Re: [ccp4bb] Peptide solubility issues
Hello Mo Wong, Some points below are good, but don't underestimate custom peptides sometimes... can be much harder/expensive than recombinant proteins. If you ordered the peptides it is good to know how they synthesized them, and how the elution profiles during purification (RP HPLC..) looked like, particularly that they are very hydrophobic. Did they use formic acid, or maybe they already dissolved them in DMSO.. Did they verify the product/bonds with MALDI-TOF for example and NMR? Furthermore, you can try 0.1-0.5 % Triton X-100. My vote would be for a final concentration of 5-25 uM (containing up to 5 % solvent), from a stock of 50-100 % solvent at 1 mM.. Btw I forgot.. for SPR (depends on the system, and especially for a 12mer), some detergents and even a minor DMSO/ethanol can affect the measurements drastically. Best wishes toufic el arnaout On Wed, Mar 5, 2014 at 12:19 AM, Mo Wong mowon...@gmail.com wrote: Hi all, Slightly off topic - but I'm having trouble solubilizing some peptides for SPR and hoped someone on the BB might have some other suggestions. The peptides are intra-chain S-S cross-linked 12mers with pIs of ~3. 10 residues are hydrophobic and 2 are acidic. Peptides have been tested with and without N- and C-terminal modifications (amidation/acetylation). I have tried: ddH2O raising (and lowering) pH (tested up to 8.5) with different buffers Including DMF as a co-solvent (I'm avoiding DMSO because of a methionine present in the peptide) - peptide still visibly precipitates out at 100uM in 5% DMF (also a 1mM stock in 50% DMF shows significant amounts of ppt) Adding a trace amount of detergent (0.005% Tween 20) I'm guessing I could try other co-solvents such as ethanol or initially solubilizing peptide in dilute NaOH before bringing the pH down with addition of a buffer (though I'm concerned about alkaline hydrolysis). Anyway, I'd rather have some insight from people before I waste any further peptide. Thanks for any suggestions.
[ccp4bb] size of a flexible pdb structure
Dear all, sorry for asking an off topic question. My protein is composed of two domains connected by a flexible linker (15aa). after 50ns simulation i came across the fact that one domain is flexible. so my question is that how could i be able to calculate the size of molecule or radius of my molecule and which conformation i should prefer to calculate the size of molecule. your any suggestion will be very helpful. thank you all in advance with best regards -- *Rajan kumar choudhary* *Senior Research Fellow* *Department of Atomic Energy(Govt.Of India)* *ACTREC TATA Memorial Center * *Kharghar Navi-Mumbai* *Mumbai-410210* *India*
Re: [ccp4bb] size of a flexible pdb structure
