Dear Nazia, My first suggestion would be to take your crystals to a synchrotron. With the same crystals, you may get a precious 0.5 Å resolution more. Concerning the processing, I agree with Jürgen that the beam center is the most frequent culprit. The second thing to do is to make sure that you do not impose any space group and let your data processing program determine the space group, which may be very different from what you expect. Next you should inspect your images for potential problems: ice/salt rings, very high mosaicity, smeared or blurred spots, bad zones etc. With poorly diffracting crystals, processing using default values may not work and you may have to fine-tune your processing parameters. Best would be to find a local expert, otherwise you should provide the bulletin board with more details like processing programs tried, expected cell parameters, assessment of the diffraction pattern etc.
Best, Herman Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von Nazia Nasir Phd2009,ProteinCrystall.Lab Gesendet: Dienstag, 1. April 2014 20:00 An: CCP4BB@JISCMAIL.AC.UK Betreff: Re: [ccp4bb] AW: [ccp4bb] Space group problem? 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 <jbos...@jhu.edu<mailto:jbos...@jhu.edu>> 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<tel:%2B1-410-614-4742> Lab: +1-410-614-4894<tel:%2B1-410-614-4894> Fax: +1-410-955-2926<tel:%2B1-410-955-2926> http://lupo.jhsph.edu On Apr 1, 2014, at 1:26 PM, Nazia Nasir Phd2009,ProteinCrystall.Lab <nazia.nasi...@nii.ac.in<mailto:nazia.nasi...@nii.ac.in>> 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 <c.z...@yale.edu<mailto:c.z...@yale.edu>> 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, <herman.schreu...@sanofi.com<mailto:herman.schreu...@sanofi.com>> 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:CCP4BB@JISCMAIL.AC.UK<mailto:CCP4BB@JISCMAIL.AC.UK>] Im Auftrag von Chen Zhao Gesendet: Montag, 31. März 2014 23:46 An: CCP4BB@JISCMAIL.AC.UK<mailto:CCP4BB@JISCMAIL.AC.UK> 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
