Re: [ccp4bb] unknown densities
Yamei, This is not unusual, particularly for many proteins that bind nucleotide derivatives, especially GDP/GTP binding proteins, as Nat said. If it is GDP that is tightly bound at high occupancy, it should be quite easy to identify because of the pyrophosphates and the guanine ring. To build into, pop in a GDP molecule from another GDP/GTP binding protein structure; there is GDP .cif files in the CCP4 and Phenix libraries. Just be aware that there are at least two major conformers seen regarding the guanine ring (syn and anti). While in GDP/GTP binding proteins the ring conformer I believe is anti (the ring not over the ribose), in GDP-sugar enzymes, it can be syn (the ring over the ribose). Michael R. Michael Garavito, Ph.D. Professor of Biochemistry Molecular Biology 603 Wilson Rd., Rm. 513 Michigan State University East Lansing, MI 48824-1319 Office: (517) 355-9724 Lab: (517) 353-9125 FAX: (517) 353-9334Email: rmgarav...@gmail.com On Dec 8, 2014, at 10:15 PM, Yamei Yu ymyux...@gmail.com wrote: Hi all, We crystallised a small GTPase expressed in E. Coli and found some densities in GDP/GTP binding site. We didn’t add any GDP/GTP or GDP/GPD homologues during protein expression, purification and crystallisation. The resolution is not high, we couldn’t tell what it is. Is there any method to detect what it is? Thanks! Best wishes! yamei Yamei Yu State Key Laboratory of Biotherapy／Collaborative Innovation Center of Biotherapy, West China Hospital, Sichuan University,Chengdu,610041, P.R.China Tel: 15882013485 Email: ymyux...@gmail.com ymyux...@163.com yamei...@scu.edu.cn
Re: [ccp4bb] unknown densities
Hi Yamei, Depending on your dataset and collection, if you have collected any anomalous data, you can check for peaks that indicate phosphorus atoms in anomalous maps. It might even be worth feeding into ANoDe ( http://shelx.uni-ac.gwdg.de/SHELX/anode.php) if you have sufficiently high multiplicity on e.g. a Cu source. (Disclaimer: I'm no expert. Others here know how this works much better than I do) Shane Caldwell McGill University On Tue, Dec 9, 2014 at 8:35 AM, R. M. Garavito rmgarav...@gmail.com wrote: Yamei, This is not unusual, particularly for many proteins that bind nucleotide derivatives, especially GDP/GTP binding proteins, as Nat said. If it is GDP that is tightly bound at high occupancy, it should be quite easy to identify because of the pyrophosphates and the guanine ring. To build into, pop in a GDP molecule from another GDP/GTP binding protein structure; there is GDP .cif files in the CCP4 and Phenix libraries. Just be aware that there are at least two major conformers seen regarding the guanine ring (syn and anti). While in GDP/GTP binding proteins the ring conformer I believe is anti (the ring not over the ribose), in GDP-sugar enzymes, it can be syn (the ring over the ribose). Michael ** *R. Michael Garavito, Ph.D.* *Professor of Biochemistry Molecular Biology* *603 Wilson Rd., Rm. 513* *Michigan State University * *East Lansing, MI 48824-1319* *Office:* *(517) 355-9724 %28517%29%20355-9724 Lab: (517) 353-9125 %28517%29%20353-9125* *FAX: (517) 353-9334 %28517%29%20353-9334 Email: rmgarav...@gmail.com garav...@gmail.com* ** On Dec 8, 2014, at 10:15 PM, Yamei Yu ymyux...@gmail.com wrote: Hi all, We crystallised a small GTPase expressed in E. Coli and found some densities in GDP/GTP binding site. We didn’t add any GDP/GTP or GDP/GPD homologues during protein expression, purification and crystallisation. The resolution is not high, we couldn’t tell what it is. Is there any method to detect what it is? Thanks! Best wishes! yamei Yamei Yu State Key Laboratory of Biotherapy／Collaborative Innovation Center of Biotherapy, West China Hospital, Sichuan University,Chengdu,610041, P.R.China Tel: 15882013485 Email: ymyux...@gmail.com ymyux...@163.com yamei...@scu.edu.cn
[ccp4bb] unknown densities
Hi all, We crystallised a small GTPase expressed in E. Coli and found some densities in GDP/GTP binding site. We didn’t add any GDP/GTP or GDP/GPD homologues during protein expression, purification and crystallisation. The resolution is not high, we couldn’t tell what it is. Is there any method to detect what it is? Thanks! Best wishes! yamei Yamei Yu State Key Laboratory of Biotherapy／Collaborative Innovation Center of Biotherapy, West China Hospital, Sichuan University,Chengdu,610041, P.R.China Tel: 15882013485 Email: ymyux...@gmail.com ymyux...@163.com yamei...@scu.edu.cn
Re: [ccp4bb] unknown densities
This is where it’s customary to include a small image or two (or better, a link thereto) which shows the density, and the masters here can tell you there best guesses—seems to be a bit of a parlour game. Also include info on what is in the crystallization condition and protein buffer if you dare. FYI, sometimes native nucleotides can make it through protein purifications if binding is tight. JPK From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Yamei Yu Sent: Monday, December 08, 2014 10:15 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] unknown densities Hi all, We crystallised a small GTPase expressed in E. Coli and found some densities in GDP/GTP binding site. We didn’t add any GDP/GTP or GDP/GPD homologues during protein expression, purification and crystallisation. The resolution is not high, we couldn’t tell what it is. Is there any method to detect what it is? Thanks! Best wishes! yamei Yamei Yu State Key Laboratory of Biotherapy／Collaborative Innovation Center of Biotherapy, West China Hospital, Sichuan University,Chengdu,610041, P.R.China Tel: 15882013485 Email: ymyux...@gmail.commailto:ymyux...@gmail.com ymyux...@163.commailto:ymyux...@163.com yamei...@scu.edu.cnmailto:yamei...@scu.edu.cn
Re: [ccp4bb] unknown densities
On Mon, Dec 8, 2014 at 7:24 PM, Keller, Jacob kell...@janelia.hhmi.org wrote: FYI, sometimes native nucleotides can make it through protein purifications if binding is tight. This is especially true for G-proteins, since tight binding to GDP is an essential part of their function. I don't know what to expect from E. coli proteins, but human Ras co-purifies with GDP at close to 1:1. At low resolution, it might be easiest to superimpose the closest nucleotide-bound homolog and see how well the density aligns with the nucleotide. -Nat