Dear Rajan kumar choudhary, could you explain why you expect the volume to depend on the conformation of the molecule? Do you have some effective volume in mind, e.g. the elution volume during size exclusion chromatography? Regards, Tim On Wed, Mar 05, 2014 at 08:42:21PM +0530, rajan kumar wrote: Dear all, sorry for asking an off topic question. My protein is composed of two domains connected by a flexible linker (15aa). after 50ns simulation i came across the fact that one domain is flexible. so my question is that how could i be able to calculate the size of molecule or radius of my molecule and which conformation i should prefer to calculate the size of molecule. your any suggestion will be very helpful. thank you all in advance with best regards -- *Rajan kumar choudhary* *Senior Research Fellow* *Department of Atomic Energy(Govt.Of India)* *ACTREC TATA Memorial Center * *Kharghar Navi-Mumbai* *Mumbai-410210* *India* -- -- Dr Tim Gruene Institut fuer anorganische Chemie Tammannstr. 4 D-37077 Goettingen GPG Key ID = A46BEE1A signature.asc Description: Digital signature
[ccp4bb] CCP4 lib file
Hi, I am trying to refine (Refmac5 on CCP4) a protein structure containing ligands that are exclusively glucose units but refinement fails because I don't have a lib file for the sugars. Can I get help on making lib files please? Thank you, Remie
Re: [ccp4bb] size of a flexible pdb structure
Hi rajan I guess calculating radius of gyration for your whole protein with time will tell you about the size variation at difeerent time points or you canm say different frames. This is how you can cluster your whole trajectory PDBs into a few clusters and then get the representative PDBs from each cluster to finally select what you want. Best Monica On Wed, Mar 5, 2014 at 8:42 PM, rajan kumar rajugunju...@gmail.com wrote: Dear all, sorry for asking an off topic question. My protein is composed of two domains connected by a flexible linker (15aa). after 50ns simulation i came across the fact that one domain is flexible. so my question is that how could i be able to calculate the size of molecule or radius of my molecule and which conformation i should prefer to calculate the size of molecule. your any suggestion will be very helpful. thank you all in advance with best regards -- Rajan kumar choudhary Senior Research Fellow Department of Atomic Energy(Govt.Of India) ACTREC TATA Memorial Center Kharghar Navi-Mumbai Mumbai-410210 India
Re: [ccp4bb] CCP4 lib file
There are lots of GLUCOSE type lib files for different sugars. One way to find what is available is: From the REfinement GUI click Monomer library sketcher You get a window with File in the top LH corner Click on that and ask for Read monomer from library Then enter a key word GLUCOSE and you get this list ADQ etc etc Select what you want and go from there.. Or do this crude method to get a complete list!: [ccp4@roo timm]$ grep -i glucose $CLIBD/monomers/*/* /y/people/ccp4/ccp4-6.4.0/lib/data/monomers/a/ADQ.cif:ADQ ADQ 'ADENOSINE-5'-DIPHOSPHATE-GLUCOSE' non-polymer61 38 . /y/people/ccp4/ccp4-6.4.0/lib/data/monomers/a/AGL.cif:AGL AGL '4,6-DIDEOXY-4-AMINO-ALPHA-D-GLUCOSE ' pyranose 24 11 . /y/people/ccp4/ccp4-6.4.0/lib/data/monomers/b/BG6.cif:BG6 BG6 'BETA-D-GLUCOSE-6-PHOSPHATE ' non-polymer27 16 . /y/people/ccp4/ccp4-6.4.0/lib/data/monomers/b/BGC.cif:BGC BGC 'BETA-D-GLUCOSE ' pyranose 24 12 . /y/people/ccp4/ccp4-6.4.0/lib/data/monomers/d/D6G.cif:D6G D6G '2-DEOXY-GLUCOSE-6-PHOSPHATE ' pyranose 26 15 . /y/people/ccp4/ccp4-6.4.0/lib/data/monomers/d/DDA.cif:DDA