[ccp4bb] Call for applications to the Membrane Protein Lab at Diamond
Call for applications to the Membrane Protein Lab at Diamond Dear Crystallographers, The Membrane Protein Laboratory (MPL) at Diamond Light Source in Oxfordshire (UK) is pleased to announce that is now open our call for proposals. The MPL is a joint venture between the Diamond Light Source and Imperial College London. It is a collaboration between Prof. So Iwata at Imperial College and Dame Prof. Louise Johnson and Dr Gwyndaf Evans at Diamond. The MPL is a state-of-the-art lab, fully equipped with purification systems and crystallisation robots for membrane proteins. The facility is open to applications from groups anywhere in the world. Users are able to visit from a few days to a more extended periods depending on their project. For more information on MPL please see our website: http://www.diamond.ac.uk/Home/MPL.html http://www.diamond.ac.uk/Home/MPL.html and for information on how to apply and application forms please follow the link: http://www.diamond.ac.uk/Home/MPL/users.html http://www.diamond.ac.uk/Home/MPL/users.html The deadline for this call is 23:59 on Friday 26th November 2010. If you have any questions or would like to discuss an application, please contact the MPL group leader Dr Isabel Moraes by email: i.mor...@imperial.ac.uk With best wishes, Isabel Moraes
Re: [ccp4bb] Removing a tight binding ligand
We often dialysis protein against charcoal to remove small molecules that tightly bind to protein. Meng-Chiao Joseph Ho, PhD Department of Biochemistry Albert Einstein College of Medicine Hi all, I am working with a substrate binding protein. The protein scavenges its endogenous ligand out of the E. coli used for expression. I need to get this ligand out for both crystallographic and kinetic studies. I have tried denaturing in urea and refolding the protein with limited success. It refolds properly according to the CD spectra but it some how manages to hold on to trace amounts of ligand despite serial dialysis (500ml to 5ml of sample) in 8M, 6M, 4M, 2M 1M urea followed by 50mM Tris. I also have a homolog that abjectly refuses to refold in either urea or guanidine, though it does turn the dialysis tubing into a lovely snow globe. There are alternative methods of performing the kinetics, but those will require destroying the protein which doesn't help on the crystallography front. I was wondering if any of you out there had experience successfully removing very tightly bound ligands by an alternative method. I didn't see any mention on the subject in the archives. I had hoped you might be able to point me in the right direction. Thanks for your time, Katherine Ph. D. candidate Department of Biochemistry and Molecular Biology College of Medicine University of Florida
Re: [ccp4bb] Removing a tight binding ligand
Hi Katherine, We had a case where we used saturating amounts of NaCl and precipitated the protein to get rid of a very tight binding ligand (Structure, 11, 677-690, 2003; look at the preparation of the apoenzyme section). Regards, Mathews -Original Message- From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of SIPPEL,KATHERINE H Sent: Thursday, October 07, 2010 6:05 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Removing a tight binding ligand Hi all, I am working with a substrate binding protein. The protein scavenges its endogenous ligand out of the E. coli used for expression. I need to get this ligand out for both crystallographic and kinetic studies. I have tried denaturing in urea and refolding the protein with limited success. It refolds properly according to the CD spectra but it some how manages to hold on to trace amounts of ligand despite serial dialysis (500ml to 5ml of sample) in 8M, 6M, 4M, 2M 1M urea followed by 50mM Tris. I also have a homolog that abjectly refuses to refold in either urea or guanidine, though it does turn the dialysis tubing into a lovely snow globe. There are alternative methods of performing the kinetics, but those will require destroying the protein which doesn't help on the crystallography front. I was wondering if any of you out there had experience successfully removing very tightly bound ligands by an alternative method. I didn't see any mention on the subject in the archives. I had hoped you might be able to point me in the right direction. Thanks for your time, Katherine Ph. D. candidate Department of Biochemistry and Molecular Biology College of Medicine University of Florida
Re: [ccp4bb] Removing a tight binding ligand
