Re: [ccp4bb] disulfide bridge in red density
Hello everyone, Thank you very much for giving me such a valuable information. I will try one by one each suggestion and then ask again if i feel more problem. thanks regards Sadaf Iqbal PhD Scholar ICCBS, University of Karachi, Pakistan. Visiting Scientist University of Hamburg, Germany. From: Sanishvili, Ruslan rsanishv...@anl.gov To: CCP4BB@JISCMAIL.AC.UK Sent: Monday, March 7, 2011 1:22:14 Subject: Re: [ccp4bb] disulfide bridge in red density Hi Sadaf, Most likely you are dealing with radiation damage. If that's the case, you would need to refine occupancies as Mario has already suggested. However, you may not find a double conformation. Instead, you may find that some of your sulfurs are gone. There is a ton of publications on this topic, take a loo. If you have high enough redundancy, you may be able to use only the part of the data, collected at the beginning. This would have suffered less from radiation. You can also use zero dose extrapolations - look up Diederichs' and Borek's publications. Good luck! Nukri -Original Message- From: CCP4 bulletin board on behalf of sadaf iqbal Sent: Sun 3/6/2011 12:57 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] disulfide bridge in red density Hello everyone, Recently, i have solved a protein structure at 1.3 Å resolution. I have encountered one problem in this structure while refining in COOT. There are three disulfide bridges in my structure and if i used 2.0 Å resolution range in refmac then the density around disulfide bridges is ok but if i used 1.3 Å resolution range in refmac then red density is appeared along with blue one. Can anybody help me in this regard? I already played by increasing temperature factor of sulfur atoms but red density is still there. Thanks in advance Sadaf Iqbal PhD Scholar ICCBS, University of Karachi, Pakistan. Visiting Scientist University of Hamburg, Germany.
Re: [ccp4bb] disulfide bridge in red density
Hi Sadaf It is also possible that the features you observe are also due to anisotropy of the ADPs. At 1.3A resolution you can perform anisotropic refinement. Give it a go and se if it helps. Best Roberto On 6 Mar 2011, at 18:57, sadaf iqbal wrote: Hello everyone, Recently, i have solved a protein structure at 1.3 Å resolution. I have encountered one problem in this structure while refining in COOT. There are three disulfide bridges in my structure and if i used 2.0 Å resolution range in refmac then the density around disulfide bridges is ok but if i used 1.3 Å resolution range in refmac then red density is appeared along with blue one. Can anybody help me in this regard? I already played by increasing temperature factor of sulfur atoms but red density is still there. Thanks in advance Sadaf Iqbal PhD Scholar ICCBS, University of Karachi, Pakistan. Visiting Scientist University of Hamburg, Germany.
Re: [ccp4bb] disulfide bridge in red density
Hi Sadaf, You may want to try to play with the occupancy. I have had a case before where the disulfide bond had double occupancy. In my case it was obvious because I could see the other density, but sometimes you can just see a major one due to disorder. I hope that helps, Mario Sanches On Sun, Mar 6, 2011 at 1:57 PM, sadaf iqbal sadaf_che...@yahoo.com wrote: Hello everyone, Recently, i have solved a protein structure at 1.3 Å resolution. I have encountered one problem in this structure while refining in COOT. There are three disulfide bridges in my structure and if i used 2.0 Å resolution range in refmac then the density around disulfide bridges is ok but if i used 1.3 Å resolution range in refmac then red density is appeared along with blue one. Can anybody help me in this regard? I already played by increasing temperature factor of sulfur atoms but red density is still there. Thanks in advance Sadaf Iqbal PhD Scholar ICCBS, University of Karachi, Pakistan. Visiting Scientist University of Hamburg, Germany. -- Mario Sanches Postdoctoral Fellow Samuel Lunenfeld Research Institute Mount Sinai Hospital 600 University Ave Toronto - Ontario Canada M5G 1X5 http://ca.linkedin.com/in/mariosanches
Re: [ccp4bb] disulfide bridge in red density
Hi Sadaf, Most likely you are dealing with radiation damage. If that's the case, you would need to refine occupancies as Mario has already suggested. However, you may not find a double conformation. Instead, you may find that some of your sulfurs are gone. There is a ton of publications on this topic, take a loo. If you have high enough redundancy, you may be able to use only the part of the data, collected at the beginning. This would have suffered less from radiation. You can also use zero dose extrapolations - look up Diederichs' and Borek's publications. Good luck! Nukri -Original Message- From: CCP4 bulletin board on behalf of sadaf iqbal Sent: Sun 3/6/2011 12:57 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] disulfide bridge in red density Hello everyone, Recently, i have solved a protein structure at 1.3 Å resolution. I have encountered one problem in this structure while refining in COOT. There are three disulfide bridges in my structure and if i used 2.0 Å resolution range in refmac then the density around disulfide bridges is ok but if i used 1.3 Å resolution range in refmac then red density is appeared along with blue one. Can anybody help me in this regard? I already played by increasing temperature factor of sulfur atoms but red density is still there. Thanks in advance Sadaf Iqbal PhD Scholar ICCBS, University of Karachi, Pakistan. Visiting Scientist University of Hamburg, Germany.