DDA '2,6-DIDEOXY-BETA-D-GLUCOSE ' pyranose 22 10 . /y/people/ccp4/ccp4-6.4.0/lib/data/monomers/g/G16.cif:G16 G16 'ALPHA-D-GLUCOSE 1,6-BISPHOSPHATE' pyranose 30 20 . /y/people/ccp4/ccp4-6.4.0/lib/data/monomers/g/G1P.cif:G1P G1P 'ALPHA-D-GLUCOSE-1-PHOSPHATE ' pyranose 27 16 . /y/people/ccp4/ccp4-6.4.0/lib/data/monomers/g/G2F.cif:G2F G2F '2-DEOXY-2FLUORO-GLUCOSE ' pyranose 23 12 . /y/people/ccp4/ccp4-6.4.0/lib/data/monomers/g/G4D.cif:G4D G4D '4-DEOXY-ALPHA-D-GLUCOSE ' pyranose 23 11 . /y/people/ccp4/ccp4-6.4.0/lib/data/monomers/g/G6D.cif:G6D G6D '6-DEOXY-ALPHA-D-GLUCOSE ' non-polymer23 11 . /y/people/ccp4/ccp4-6.4.0/lib/data/monomers/g/G6P.cif:G6P G6P 'ALPHA-D-GLUCOSE-6-PHOSPHATE ' pyranose 27 16 . /y/people/ccp4/ccp4-6.4.0/lib/data/monomers/g/G6Q.cif:G6Q G6Q 'GLUCOSE-6-PHOSPHATE ' non-polymer27 16 . /y/people/ccp4/ccp4-6.4.0/lib/data/monomers/g/GDA.cif:GDA GDA '4-DEOXY-4-AMINO-BETA-D-GLUCOSE ' non-polymer25 12 . /y/people/ccp4/ccp4-6.4.0/lib/data/monomers/g/GFP.cif:GFP GFP '2-DEOXY-2-FLUORO-ALPHA-D-GLUCOSE-1-P' pyranose 26 16 . /y/people/ccp4/ccp4-6.4.0/lib/data/monomers/g/GLC-B-D.cif:GLC-b-D GLC 'beta_D_glucose ' D-pyranose 24 12 . /y/people/ccp4/ccp4-6.4.0/lib/data/monomers/g/GLC.cif:GLC GLC 'ALPHA-D-GLUCOSE ' pyranose 24 12 . /y/people/ccp4/ccp4-6.4.0/lib/data/monomers/g/GLD.cif:GLD GLD '4,6-DIDEOXYGLUCOSE ' pyranose 22 10 . /y/people/ccp4/ccp4-6.4.0/lib/data/monomers/g/GLF.cif:GLF GLF '1-FLUORO-ALPHA-1-DEOXY-D-GLUCOSE' pyranose 23 12 . /y/people/ccp4/ccp4-6.4.0/lib/data/monomers/g/GLO.cif:GLO GLO 'D-GLUCOSE IN LINEAR FORM' non-polymer24 12 . /y/people/ccp4/ccp4-6.4.0/lib/data/monomers/g/GLT.cif:GLT GLT '5-DEOXY-5-THIO-ALPHA-D-GLUCOSE ' non-polymer24 12 . /y/people/ccp4/ccp4-6.4.0/lib/data/monomers/g/GMM.cif:GMM GMM 'GLUCOSE MONOMYCOLATE' non-polymer 200 74 . /y/people/ccp4/ccp4-6.4.0/lib/data/monomers/g/GUD.cif:GUD GUD 'GLUCOSE-URIDINE-C1,5'-DIPHOSPHATE ' non-polymer58 36 . /y/people/ccp4/ccp4-6.4.0/lib/data/monomers/i/ISX.cif:ISX ISX 'GLUCOSE BETA-1,3-ISOFAGAMINE' non-polymer44 21 . /y/people/ccp4/ccp4-6.4.0/lib/data/monomers/k/KBG.cif:KBG KBG '2-KETO-BETA-D-GLUCOSE ' non-polymer22 12 . /y/people/ccp4/ccp4-6.4.0/lib/data/monomers/list/mon_lib_list.cif:ADQ ADQ 'ADENOSINE-5'-DIPHOSPHATE-GLUCOSE' non-polymer61 38 . /y/people/ccp4/ccp4-6.4.0/lib/data/monomers/list/mon_lib_list.cif:AGL AGL '4,6-DIDEOXY-4-AMINO-ALPHA-D-GLUCOSE ' pyranose 24 11 . /y/people/ccp4/ccp4-6.4.0/lib/data/monomers/list/mon_lib_list.cif:BG6 BG6 'BETA-D-GLUCOSE-6-PHOSPHATE ' non-polymer27 16 . /y/people/ccp4/ccp4-6.4.0/lib/data/monomers/list/mon_lib_list.cif:BGC BGC 'BETA-D-GLUCOSE ' pyranose 24 12 . /y/people/ccp4/ccp4-6.4.0/lib/data/monomers/list/mon_lib_list.cif:D6G D6G '2-DEOXY-GLUCOSE-6-PHOSPHATE ' pyranose 26 15 . /y/people/ccp4/ccp4-6.4.0/lib/data/monomers/list/mon_lib_list.cif:DDA DDA '2,6-DIDEOXY-BETA-D-GLUCOSE ' pyranose 22 10 . /y/people/ccp4/ccp4-6.4.0/lib/data/monomers/list/mon_lib_list.cif:G16 G16 'ALPHA-D-GLUCOSE 1,6-BISPHOSPHATE' pyranose 30 20 . /y/people/ccp4/ccp4-6.4.0/lib/data/monomers/list/mon_lib_list.cif:G1P G1P 'ALPHA-D-GLUCOSE-1-PHOSPHATE ' pyranose 27 16 .