Hi, It could help if you said what your ligand is. Nadir Pr. Nadir T. Mrabet Structural Molecular Biochemistry Nutrigenex - INSERM U-954 Nancy University, School of Medicine 9, Avenue de la Foret de Haye, BP 184 54505 Vandoeuvre-les-Nancy Cedex France Phone: +33 (0)3.83.68.32.73 Fax: +33 (0)3.83.68.32.79 E-mail: Nadir.Mrabetat medecine.uhp-nancy.fr On 08/10/2010 03:05, SIPPEL,KATHERINE H wrote: Hi all, I am working with a substrate binding protein. The protein scavenges its endogenous ligand out of the E. coli used for expression. I need to get this ligand out for both crystallographic and kinetic studies. I have tried denaturing in urea and refolding the protein with limited success. It refolds properly according to the CD spectra but it some how manages to hold on to trace amounts of ligand despite serial dialysis (500ml to 5ml of sample) in 8M, 6M, 4M, 2M 1M urea followed by 50mM Tris. I also have a homolog that abjectly refuses to refold in either urea or guanidine, though it does turn the dialysis tubing into a lovely snow globe. There are alternative methods of performing the kinetics, but those will require destroying the protein which doesn't help on the crystallography front. I was wondering if any of you out there had experience successfully removing very tightly bound ligands by an alternative method. I didn't see any mention on the subject in the archives. I had hoped you might be able to point me in the right direction. Thanks for your time, Katherine Ph. D. candidate Department of Biochemistry and Molecular Biology College of Medicine University of Florida
Re: [ccp4bb] Removing a tight binding ligand
It is just like the regular dialysis but the deserved buffer contains charcol powder. Meng-Chiao Joseph Ho How do you do this ? I have not heard of this, but I also never had to deal with getting rid of a ligand. However I would be interested to learn more about this method. Thanks, Jürgen - Jürgen Bosch Johns Hopkins Bloomberg School of Public Health Department of Biochemistry Molecular Biology Johns Hopkins Malaria Research Institute 615 North Wolfe Street, W8708 Baltimore, MD 21205 Phone: +1-410-614-4742 Lab: +1-410-614-4894 Fax: +1-410-955-3655 http://web.mac.com/bosch_lab/ On Oct 8, 2010, at 11:01 AM, Joseph Ho wrote: We often dialysis protein against charcoal to remove small molecules that tightly bind to protein. Meng-Chiao Joseph Ho, PhD Department of Biochemistry Albert Einstein College of Medicine Hi all, I am working with a substrate binding protein. The protein scavenges its endogenous ligand out of the E. coli used for expression. I need to get this ligand out for both crystallographic and kinetic studies. I have tried denaturing in urea and refolding the protein with limited success. It refolds properly according to the CD spectra but it some how manages to hold on to trace amounts of ligand despite serial dialysis (500ml to 5ml of sample) in 8M, 6M, 4M, 2M 1M urea followed by 50mM Tris. I also have a homolog that abjectly refuses to refold in either urea or guanidine, though it does turn the dialysis tubing into a lovely snow globe. There are alternative methods of performing the kinetics, but those will require destroying the protein which doesn't help on the crystallography front. I was wondering if any of you out there had experience successfully removing very tightly bound ligands by an alternative method. I didn't see any mention on the subject in the archives. I had hoped you might be able to point me in the right direction. Thanks for your time, Katherine Ph. D. candidate Department of Biochemistry and Molecular Biology College of Medicine University of Florida
[ccp4bb] unknown ligand density
Hello all, I have a structure of my protein at 1.3 Ang. There is a clear density in the binding site but I can't seem to figure out what the ligand is. The crystalization conditions contain PEG, sulfate, TMAO and acetate buffer. The protein was purified by a Ni-NTA column and went into phosphate buffer after that. The ligand appears is interacting with a histidine and a water atom and is most likely acetate. Pictures can be viewed at www.xtalmerski.tumblr.com. However, there seems to be an additional atom on the acetate, which would be the easy answer but the buffer was of the 99+% purity (admittedly from Sigma) so I doubt that enough proprionic acid would make it through to account for 100% ligand occupancy. I also looked at TMAO decomposition by NMR under the same conditions for twice as long as the time it took to grow crystals and there was no apparent decomposition (which is 95% by NMR of course). A possible precendent in the literature for TMAO rearrangement requires a homolytic decomposition to formaldehyde and then formation of the new product to get oxygen migration which may be a bit of a stretch to suggest as a new enzyme catalyzed reaction Plus, refinement with that product isn't perfect either as the nitrogen has too many electrons to be in the tertiary center to fit this density. So, has anyone seen or heard or can imagine a way to get an extra atom on acetate? Thanks for any help you can give. Matthew Merski Post-doc Shoichet Group UCSF
