Re: [ccp4bb] SP Sep HP tight binding of proteins
Hi Meg, I think with your pi you may also test a Q-Sepharose at neutral to slightly alkaline pH. Maybe your protein is more comfortable with this condition compared to a pH of 4.5. As mentioned befor your protein is most likely precipitated on the column. Best regards Christian Am Dienstag 04 Januar 2011 10:34:26 schrieb megha goyal: Dear All, We used SP sepharose high performance as second stage Ion exchange chromatography for polishing the product. We did get pure product but yield obtained was mere 25%. Our protein has a pI of 5.5 - 6.0 and we had used 25 mM Na Acetate buffer pH 4.5 for loading and same buffer with 1M NaCl for elution, 25 C.V. linear gradient. Can you suggest some changes that i can incorporate to increase the yield i.e additives to be added or some change in pH etc. I tried elution with arginine HCL as elution buffer as was recommended in one paper, but the yield obtained was even less. On washing with 2M NaCl ther is not much peak appearing but on washing with 1M NaOH substantial peak appears. Kindly help me through this. meg
[ccp4bb] crystallographer position available
To the CCP4BB members: We are seeking to hire a protein crystallographer to work on a new project in our group. Funding is currently available to support the position for one year, with the possibility of renewal at the end of this time. We are looking for someone with 2-5 years of experience beyond their Ph.D. - a newly minted doctorate will likely not have enough experience for the job, while someone with many years of experience in the field would probably not be interested in the salary range we are considering. Also, please note that, as a small non-profit organization, we are unable to provide relocation costs, so applicants from beyond the New York area should keep this in mind. Applications should be directed to me (mfrank...@nysbc.org). The text of the job advertisement follows: The New York Structural Biology Center is a shared research organization, owned and operated by ten universities and medical schools in the New York area, and devoted to the structural analysis of biological processes through the use of NMR, X-ray crystallography, and electron microscopy. We are currently expanding our crystallography lab to begin work on a new project involving the large-scale determination of many protein structures. We are seeking an experienced crystallographer to be an integral part of this work. The successful candidate will work with two other crystallographers and three research assistants, collaborating on all aspects of the structure determination pipeline, from large-scale clone design and construction, to protein expression and purification, to synchrotron data collection, structure determination and PDB submission, to writing research articles describing the significant findings. Applicants should have a Ph.D. in protein crystallography, with 2 - 5 years of postdoctoral experience. A proven track record of multiple successful structure determinations is required. Previous experience with protein expression and purification, and/or cell culture, is highly desirable. Some managerial experience, while not necessary, would be helpful. Salary level is commensurate with qualifications and experience. Thank you for your attention to this. - Matt Franklin -- Matthew Franklin, Ph. D. Senior Research Scientist New York Structural Biology Center 89 Convent Avenue, New York, NY 10027 (646) 275-7165
[ccp4bb] A quick question - monomer lib cif
Dear all experts, Just a simple question, how can I obtain a monomer library CIF (_lib.cif) of a new small molecule that could be recognized by Refmac5? If i have a CCDC cif file. many thanks stephen -- Dr. Stephen Sin-Yin Chui (徐先賢) Assistant Professor, Department of Chemistry, The University of Hong Kong, Pokfulam Road, Hong Kong SAR, China. Tel: 22415814 (Office), 22415818 (X-ray Diffraction Laboratory)
Re: [ccp4bb] A quick question - monomer lib cif
Dear Stephen, First you change cife file to pdb file of your structure using CCDC mercury program. It's freely available in the CCDC site. Then use CCP4 Sketcher program to create the monomer library file with the pdb file of your structure. Wishes, Sampath N 2011/1/5 Dr. STEPHEN SIN-YIN, CHUI chui...@hkucc.hku.hk Dear all experts, Just a simple question, how can I obtain a monomer library CIF (_lib.cif) of a new small molecule that could be recognized by Refmac5? If i have a CCDC cif file. many thanks stephen -- Dr. Stephen Sin-Yin Chui (徐先賢) Assistant Professor, Department of Chemistry, The University of Hong Kong, Pokfulam Road, Hong Kong SAR, China. Tel: 22415814 (Office), 22415818 (X-ray Diffraction Laboratory) -- Dr. N.Sampath Assistant Professor Dept. of Advanced Technology Fusion Konkuk University 1 Hwayang-dong, Gwangjin-gu, Seoul, 143-701, Korea Tel: 82-2-450-4151 Fax: 82-2-444-6707 E-mail: samp...@konkuk.ac.kr
Re: [ccp4bb] A quick question - monomer lib cif
Dear Stephen, a cif from CCDC is something totally different from a refmac CIF. You need to convert to PDB (you can also use MOE software if available). When you have built in your small molecule PDB into your whole model you just run refmac. Refmac will fail but write out a suggestion for a molecular description cif file. You simply edit this cif file using bond lengths and angles from the CCDC structure (i use pymol for analysis). Through out wrong or weird info (often torsion restraints and stereoisomer info) and use the edited cif file as library input in a new Refmac run. Good luck, Matthias Am 06.01.2011 04:30, schrieb Dr. STEPHEN SIN-YIN, CHUI: Dear all experts, Just a simple question, how can I obtain a monomer library CIF (_lib.cif) of a new small molecule that could be recognized by Refmac5? If i have a CCDC cif file. many thanks stephen -- Dr. Matthias Zebisch Universität Leipzig Biotechnologisch-Biomedizinisches Zentrum Strukturanalytik von Biopolymeren Deutscher Platz 5 04103 Leipzig Germany Phone: 0049-341-97-31323 (lab) -31312 (office) Fax : 0049-341-97-31319 email: matthias.zebi...@bbz.uni-leipzig.de
