Re: [ccp4bb] A question on protein microheterogenity for crystalization
Hi Acoot, There are a lot of great suggestions here already. I've also run into this phenomenon in couple of cases. The first was a binding protein in mixed bound and unbound forms. The second was a case of heterogeneous post-translational modification. In both cases I could only get crystals from purified peaks and not pooled protein. If protein is precious I'd second Mark's suggestion to try screening pooled protein while you scale up your prep. Cheers, Katherine On Sat, Dec 14, 2013 at 6:16 AM, Acoot Brett 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 -- Nil illegitimo carborundum* - *Didactylos
Re: [ccp4bb] A question on protein microheterogenity for crystalization
It might be that the protein is innocent and the FPLC is guilty. If FPLC pump needs maintenance and is stuttering a bit when running at high pressure, the salt gradient won't be as smooth as you'd like. If you have an ionic strength detector, that trace will tell you. Otherwise, see if other people's proteins are also looking a bit iffy under similar circumstances. Phoebe From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Katherine Sippel [katherine.sip...@gmail.com] Sent: Sunday, December 15, 2013 11:53 AM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] A question on protein microheterogenity for crystalization Hi Acoot, There are a lot of great suggestions here already. I've also run into this phenomenon in couple of cases. The first was a binding protein in mixed bound and unbound forms. The second was a case of heterogeneous post-translational modification. In both cases I could only get crystals from purified peaks and not pooled protein. If protein is precious I'd second Mark's suggestion to try screening pooled protein while you scale up your prep. Cheers, Katherine On Sat, Dec 14, 2013 at 6:16 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 -- Nil illegitimo carborundum - Didactylos
[ccp4bb] Heavy atom sites
I have some SIRAS data of a known structure. I want to get the isomorphous and anomalous occupancy and phasing power from my data. What's the best software to do this? -- === All Things Serve the Beam === David J. Schuller modern man in a post-modern world MacCHESS, Cornell University schul...@cornell.edu
Re: [ccp4bb] A question on protein microheterogenity for crystalization
We had this happen with RPA14/32 heterodimer, we kept the two peaks separate, and then grew crystals from each but in different space groups and diffraction resolution. See Habel, J. E., Ohren, J. F. and Borgstahl, G. E. O. Dynamic light scattering analysis of full-length, human RPA14/32 dimer: purification crystallization and self-association *Acta Cryst.*D57, 254-259 (2001). On Sun, Dec 15, 2013 at 2:33 PM, Phoebe A. Rice pr...@uchicago.edu wrote: It might be that the protein is innocent and the FPLC is guilty. If FPLC pump needs maintenance and is stuttering a bit when running at high pressure, the salt gradient won't be as smooth as you'd like. If you have an ionic strength detector, that trace will tell you. Otherwise, see if other people's proteins are also looking a bit iffy under similar circumstances. Phoebe -- *From:* CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Katherine Sippel [katherine.sip...@gmail.com] *Sent:* Sunday, December 15, 2013 11:53 AM *To:* CCP4BB@JISCMAIL.AC.UK *Subject:* Re: [ccp4bb] A question on protein microheterogenity for crystalization Hi Acoot, There are a lot of great suggestions here already. I've also run into this phenomenon in couple of cases. The first was a binding protein in mixed bound and unbound forms. The second was a case of heterogeneous post-translational modification. In both cases I could only get crystals from purified peaks and not pooled protein. If protein is precious I'd second Mark's suggestion to try screening pooled protein while you scale up your prep. Cheers, Katherine On Sat, Dec 14, 2013 at 6:16 AM, Acoot Brett 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 -- Nil illegitimo carborundum* - *Didactylos
Re: [ccp4bb] Wrong Space Group?
