One practically HAS to do what Dale describes. The sigma levels of a difference map peak depends on how much it is above the mean noise level of the map. With high resolution (and given a cooperating molecule) it is very likely that the model is very good and complete, thus low mean map noise level, and anything missing will create huge peaks. A forgotten water molecule then can easily have difference peak (sigma not absolute) levels in the 20s...
Best, BR -----Original Message----- From: CCP4 bulletin board <CCP4BB@JISCMAIL.AC.UK> On Behalf Of Dale Tronrud Sent: Tuesday, August 6, 2019 00:24 To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Extra density close to phosphate bound to Zn2+ One thing to remember is that Li+ only has two electrons. It should be a little more than twice the density of an hydrogen (because the charge pulls in the electron cloud). If you are seeing lithium at 8 "sigma" you should be seeing the well located hydrogen atoms at 4 "sigma". Are you? I always like controls. Take out one of the near by water molecules and see how that, known, difference peak compares to your mystery peak. A Li+ should have about 1/5th the height of an H2O. If you like the hypothesis of two orientations of the PO4 group, the relative height of the two peaks will give insight to the occupancy ratio. Remember, if the PO4 has two orientations in the crystal, the water molecules it (they?) is/are bound to will likely also have alternatives. Dale Tronrud On 8/5/2019 6:05 AM, Maria Håkansson wrote: > Dear CCP4 bulletin board, > > I am working with some lytic enzymes called endolysines, which bind > Zn2+ in the active site. I have three homologues protein determined to > 1.2 Å each where the Zn2+ is bound to a cystein, two histidines and > one phosphate ion added (1.9-2.3 Å binding distances) in the > crystallization experiment. > > Now to my question. Close to the phosphate (B-factor=20Å2) ion a 8 > sigma peak is present in all three endolysines, see below. > I have modeled it to a Na+ (B-factor= 30 Å2) or a Li+ (B factor = > 13Å2) ion. > Sodium has benn added in the crystallization experiments since sodium > potassium phosphate salt has been used. The only reason for including > Li+ is that I think the binding distances (1.7-2.0 Å) are too short > for Na+. > > I have also tried to make a model with the phosphate in two different > conformations but it does not fit. > > Have anyone seen something similar before? What is the most correct > way of dealing with unknown densities? > It is difficult to disregard +8 sigma difference density close to the > active site. > > Thanks in advance for any help! > > Best regards, > Maria > > > > Maria Håkansson, PhD, Crystallization Facility Manager Principal > Scientist > > SARomics Biostructures AB > Medicon Village > SE-223 81 Lund, Sweden > > Mobile: +46 (0)76 8585706 > Web: www.saromics.com <http://www.saromics.com> > > > > > > ---------------------------------------------------------------------- > -- > > 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 ######################################################################## To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB&A=1
