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