Its possible you are in a lower space group, perhaps with some twinning, but your search model is different enough to only find a solution when things are over-merged. Try refining your P6522 model against data merged in P65. If the other copy (symmetry mate in P6522) does not show up, you may be in trouble (wrong MR solution). I'd also try refinement/building in the other triogonal/hexagonal space groups, but again, start with the PDB file that you got for P6522. Just change the space group in the header, and switch out the MTZ file. You will need to merge your data in each space group and also check the a-b flip re-indexing for most of them. Have a look at the CCP4 reindexing list for the h,k,l operators to try: http://www.ccp4.ac.uk/html/reindexing.html note how similar they are to the twinning operators: http://www.ccp4.ac.uk/html/twinning.html If I have counted right, that means you have 36 jobs to run. I'd also recommend turning the TWIN option in refmac off and on for each of these cases. This will always give you a lower R factor, because of the dynamic range compression you get with twinning, but if one particular combination of twinning with a particular space group and axis reindexing is markedly better than all the others, then you have just found your right space group. So, now we are up to 72 jobs, but hardly a lot of work compared to growing the crystals in the first place. You might also want to try being clever and generating the symmetry mates of your P6522 model and refine these partners as separate molecules as you reduce the symmetry of the data. It's tricky, but think of it as an exercise. Which real-space operator becomes what reciprocal-space operator? You can check your answer by loading it up in coot and seeing if symmetry mates clash with the input coordinates. Yes, its a lot of work to try all these combinations, but that's the annoying thing about twinning, it opens up a lot of ambiguities. Good luck! -James Holton MAD Scientist On 12/14/2013 6:44 AM, D Bonsor wrote: 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] Please apply for RapiData 2014, a course on Data Collection and Structure Solving at the NSLS.
We are offering RapiData 2014, the sixteenth offering of our popular course: Rapid Data Collection and Structure Solving at the NSLS: A Practical Course in Macromolecular X-Ray Diffraction Measurement The course will be held 27 April - 2 May 2013: http://www.bnl.gov/RapiData/. Students could be at any level from advanced undergraduate to full professor. The course should accommodate 48 students total. We encourage all students to bring their own specimens for data collection, and to bring old data for the data-reduction and structure-solving tutorials. Please read the Course Announcement at http://www.bnl.gov/RapiData/. You'll see that many experts in the field will be available for lectures and tutorials. In addition to MX there will be opportunity for learning about correlated MX and spectroscopy, and SAXS. You'll find the application materials on the Course Application tab at this site. For the twelfth time we will hold a short lecture course on the fundamentals of crystallography for roughly five hours on Sunday 27 April. The body of the RapiData course really requires that students have a healthy knowledge of crystallography. For potential students who have some experience but are shaky about fundamentals, this course will help. There will be an additional fee for the fundamentals course, to pay for Saturday night accomodations and food on Sunday morning and noon. Latin American Scientists: Several scholarships are available, from the International Union of Crystallography, to pay partial travel and subsistence costs for Latin-American students and junior faculty (no more than 30 yrs). Please apply for the course, and then contact R. Sweet (sw...@bnl.gov) if you are interested in applying for a scholarship. In accordance with the standards of the International Union of Crystallography, we observe the basic policy of non-discrimination, affirming the right and freedom of scientists to associate in international scientific activity without regard to such factors as citizenship, religion, creed, political stance, ethnic origin, race, colour, language, age, or gender, in accordance with the Statutes of the International Council for Science. At this course no barriers will exist beyond the application procedure that would prevent the participation of bona fide scientists. Please apply yourself, or send your students to our course, Bob Sweet, Sonya Kiss, and Alex Soares Course Announcement at http://www.bnl.gov/RapiData/ = Robert M. Sweet E-Dress: sw...@bnl.gov Group Leader, PXRR: Macromolecular ^ (that's L Crystallography Research Resource at NSLSnot 1) http://px.nsls.bnl.gov/ Photon Sciences and Biosciences Dept Office and mail, Bldg 745, a.k.a. LOB-5 Brookhaven Nat'l Lab. Phones: Upton, NY 11973631 344 3401 (Office) U.S.A. 631 344 2741 (Facsimile) =
