Re: [ccp4bb] Wrong Space Group?
On Fri, 13 Dec 2013 19:44:44 +, D Bonsor dbon...@ihv.umaryland.edu wrote: Dear D, I agree with Tim and Jürgen that a) the map after Phaser, and before refinement is the most unbiased and should be used for sequence assignment. b) there may be a sequence register shift error that is responsible for the high R-values, and is masked by overfitting So I would try a) To stay on the safe side, you could chop the Phaser model into secondary structure elements and do rigid-body refinement. This yields maps that are largely unbiased. b) sharpening (very easy in coot) and inspection of these unbiased maps, to confirm the sequence register c) submit the Phaser model to Arp/wArp re-building, and also try the buccaneer/refmac iterative re-building, and maybe phenix.autobuild But the problem may also be your data. a) Maybe every second reflection is not integrated because it is weak? That is easy to check with XDSGUI using Tools/show frame with predicted spots b) other pathologies like spot overlap or experimental instability (what is the value of ISa in CORRECT.LP ?) - you could post FRAME.cbf and IDXREF.LP, INTEGRATE.LP and CORRECT.LP and pointless/xtriage statistics If the true space group is P6x22, then the data cannot be twinned. But if the true space group has lower symmetry the data may appear to be P6x22 . HTH, Kay P.S. XDSGUI latest version can be obtained from http://strucbio.biologie.uni-konstanz.de/xdswiki/index.php/XDSGUI Dear all, I have collected ~160 degrees of data on a new crystal form of a protein which has already been solved. Data was processed with XDS and reindex, scaled and truncated with Aimless. Both XDS and Pointless suggested a Laue group of P6/mmm with a possible space group of P6122 or P6522. Stats showed an overall Rmerge of 0.131 but an Rpim of 0.041 (multiplicity/redundancy of 19.1), a completeness of 99.1% and resolution of 2.8Ang. With cell dimensions of 63.1 63.1 243, only one protein chain can be found in the asymmetric unit (two copies would leave a solvent content of 8%). I ran phaser with all alternative space groups and a single solution in P6522 with a TFZ of 10.0. I then performed 20 Refmac cycles ending up with an R/Rfree of 35.5/45.5. I open the structure and map in Coot and could see that there was a large conformational change of helix-turn-helix actually becoming just a long helix (https://www.dropbox.com/s/4s6g8apatsi5xcg/Before_Building.png) and then dimerizing through the long helix with one of the symmetry mates. This section was rebuilt (https://www.dropbox.com/s/5j7tv0i5yq3mxxx/After_Building.png) and ran through Refmac again resulting in an R/Rfree of 35.5/44.3. Looking through the rest of the structure I see nothing else really to be modeled. Nothing that could bring the Rfactors down to a reasonable range. I have therefore tried several things. I ran the structure through Zanuda server to look at other space group possibilities. The server suggested I was in the correct space group. However I did reprocess the data to P6, P3, P312, P321, C2221, P2 and C2, and reran phaser in search all alternative space groups using the original search model but found no solutions. I did reprocess the data in P1, though I did not collect enough data. Twinning tests show no twinning. Although that does not mean there is no twinning, I can see that P6522 has no twin laws. Does that mean no twinning can occur in P6522 or that it can occur but there is no law to be able to separate the amplitudes? I also collected data on a single point mutation of the protein. Although this diffracted to a slightly weaker resolution (3.2Ang), I also observe the same problem of good maps in P6522 but no solution in the groups described above, a clear indication that this helix has elongated but terrible Rfactors. Based upon that the maps look good in P6522 do you believe that I have solved the structure in the correct space group but my data collection is at fault or in fact that I have some form of pseudosymmetry or something else going on and that the space group has lower symmetry but not in the space groups I have checked. Or is it something else. Any suggestions, criticisms or you need further information please contact me and enjoy your weekend.
