[ccp4bb] Using intrinsically bound Zn atoms for phasing
Hi I am trying to solve the structure of an engineered protein.The protein is crystallized with Zn bound to it .We collected a 1.5A0 data. Molecular Replacement didn't yield a good match for the protein. I want to try MAD taking advantage of the Zn atoms in protein. I am not sure what wavelength should i use to collect the diffraction data for Zn. any suggestions? Thank You Deepthi -- Deepthi
Re: [ccp4bb] Using intrinsically bound Zn atoms for phasing
http://skuld.bmsc.washington.edu/scatter/AS_form.html Maybe useful to you. However, I would advise to do a fluorescence scan over the range given in the graph and then use chooch to provide the precise energies for your peak and inflection. If you have a large crystal don't expose all of it when you do the fluorescence scan but rather reserve a 'fresh' piece to do your actual data collection. F On Mar 6, 2012, at 1:09 PM, Deepthi wrote: Hi I am trying to solve the structure of an engineered protein.The protein is crystallized with Zn bound to it .We collected a 1.5A0 data. Molecular Replacement didn't yield a good match for the protein. I want to try MAD taking advantage of the Zn atoms in protein. I am not sure what wavelength should i use to collect the diffraction data for Zn. any suggestions? Thank You Deepthi -- Deepthi
Re: [ccp4bb] Using intrinsically bound Zn atoms for phasing
Since you've collected the data already use your favourite data processing program and treat the Friedel pairs separately. I'd suggest to try HKL2map in conjunction with SHELX C/D/E (sorry for the non CCP4 advertisement here) for solving the heavy atom sites. You can in parallel also try SnB or BnP to find a substructure solution. Depending how bad you resulting density looks like you might want to improve your phases via Sharp. If you want to stay in the CCP4 protected sandbox, then give Crank a try. Jürgen On Mar 6, 2012, at 3:24 PM, Francis E Reyes wrote: http://skuld.bmsc.washington.edu/scatter/AS_form.html Maybe useful to you. However, I would advise to do a fluorescence scan over the range given in the graph and then use chooch to provide the precise energies for your peak and inflection. If you have a large crystal don't expose all of it when you do the fluorescence scan but rather reserve a 'fresh' piece to do your actual data collection. F On Mar 6, 2012, at 1:09 PM, Deepthi wrote: Hi I am trying to solve the structure of an engineered protein.The protein is crystallized with Zn bound to it .We collected a 1.5A0 data. Molecular Replacement didn't yield a good match for the protein. I want to try MAD taking advantage of the Zn atoms in protein. I am not sure what wavelength should i use to collect the diffraction data for Zn. any suggestions? Thank You Deepthi -- Deepthi .. 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://web.mac.com/bosch_lab/
Re: [ccp4bb] Using intrinsically bound Zn atoms for phasing
A reference for a real MAD phasing with Zinc (worked very well): Ennifar et al. MAD phasing replacing magnesium with zinc. Acta Cryst. (2001). D57, 330 Philippe Dumas Bosch, Juergen jubo...@jhsph.edu a écrit : Since you've collected the data already use your favourite data processing program and treat the Friedel pairs separately. I'd suggest to try HKL2map in conjunction with SHELX C/D/E (sorry for the non CCP4 advertisement here) for solving the heavy atom sites. You can in parallel also try SnB or BnP to find a substructure solution. Depending how bad you resulting density looks like you might want to improve your phases via Sharp. If you want to stay in the CCP4 protected sandbox, then give Crank a try. Jürgen On Mar 6, 2012, at 3:24 PM, Francis E Reyes wrote: http://skuld.bmsc.washington.edu/scatter/AS_form.html Maybe useful to you. However, I would advise to do a fluorescence scan over the range given in the graph and then use chooch to provide the precise energies for your peak and inflection. If you have a large crystal don't expose all of it when you do the fluorescence scan but rather reserve a 'fresh' piece to do your actual data collection. F On Mar 6, 2012, at 1:09 PM, Deepthi wrote: Hi I am trying to solve the structure of an engineered protein.The protein is crystallized with Zn bound to it .We collected a 1.5A0 data. Molecular Replacement didn't yield a good match for the protein. I want to try MAD taking advantage of the Zn atoms in protein. I am not sure what wavelength should i use to collect the diffraction data for Zn. any suggestions? Thank You Deepthi -- Deepthi .. 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://web.mac.com/bosch_lab/
Re: [ccp4bb] Using intrinsically bound Zn atoms for phasing
Self-referentially: I once used the structural Zn of p53 to do a Zn MAD structure of a p53:53BP1 complex at 2.5 Angstrom with one zinc per 450 residues. Apparently using 1.283, 1.282 and 1.262 Angstroms (i.e. the Zinc edge). http://genesdev.cshlp.org/content/16/5/583.long But of course do your own fluorescence scan. The advantage of structural metals is full occupancy and relatively lower B-factor. That map was actually pretty good, and since it came out of MLPHARE I don't doubt modern programs like SHARP could make it quite a lot better. Phil Jeffrey Princeton On 3/6/12 3:09 PM, Deepthi wrote: Hi I am trying to solve the structure of an engineered protein.The protein is crystallized with Zn bound to it .We collected a 1.5A0 data. Molecular Replacement didn't yield a good match for the protein. I want to try MAD taking advantage of the Zn atoms in protein. I am not sure what wavelength should i use to collect the diffraction data for Zn. any suggestions? Thank You Deepthi -- Deepthi
Re: [ccp4bb] Using intrinsically bound Zn atoms for phasing
Hi, 1. You don't mention how many Zn sites you have, or how big your protein is - as Phil mentioned, these are factors. 2. I'll add to the chorus - pick your wavelength(s) based on a fluorescence scan. 3. If 1.5A0 is your wavelength, not your resolution, you may still have some anomalous signal - I've had a dataset collected on a copper rotating anode (wavelength 1.54) with detectable zinc anomalous signal. You'll get better signal closer to the anomalous peak; but if your Zn/protein ratio is large enough it may not be necessary. Pete Deepthi wrote: Hi I am trying to solve the structure of an engineered protein.The protein is crystallized with Zn bound to it .We collected a 1.5A0 data. Molecular Replacement didn't yield a good match for the protein. I want to try MAD taking advantage of the Zn atoms in protein. I am not sure what wavelength should i use to collect the diffraction data for Zn. any suggestions? Thank You Deepthi -- Deepthi
