Positive difference map features indicate a likelihood that atoms should be placed in that location. Putting atoms in that density, any atoms, will lower the R value. This does not mean that your interpretation is correct.
The fact that you, the person who has seen this map most clearly, can't decide between a PO4 and a Ade means that this density is ambiguous. These are two very different shapes! A constellation of isolated blobs is about the worst thing to figure out. It is quite possible that you have a partially occupied something with ordered water molecules when that thing is not present. When looking for partially occupied things you have to contour the map at a much lower level, but not take what you see too seriously. Remember you are looking for something at, say, 50% occupancy, with 50% occupied water molecules sitting on top. That said, I don't understand you difference maps. You built a ADN into your blobs, and that molecule is quite a bit larger than your blobs, yet you have a huge amount of positive difference density covering your ADN. How is this possible? The goal of refinement is to make the difference map go to zero at the location of atoms. There is something about these maps that you are not telling us. When interpreting blobs the first and most important thing to consider is what is in the crystal. If it doesn't contain PO4 there is no reason to test PO4 as a possibility. If you didn't add ADN then you are hypothesizing that it was carried all the way through purification which means that has to bind fairly strongly to survive even at partial occupancy. Is this location on your protein a nucleotide binding site? These things are easily recognized just from the structure of the protein. Does the hydrogen bonding and charge-charge interactions of your model make sense? It is hard to tell in a flat picture, but I don't see many hydrogen bonds to your ADN model. If I compare your PO4 model to the structure I see in the ADN map, I see that there is a ASP right next to that blob. You can't put a PO4 next to an ASP. Since the map is confusing you have to use as much information from it as possible (lower contours) but add in as much information from other sources as possible. What's in the crystal? What is your cryoprotectant? Is this part the the protein a known binding motif for something? What is the function of this protein and what sorts of compounds might be expected to bind to it? Is this an enzyme and is the spot anywhere near the active site? Do you know where the active site is? Once you build a model you have to test it. You should be your worst critic! As I said, a drop in R value is meaningless. Does the chemistry make sense? Can you explain why that molecule is there? Does it have a purpose? Can you perform an experiment that confirms your model? Can you soak more of that compound into the crystal and see an increase in occupancy? Can you analyze the crystal by some other means to detect the compound? Mass Spec? If PO4, can you detect the presence of Phosphorus? The validation has to be designed based on your model. You can get better maps than simple Fo-Fc maps. I really like the maps produced by Buster and have great success with getting better views that way. It is possible that the "Polder" map from Phenix might help - I'm not too clear on the difference between it and the Buster map. Load as much information into your head as you can, get the best possible map(s), and start running though the possibilities! And be willing to accept that you may never figure it out. Don't build a model you don't believe. I once spent about twenty years trying to figure out a blob (not full time!). I got a nice paper about it in the end. Dale Tronrud On 8/5/2018 7:00 AM, Preeti Preeti wrote: > Dear CCP4 member > > I am solving a protein structure with Resolution 2.2 Angstrom. I could > see some blobs (2fo-fc @1sigma) and fo-fc @ 2.5 sigma) and need your > suggestions on these extra electron densities. In addition to this, in > one of the large blob I have added the phosphate group. > This is a nucelotide binding protein however structure I am showing > here was of native protein crystal without any nucleotide soaking and > also no phosphate buffer was used at any time during the purification as > well as crystallization process. > Furthermore in one such blob I tried fitting the adenine moiety though > it is not fitting exactly in the map, it decreased the Rfree value > significantly, > > kindly suggest me what it should be corresponding to? > > Also please let me know if any other structure information required > regarding this protein data or this blob density > > Thanks a lot in advance > > > > > > > > Preeti > > > > ------------------------------------------------------------------------ > > 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
