2022, 14:17, Clemens Vonrhein wrote: > Maybe a crazy idea, but couldn't one use various model/geometry validation > tools to figure out some of those ambiguities? As a test one could take a > very good 1.7A structure and do some random ASN->ASP, THR->VAL etc mutations > followed by refinement (including hydrogens). Wouldn't some validation tool > pick up unfavourable conformations, poor rotamers and/or hydrogen clashes and > poor H-networks (compared to the initial, correct sequence)? Maybe there is > some kind of "fingerprint" in validation results for such incorrect residue > assignments that can distinguish correct from incorrect sequences ... Or put > another way, if model validation can not pick up such sequence errors: should > we be worried about the reliability of our validation criteria? A large scale > re-refinement of deposited structures with (1) the current/correct sequence > and (2) those ASN/ASP, THR/VAL etc ambiguities artificially introduced, could > provide a clever algorithm (AI?) with the data basis to figure out those > "fingerprints". Maybe even for the ASN/GLN/HIS side-chain orientations when > the sequence is actually correct. Cheers Clemens On Fri, Jul 29, 2022 at > 12:08:58PM +0000, Jon Cooper wrote: > Thank you so much for your replies. I > apologise for being unclear. The protein is purified from a plant that hasn't > had its genome sequence determined. We know the enzyme family of the protein > and therefore the structure was originally solved by MR. The 'X-ray sequence' > we have is just determined from looking at the 1.7 Angstrom density, which is > good, over several refinement and rebuilding rounds. The resulting sequence > has been run through blast and it is up to 58% identical with other family > members. To me this seemed low but that degree of identity is typical of > other family members. The postgrad who did the work did obtain some peptide > sequences and prior to that about 20% of the sequence was determined by the > Edman method with the usual Asp/Asn and Glu/Gln ambiguity. However, there > isn't any prospect of us doing further experimental work, sorry, but that's > the way it is!! > > Best wishes, Jon.C. > > Sent from ProtonMail mobile > > > -------- Original Message -------- > On 29 Jul 2022, 12:23, Jan Dohnalek > wrote: > > > If you know at least something about your protein, organism, > type of molecule, ..., you could try mass spectrometry peptide mapping to > known sequences, this may give you some answers for the ambiguities you might > be seeing, if nothing else .. > > > > Jan > > > > On Fri, Jul 29, 2022 at > 12:15 PM Jon Cooper wrote: > > > >> Hello, I am looking for suggestions of > ways to check a 1.7 Angstrom X-ray sequence for a protein where it is > impractical to do experimental sequencing, protein or DNA. The structure > refines to publishable R/R-free and the main ambiguities seem to be Thr/Val, > Asp/Asn and Glu/Gln where alternative H-bonding networks are possible. > Running alpha-fold seems an interesting option? Any suggestions much > appreciated. > >> > >> Cheers, Jon.C. > >> > >> Sent from ProtonMail mobile > > >> > >> --------------------------------------------------------------- > >> > > >> To unsubscribe from the CCP4BB list, click the following link: > >> > https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1 > > > > -- > > > > > Jan Dohnalek, Ph.D > > Institute of Biotechnology > > > > Academy of > Sciences of the Czech Republic > > Biocev > > Prumyslova 595 > > 252 50 > Vestec near Prague > > Czech Republic > > Tel. +420 325 873 758 > > > ######################################################################## > > > 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/ -- > *-------------------------------------------------------------- * Clemens > Vonrhein, Ph.D. vonrhein AT GlobalPhasing DOT com * Global Phasing Ltd., > Sheraton House, Castle Park * Cambridge CB3 0AX, UK www.globalphasing.com > *-------------------------------------------------------------- > ######################################################################## 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/ ######################################################################## 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/
