By the way, I have another question related to the number of sites. I just rethought about what Herman mentioned, and I just realized that the number of sites, at least strong sites, could be guessed from an anomalous Patterson map. Therefore I looked at the anomalous Patterson of my data. The map indicates that the signal is very weak, and there are only 4 non-origin positive peaks (3 effective peaks, because 2 are symmetry equivalent), indicating there are at most 3 heavy atoms (3x2=6) if all of them contribute to the map. However, in shelxd, I can get optimal results from 6 sites out of the same data set. I am not sure whether this is because the anomalous Patterson is not sensitive enough. Now I am more confused about the criteria in choosing the parameters when running shelx and all other programs. What should be the general practice? Should I rely more on the anomalous Patterson or more on the better scores reported? When I tried to optimize the results by changing the parameters (either when locating sites or when phasing), am I more heading for the real signal or more simply tweaking the parameters? Maybe the golden standard would be whether you could solve the structure.
Another question is, although from the Euler's equation I could understand negative Patterson peaks (maybe I am wrong), I am still not clear what negative Patterson peaks represent and why people generally don't talk about it. In the specific data I am talking about above, there is a negative Patterson peak at the same position as a positive Patterson peak with the same height. Is there any indication about this? Thank you so much for your attention! On Tue, Apr 1, 2014 at 2:00 PM, Nazia Nasir Phd2009,ProteinCrystall.Lab < [email protected]> wrote: > Dear Jurgen, > > The beam position is fine. we have collected many data sets before and > after this data. Moreover, we the Technical scientist always checks the > beam position before we mount the crystals. > > Thanks > > > On Tue, Apr 1, 2014 at 11:23 PM, Jurgen Bosch <[email protected]> wrote: > >> check your beam position >> ...................... >> Jürgen Bosch >> Johns Hopkins University >> Bloomberg School of Public Health >> Department of Biochemistry & Molecular Biology >> Johns Hopkins Malaria Research Institute >> 615 North Wolfe Street, W8708 >> Baltimore, MD 21205 >> Office: +1-410-614-4742 >> Lab: +1-410-614-4894 >> Fax: +1-410-955-2926 >> http://lupo.jhsph.edu >> >> On Apr 1, 2014, at 1:26 PM, Nazia Nasir Phd2009,ProteinCrystall.Lab < >> [email protected]> wrote: >> >> Dear all, >> >> I am just taking advantage of this particular thread to add my query >> also. I hope you don't mind Chen. >> >> We haven't solved any structure in our lab using SAD phasing, so pardon >> me for sounding naive. >> >> I have a 6.5 A data of anomalous scattering with a 3.5 A data using the >> Cu anode.My crystals dont grow any better. I have around 10 Met distributed >> fairly evenly through out the sequence. So is it possible to get any >> anomalous signals form this data? >> Moreover, I am not able to index my 3.5A data at all. The spots don't fit >> well. What could be the problem? >> >> Hope this thread can be of benefit for both me and Chen. >> >> Thanks >> >> >> On Tue, Apr 1, 2014 at 8:21 PM, Chen Zhao <[email protected]> wrote: >> >>> Dear Herman, >>> >>> Thank you so much for your suggestions. The density that passes through >>> the rotational axis is so strong and extended that can be considered as a >>> significant portion of the molecule. However, some density in the middle >>> might show some features. I have no experience and this could be only >>> artifact. >>> >>> The unit cell seems to be able to fit 1-2 molecules. But if there is >>> one molecule/ASU, the solvent content is about 89%, which is possible but >>> unlikely. Although the resolution of the crystal is not high, it is rather >>> rigid and easy to handle, which might indicate a not-too-high solvent >>> content. I soaked the native crystal with heavy atom compounds and I have >>> no clear idea of the relationship between metal binding and sequence, so I >>> don't know how many sites to expect. >>> >>> Best, >>> Chen >>> >>> >>> On Tue, Apr 1, 2014 at 9:42 AM, <[email protected]> wrote: >>> >>>> Dear Chen, >>>> >>>> I am not an expert on SAD and MAD. However, at this stage I would not >>>> worry too much about density going through the 2-fold axis. There might be >>>> a sulfate ion or some other buffer component present at that position, or >>>> it may just be an artifact that will go away once the structure has been >>>> built and refined. >>>> >>>> >>>> >>>> The questions I would worry about is: how much too small is your unit >>>> cell? Is it just crowded, say 25-30% solvent, or would your protein >>>> molecule not fit at all? Does the amount of solvent as estimated from your >>>> SAD/MAD maps agree with the amount of solvent obtained from the calculation >>>> of the Matthews volume? How many (SeMet?) sites do you expect, 6, more, >>>> less? If everything looks ok except that the unit cell is rather crowded, I >>>> would go ahead and try to build the structure. >>>> >>>> However, if even a single protein molecule would not fit in your unit >>>> cell, or you find many more sites than you can explain, you should start >>>> worrying about twinning. Even than the structure can probably be solved, >>>> but then you need some real experts! >>>> >>>> >>>> >>>> My 2 cents, >>>> >>>> Herman >>>> >>>> >>>> >>>> >>>> >>>> >>>> >>>> *Von:* CCP4 bulletin board [mailto:[email protected]] *Im Auftrag >>>> von *Chen Zhao >>>> *Gesendet:* Montag, 31. März 2014 23:46 >>>> *An:* [email protected] >>>> *Betreff:* [ccp4bb] Space group problem? >>>> >>>> >>>> >>>> Dear all, >>>> >>>> I am now trying to phase a structure in C2 using anomalous scattering >>>> at 5-6 A. It is hard to improve the derivative resolution at the moment. >>>> Shelxd is able to locate 6 sites with a distinct CC and FOM. After density >>>> modification in shelxe, the contrast of the two enantiomers is 0.59/0.38 >>>> for SAD and 0.7/0.3 for MAD. When I looked at the electron density, the >>>> maps from SAD and MAD are similar, and the solvent boundary is quite clear. >>>> However, the problem is that the electron density blob passes through the >>>> 2-fold rotation axis, even at 3 rmsd contour level. Also, the unit cell >>>> seems to be too small for the molecule. I am afraid that the space group >>>> assignment is wrong, but I am a beginner so I nearly have no clue. I did >>>> reprocess the data in P1 and looked at the self-rotation function with a >>>> radius at 200 A. From the list it seems that there is only one 2-fold >>>> rotation axis. I am quite confused. Could anybody give me some hint of this >>>> problem? >>>> >>>> Thanks a lot in advance! >>>> >>>> Sincerely, >>>> >>>> Chen >>>> >>> >>> >> >> >> -- >> Nazia Nasir >> PhD Scholar >> Protein Crystallography Lab >> National Institute of Immunology >> New Delhi >> >> >> > > > -- > Nazia Nasir > PhD Scholar > Protein Crystallography Lab > National Institute of Immunology > New Delhi >