Re: [ccp4bb] Removing a tight binding ligand
I would like to thank all of you for your replies. I very much appreciate your time and I've got some great starting points to try out. I will put together a recap of the off- and on-board comments in the next day or so for archive posterity. For those looking for more details... The protein is specifically a monomeric substrate binding protein (very roughly similar to MBP) and binds a water-soluble vitamin. It's purified exclusively by ion exchange so no tags though it is shy a bit of the N-terminal domain to remove a lipo- moiety. I have no idea what the Kd is at the moment, that was what I was hoping to calculate. I do have the bound structure, it is not covalently attached to the protein. The interactions are mostly hydrophobic with only a couple of hydrogen bonds. Once again, thank you all, Katherine On Fri, Oct 8, 2010 at 12:20 PM, Joseph Ho j...@medusa.bioc.aecom.yu.edu wrote: It is just like the regular dialysis but the deserved buffer contains charcol powder. Meng-Chiao Joseph Ho How do you do this ? I have not heard of this, but I also never had to deal with getting rid of a ligand. However I would be interested to learn more about this method. Thanks, J??rgen - J??rgen Bosch Johns Hopkins Bloomberg School of Public Health Department of Biochemistry Molecular Biology Johns Hopkins Malaria Research Institute 615 North Wolfe Street, W8708 Baltimore, MD 21205 Phone: +1-410-614-4742 Lab: +1-410-614-4894 Fax: +1-410-955-3655 http://web.mac.com/bosch_lab/ On Oct 8, 2010, at 11:01 AM, Joseph Ho wrote: We often dialysis protein against charcoal to remove small molecules that tightly bind to protein. Meng-Chiao Joseph Ho, PhD Department of Biochemistry Albert Einstein College of Medicine Hi all, I am working with a substrate binding protein. The protein scavenges its endogenous ligand out of the E. coli used for expression. I need to get this ligand out for both crystallographic and kinetic studies. I have tried denaturing in urea and refolding the protein with limited success. It refolds properly according to the CD spectra but it some how manages to hold on to trace amounts of ligand despite serial dialysis (500ml to 5ml of sample) in 8M, 6M, 4M, 2M 1M urea followed by 50mM Tris. I also have a homolog that abjectly refuses to refold in either urea or guanidine, though it does turn the dialysis tubing into a lovely snow globe. There are alternative methods of performing the kinetics, but those will require destroying the protein which doesn't help on the crystallography front. I was wondering if any of you out there had experience successfully removing very tightly bound ligands by an alternative method. I didn't see any mention on the subject in the archives. I had hoped you might be able to point me in the right direction. Thanks for your time, Katherine Ph. D. candidate Department of Biochemistry and Molecular Biology College of Medicine University of Florida -- SIPPEL,KATHERINE H Ph. D. candidate Department of Biochemistry and Molecular Biology College of Medicine University of Florida
[ccp4bb] Differences between refmac_5.5 and refmac_5.2
Hi, I found there are many changes between refmac_5.5 and refmac_5.2. For example, the key word REFI BREF OVER will result in totally different results under these 2 versions. Based on my input PDB with anisotropic B pre-refined, refmac_5.5 gave a much higher R/Rfree than refmac_5.2. Can somebody explain the difference and give me some suggestions on how to modfiy the keyword in refmac_5.5 to match what refmac_5.2 have done by using REFI BREF OVER? Thanks!
Re: [ccp4bb] Differences between refmac_5.5 and refmac_5.2
Hi the reason could be that refmac converts all aniso B to iso B when you do overall B value refinement. Without data it would be difficult to test and give definite answer. regards Garib On 9 Oct 2010, at 00:15, Hailiang Zhang wrote: Hi, I found there are many changes between refmac_5.5 and refmac_5.2. For example, the key word REFI BREF OVER will result in totally different results under these 2 versions. Based on my input PDB with anisotropic B pre-refined, refmac_5.5 gave a much higher R/Rfree than refmac_5.2. Can somebody explain the difference and give me some suggestions on how to modfiy the keyword in refmac_5.5 to match what refmac_5.2 have done by using REFI BREF OVER? Thanks!
