Hello, happy Boxing day. Re: "The inappropriate identification of an ion as PO4(-3) will significantly degrade the quality of any electrostatic potential one calculates from the model."
A couple of comments. I always deleted solvents and counter-ions, etc, when doing this sort of thing, given all the uncertainties about their ionisation state. Not being clever enough to do electrostatic calculations properly (evidently), let alone write an FFT refinement program ;-0, I thought that was the best way to look at the charge on the protein itself. I agree it is very honourable to make an informed decision about a ligand's protonation state and to deposit one's preferred model, but there are an awful lot of other hydrogens that are not considered in a typical X-ray structure analysis ;-0 Maybe this is where neutron and electron diffraction can/will be able to help. Best wishes, Jon Cooper. [email protected] Sent from Proton Mail mobile -------- Original Message -------- On 25 Dec 2023, 09:52, Dale Tronrud wrote: > 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 on behalf of Arpita > Goswami > *Sent:* Sunday, > December 17, 2023 9:46 PM > *To:* [email protected] > *Subject:* > [ccp4bb] Query on density fitting to phosphate > > You don't often get email > from [email protected]. Learn why this is > important > > > 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 > > > > 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/ > ). > > > 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 > > > > > ------------------------------------------------------------------------ > > > 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/ ######################################################################## 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/
