Re: [ccp4bb] The phase problem
Hi Giulliana, What is the percent identity of your best search model with your target? At what resolution does your crystal diffract? If you use PHASER without choosing the space group, it might help find you a solution in a different space group than you expect. There are also some tricks on making a good search model to get more positive MR, as well. hope this helps, Ivan On Wed, Nov 5, 2014 at 12:20 PM, Giulliana Rangel giulliana.ran...@gmail.com wrote: Dear all, I would like to known if someone could help me with some idea about the phase problem. I am a beginner in crystallography and the first time I tryed to solve the structure by Molrep, amore, mr. Bump and I didnt find anything for molecular replacement. Thus, currently I've tried to soak heavy-atom ( Hg, Pt, I, Pb) and the diffraction was good, the results in XDS didnt show so good. I didnt look the heavy atom in density. I appreciate any suggestion and ideas what I could do. Best regards, -- Giulliana Rangel Mestranda PPG-Biotecnologia UNIFESP Laboratório de Biologia Estrutural Tel.: (12) 3309-9698 Rua Talim 330, Vila Nair CEP 12231-280 São José dos Campos - SP
Re: [ccp4bb] The phase problem
Hi everyone, I'd like to add that tunable synchrotron beamlines are tending more and more to support Sufur-SAD on native proteins - in particular, there are dedicated long-wavelength beamlines (like I23 at Diamond). And if radiation damage is the main concern, availability of multiple crystals (if one is in the lucky situation of getting many isomorphous ones of sufficient quality), can be exploited to get high multiplicity data by merging their anomalous diffraction intensities, see e.g.: Liu et al. (2012). Science 336, 1033. El Omari et al. (2014). Acta D 70, 2197. Best regards, Fabio Hi Giuliana, if your crystal diffracts to 3 Å or better and you have at least one Cys/Met per 25 residues, put it on the home source and collect until the sulfur signal rises above the noise. Then solve by SAD. http://www3.imperial.ac.uk/xraycrystallography/learning/sulfur_sad http://www3.imperial.ac.uk/xraycrystallography/learning/sulfur_sad Andreas -- Dr. rer. nat. Fabio Dall'Antonia European Molecular Biology Laboratory c/o DESY Notkestraße 85, Bldg. 25a D-22603 Hamburg phone: +49 (0)40 89902-178 fax:+49 (0)40 89902-149 e-mail: fabio.dallanto...@embl-hamburg.de
[ccp4bb] The phase problem
Dear all, I would like to known if someone could help me with some idea about the phase problem. I am a beginner in crystallography and the first time I tryed to solve the structure by Molrep, amore, mr. Bump and I didnt find anything for molecular replacement. Thus, currently I've tried to soak heavy-atom ( Hg, Pt, I, Pb) and the diffraction was good, the results in XDS didnt show so good. I didnt look the heavy atom in density. I appreciate any suggestion and ideas what I could do. Best regards, -- Giulliana Rangel Mestranda PPG-Biotecnologia UNIFESP Laboratório de Biologia Estrutural Tel.: (12) 3309-9698 Rua Talim 330, Vila Nair CEP 12231-280 São José dos Campos - SP
Re: [ccp4bb] The phase problem
Screen for heavy atoms with native gel. Run protein with variety of heavy atoms. If you see a discreet shift in the native gel, then you will get derivative. If you don't see a shift, don't bother soaking. It's worked for me every time. Also, ammonium sulfate will mess up your heavy atoms, so if that's in your condition, you need to substitute AmSulf with LiSO4 in your soak. Nick On Wed, Nov 5, 2014 at 2:20 PM, Giulliana Rangel giulliana.ran...@gmail.com wrote: Dear all, I would like to known if someone could help me with some idea about the phase problem. I am a beginner in crystallography and the first time I tryed to solve the structure by Molrep, amore, mr. Bump and I didnt find anything for molecular replacement. Thus, currently I've tried to soak heavy-atom ( Hg, Pt, I, Pb) and the diffraction was good, the results in XDS didnt show so good. I didnt look the heavy atom in density. I appreciate any suggestion and ideas what I could do. Best regards, -- Giulliana Rangel Mestranda PPG-Biotecnologia UNIFESP Laboratório de Biologia Estrutural Tel.: (12) 3309-9698 Rua Talim 330, Vila Nair CEP 12231-280 São José dos Campos - SP -- [This e-mail message may contain privileged, confidential and/or proprietary information of H3 Biomedicine. If you believe that it has been sent to you in error, please contact the sender immediately and delete the message including any attachments, without copying, using, or distributing any of the information contained therein. This e-mail message should not be interpreted to include a digital or electronic signature that can be used to authenticate an agreement, contract or other legal document, nor to reflect an intention to be bound to any legally-binding agreement or contract.]
Re: [ccp4bb] The phase problem
Hi Giuliana, if your crystal diffracts to 3 Å or better and you have at least one Cys/Met per 25 residues, put it on the home source and collect until the sulfur signal rises above the noise. Then solve by SAD. http://www3.imperial.ac.uk/xraycrystallography/learning/sulfur_sad Andreas On 05/11/2014 7:20, Giulliana Rangel wrote: Dear all, I would like to known if someone could help me with some idea about the phase problem. I am a beginner in crystallography and the first time I tryed to solve the structure by Molrep, amore, mr. Bump and I didnt find anything for molecular replacement. Thus, currently I've tried to soak heavy-atom ( Hg, Pt, I, Pb) and the diffraction was good, the results in XDS didnt show so good. I didnt look the heavy atom in density. I appreciate any suggestion and ideas what I could do. Best regards, -- Giulliana Rangel Mestranda PPG-Biotecnologia UNIFESP Laboratório de Biologia Estrutural Tel.: (12) 3309-9698 Rua Talim 330, Vila Nair CEP 12231-280 São José dos Campos - SP -- Andreas Förster X-ray Crystallograhpy Facility Manager Centre for Structural Biology Imperial College London
Re: [ccp4bb] The phase problem
Hi Giulliana, I would (re)check for issues during the assignment of the space group prior to MR. Many people in the list can help you with this if you provide additional information regarding your data. In case you have a structure that can not be solved by MR, I would give a chance for the so-called quick cryo soaking. There are many successful histories of phasing with this approach. Some references that might be useful: http://scripts.iucr.org/cgi-bin/paper?gr0995 (*Acta Cryst.* (2000). D*56*, 232-237) http://scripts.iucr.org/cgi-bin/paper?dz5043 (*Acta Cryst.* (2005). D*61*, 1022-1030) http://www.sciencedirect.com/science/article/pii/S0959440X0200372X (Current Opinion in Structural Biology,Volume 12, Issue 5, 1 October 2002, Pages 674–678) HTH, -Alessandro [ ]s --alessandro 2014-11-05 17:20 GMT-02:00 Giulliana Rangel giulliana.ran...@gmail.com: Dear all, I would like to known if someone could help me with some idea about the phase problem. I am a beginner in crystallography and the first time I tryed to solve the structure by Molrep, amore, mr. Bump and I didnt find anything for molecular replacement. Thus, currently I've tried to soak heavy-atom ( Hg, Pt, I, Pb) and the diffraction was good, the results in XDS didnt show so good. I didnt look the heavy atom in density. I appreciate any suggestion and ideas what I could do. Best regards, -- Giulliana Rangel Mestranda PPG-Biotecnologia UNIFESP Laboratório de Biologia Estrutural Tel.: (12) 3309-9698 Rua Talim 330, Vila Nair CEP 12231-280 São José dos Campos - SP
