Back to the value of an anomalous map - IF the anomalous data is good enough to give a significant peak at a sulphur position, you might expect to get a peak at a well ordered phosphate- if no sulphur peaks not much hope...
On Mon, 25 Dec 2023 at 19:51, Tom Peat < 0000b7e4a7a8af49-dmarc-requ...@jiscmail.ac.uk> wrote: > Hello Dale, > > Thank you for the correction/ clarification. > I think this is still a tricky question, as in solution, this is an > average state and one doesn't have a stable hydrogen (or two) sitting > discretely on the phosphate. More specifically, the hydrogens are coming > off and popping back on constantly (just the ratios change depending on the > pH). It is likely that the phosphate is also moving in and out of the > binding site of a protein in solution. What state is captured in a crystal > structure and is that consistent across all of the proteins in that > crystal? > As you say, one needs very high resolution to determine the bond length > difference between those oxygens with and without a potential hydrogen > attached to orient a phosphate correctly in a structure, assuming that > there is only a single preferred orientation to start with. > I believe the original question was whether in fact the density supported > a phosphate ion, and I still believe that looking for some anomalous signal > may be a good way to approach that question. > Nonetheless, I stand corrected and there is likely to be some hydrogen on > phosphate ions found in crystal structures. > Happy holidays to all, tom > ------------------------------ > *From:* Dale Tronrud <de...@daletronrud.com> > *Sent:* Monday, December 25, 2023 8:52 PM > *To:* Tom Peat <t.p...@unsw.edu.au>; CCP4BB@JISCMAIL.AC.UK < > CCP4BB@JISCMAIL.AC.UK> > *Subject:* Re: [ccp4bb] Query on density fitting to phosphate > > [You don't often get email from de...@daletronrud.com. Learn why this is > important at https://aka.ms/LearnAboutSenderIdentification ] > > > Hi, > > I wanted to correct a statement by Prof. Peat about the ionic state > of phosphate in solution. Phosphate has four states differing by the > number of attached hydrogen atoms. The number of hydrogen atoms depends > on the pH, or maybe it is the other way around since phosphate is used > as a buffer. I've attached a plot of the fraction of each species as a > function of pH (Citation: "By Clarolux - Own work, CC BY-SA 4.0, > https://commons.wikimedia.org/w/index.php?curid=90586171";). There you > can see that for all pH's usually seen in mother liquors the solution is > almost completely either HPO4(-2) and H2PO4(-1). A binding site may, of > course, prefer a species that is present in low concentration but such a > protein will be fighting entropy to fill its pocket. > > Unless your mother liquor has an extreme pH you should expect that > the phosphate species you are seeing in your crystal has either one or > two hydrogen atoms attached. Their presence will affect both the nature > of the hydrogen bonding of the protein to the phosphate and will change > the length of the P-O bonds (with the P-O-H bond being about 0.05 A > longer than the P=O bond). The two lengths will only be distinguishable > given very high resolution diffraction data but there are examples in > the PDB where the differences are clear. You can determine the presence > of an hydrogen atom at much lower resolution if the hydrogen bond is > made with an obligate hydrogen bond acceptor. > > The inappropriate identification of an ion as PO4(-3) will > significantly degrade the quality of any electrostatic potential one > calculates from the model. > > I did a quick-and-dirty search of the PDB for the various species of > phosphates in PDB entries. While I found 5979 models with PO4(-3) (ID: > PO4) I only found 42 with HPO4(-2) (ID: PI) and 27 with H2PO4(-1) (ID: > 2HP). I didn't find any H3PO4 and could not find an ID code for that > molecule. (This search was done quite a while ago.) I believe > depositors are mostly assuming the ID PO4 indicates any protonation of a > phosphate ion but that is not correct. I am unaware of any ID that is > defined as a phosphate ion with unknown protonation state. To conform > to the wwPDB standards a depositor must do their best, using the limited > data available to them, to choose one species of phosphate when they > identify the presence of one, but almost certainly that choice should > not be PO4. > > As usual, just causing trouble, > Dale E. Tronrud > > On 12/17/2023 1:05 PM, Tom Peat wrote: > > Dear Arpita, > > > > The hydrogens on phosphate, just like sodium and potassium, will come > > off the oxygens in water. > > To be more explicit, you don't have mono- or di-hydrogen phosphate in > > water (except transiently), you just have phosphate, depending somewhat > > on the pH of course. At 2.5 Angstrom resolution, there is no way to > > 'see' hydrogens with X-rays. > > Depending on the wavelength you used for your data collection, you could > > try doing an anomalous map and see if you have any anomalous signal at > > this position, which may help in identifying what the density is. > > Best of luck, tom > > > > ------------------------------------------------------------------------ > > *From:* CCP4 bulletin board <CCP4BB@JISCMAIL.AC.UK> on behalf of Arpita > > Goswami <bt.arp...@gmail.com> > > *Sent:* Sunday, December 17, 2023 9:46 PM > > *To:* CCP4BB@JISCMAIL.AC.UK <CCP4BB@JISCMAIL.AC.UK> > > *Subject:* [ccp4bb] Query on density fitting to phosphate > > > > You don't often get email from bt.arp...@gmail.com. Learn why this is > > important <https://aka.ms/LearnAboutSenderIdentification> > > > > > > Dear All, > > > > Hope you all are doing well. > > > > The density in the image (in link below) is fitted with PO4 ion, > > although the crystallization condition has both mono and dihydrogen > > phosphate which is not fitting without hydrogen. But the resolution is > > 2.5 A, so hydrogen may not be put in, or is there any way to do so? > > Otherwise placing water is the final option. > > > > https://i.postimg.cc/4N7q2K0p/Screenshot-from-2023-12-17-16-07-07.png > > <https://i.postimg.cc/4N7q2K0p/Screenshot-from-2023-12-17-16-07-07.png> > > > > Also the density is quite close to Aspartate, so PO4 may not be right. > > Can it be dihydrogen phosphate as two positively charged residues > > (Specially the lysine) are also nearby to neutralize positive charge? > > Other ions in the crystallization condition are Cl-, K+ and Na+. These > > are not put as both aspartate and lysine are at comparable distances > > from the density. The pH is 6.2 in which dihydrogen phosphate is > > reported to interact with aspartate > > (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5855859/ > > <https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5855859/>). > > > > Waiting eagerly for your reply. > > -- > > Thanks and Merry Christmas in advance. > > Best Regards, > > Arpita > > > > ------------------------------------------------------------------------ > > > > To unsubscribe from the CCP4BB list, click the following link: > > https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1 > > <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 > > <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 > ######################################################################## 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/