[ccp4bb] A question on protein microheterogenity for crystalization
Dear All, When I purified my protein by ion exchange chromatography for crystallization, there were several peaks containing the target protein as analyzed by SDS-PAGE. All these peaks have the same MW as determined by gel filtration coupled MALLS. For crystallization purpose, can I merge the corresponding ion exchange chromatography peaks together? Otherwise the protein yield will be too low. And how to explain the heterogeneity by ion exchange chromatography in this situation? I am looking forward to getting a reply from you. Acoot
Re: [ccp4bb] A question on protein microheterogenity for crystalization
Hi Acoot, since they behave differently on IEX, they are different - you would introduce heterogeneity into your crystallization setup, which usually is not a good idea for crystallization in general. Are you using Benzonucleases by any chance in your preparation and if not, that may explain the different populations of your protein. Your protein may bind some short DNA chunks carried over during your lysis process leading to differently charged species on the IEX column. On an SDS gel these complexes are separated and your protein runs at an identical molecular weight. An alternative explanation would be multiple folded states that may be at equilibrium and under certain conditions e.g. pH, salt concentration may be pushed in one predominant species. Also is several 2 or 10 peaks ? If you have e.g. an N-terminal His6-tag you might end up with truncation products and say your protein is rather large e.g. 80 kDa you might not be able to see the molecular weight difference either by SEC or SDS-PAGE if you are running a low percentage gel. A protein of 72 kDa might look like 80 kDa but you most likely will have charge differences distinguishable in IEX. If you have a His6-tag, run a Western on it to identify single or multiple bands. Good luck, Jürgen On Dec 14, 2013, at 7:16 AM, Acoot Brett acootbr...@yahoo.commailto:acootbr...@yahoo.com wrote: Dear All, When I purified my protein by ion exchange chromatography for crystallization, there were several peaks containing the target protein as analyzed by SDS-PAGE. All these peaks have the same MW as determined by gel filtration coupled MALLS. For crystallization purpose, can I merge the corresponding ion exchange chromatography peaks together? Otherwise the protein yield will be too low. And how to explain the heterogeneity by ion exchange chromatography in this situation? I am looking forward to getting a reply from you. Acoot .. 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
Re: [ccp4bb] A question on protein microheterogenity for crystalization
Dear Brett, We have seen this behaviour several times for different adenovirus fibre head proteins and don't really have an explanation for it. We have always set the peaks up separately when we had enough protein. For this purification, as you don't have enough protein to pool them separately, I would pool them together and do a first crystallisation screen. But I would also immediately start a larger scale purification so you in the next crystallisation trial you can set the peaks up separately. If you are lucky, by the time you have done the second prep, from the first screen you may have some conditions to optimise. If not, the first screen should at least give some ideas about which precipitants, pHs and perhaps additives are most suitable. Mark On 14 Dec 2013, at 13:16, Acoot Brett wrote: Dear All, When I purified my protein by ion exchange chromatography for crystallization, there were several peaks containing the target protein as analyzed by SDS-PAGE. All these peaks have the same MW as determined by gel filtration coupled MALLS. For crystallization purpose, can I merge the corresponding ion exchange chromatography peaks together? Otherwise the protein yield will be too low. And how to explain the heterogeneity by ion exchange chromatography in this situation? I am looking forward to getting a reply from you. Acoot
Re: [ccp4bb] A question on protein microheterogenity for crystalization
Hi Acoot: Can your protein form some kind of dynamic self oligomers? When you test by using the gel-filtration column, if the peak is symmetry? Sometime if the target protein can have week self interaction, you can observe a tailed peak. If the protein can have a strong self interaction, maybe you can observe isolated peaks, each peak is corresponding to different amount of the oligomersation state of the protein. You also can test your protein by using native or IEF gels. Dee Date: Sat, 14 Dec 2013 19:01:09 +0100 From: mjvanra...@cnb.csic.es Subject: Re: [ccp4bb] A question on protein microheterogenity for crystalization To: CCP4BB@JISCMAIL.AC.UK Dear Brett, We have seen this behaviour several times for different adenovirus fibre head proteins and don't really have an explanation for it. We have always set the peaks up separately when we had enough protein. For this purification, as you don't have enough protein to pool them separately, I would pool them together and do a first crystallisation screen. But I would also immediately start a larger scale purification so you in the next crystallisation trial you can set the peaks up separately. If you are lucky, by the time you have done the second prep, from the first screen you may have some conditions to optimise. If not, the first screen should at least give some ideas about which precipitants, pHs and perhaps additives are most suitable.Mark On 14 Dec 2013, at 13:16, Acoot Brett wrote:Dear All, When I purified my protein by ion exchange chromatography for crystallization, there were several peaks containing the target protein as analyzed by SDS-PAGE. All these peaks have the same MW as determined by gel filtration coupled MALLS. For crystallization purpose, can I merge the corresponding ion exchange chromatography peaks together? Otherwise the protein yield will be too low. And how to explain the heterogeneity by ion exchange chromatography in this situation? I am looking forward to getting a reply from you. Acoot