[ccp4bb] Post-doctoral Fellowships at the University of Texas
Macromolecular Crystallography, Biophysics and Biochemistry A post-doctoral fellowship position is available at the University of Texas Medical Branch (UTMB) located in the greater Houston area in the research team of Dr. Gabrielle Rudenko. We study molecules that mediate the formation, maintenance and plasticity of synapses, the contact and communication point between neurons. Our proteins of interest organize and regulate protein interaction networks in the synaptic cleft and impact how neurons communicate with each other at synapses. Many of the proteins we study are implicated in neurological disease and serve as potential novel therapeutic targets. We are looking for highly motivated, enthusiastic, hard working individuals who are interested in combining the fields of structural biology and neuroscience. Research projects involve protein over-expression and purification, x-ray crystallography, biochemical and biophysical techniques, proteomics, chemical biology and in collaboration electron microscopy and NMR. Our laboratory is part of the endowed Sealy Center for Structural Biology and Molecular Biophysics and the Department of Pharmacology/Toxicology at UTMB. The Sealy Center provides access to exceptional facilities, training and experimental assistance. X-ray facilities include a high-brilliance FR-E Superbright x-ray generator, a Rigaku R-AXIS-IV dual 30cm Imaging Plate detector, a Bruker SMART 2K CCD and a Rigaku BioSAXS-1000 2D-Kratky camera (for solution scattering data). Biophysics facilities include equipment for analytical ultracentrifugation, fluorescence, MALDI-TOF mass spectrometry, dynamic light scattering, surface plasmon resonance, titration micro-calorimetry, and high throughput screening with the ThermoFluor HTS apparatus. In addition, the Electron Microscopy facilities house three modern JEOL cryo-electron microscopes (a 200 keV JEOL 2200FS cryo-EM with in-column electron energy filter and field emission gun, a 200 keV JEOL 2100 EM, and a 120 keV JEM1400 microscope) and the NMR facilities include three NMR spectrometers (Bruker Avance III 800, 750 and 600 MHz with cryoprobes). See http://www.scsb.utmb.edu/resources/ UTMB is part of the Texas Medical Center, the world’s largest medical center located in greater Houston. UTMB has research facilities situated on Galveston Island offering a pleasant subtropical climate and miles of relaxing beaches. UTMB is located within commuting distance of Houston, the fourth largest city in the United States and a leader in the arts, education, and health care. Qualifications: Applicants should have received a Ph.D. degree in a field relevant to structural biology within in the last three years and have a strong publication record. Applicants with experience in cloning, bacterial/insect cell culture, protein purification and crystallization, and macromolecular structure determination are preferred. However, recent graduates with a strong background in protein over-expression and purification, and an exceptional interest in learning crystallography are also encouraged to apply. Good written and oral communication skills in English are essential as well as the ability and desire to work as part of a highly motivated, collaborative team. To apply please e-mail the following to rudenkolab.applicat...@gmail.commailto:rudenkolab.applicat...@gmail.com: CV, contact information for three references (address, phone and e-mail), a list of your experimental expertise and skills relevant to this position and a research statement describing your past research experience and future goals (1-2 pages). Visa sponsorship is available for non-US nationals. For questions, please contact Dr. Gabrielle Rudenko at gabrielle.rude...@utmb.edu For more information please visit http://scsb.utmb.edu/labgroups/rudenko/ The University of Texas is a non-discriminatory, affirmative action employer. Gabrielle Rudenko, PhD Dept of Pharmacology and Toxicology Sealy Center for Structural Biology Biophysics University of Texas Medical Branch 301 University Boulevard Basic Sciences Building Rm. 5.114B Galveston Texas 77555
Re: [ccp4bb] size of a flexible pdb structure
Dear Rajan, Since you raised your question about calculating the radius of your molecule. here my suggestion, a bit long back I heard about the program HYDROPRO I am not sure its going to be work for you, but you may give a try. I think the program calculates the radius of gyration ! best wishes, Dr. S. M. Jaimohan On Wednesday, 5 March 2014 8:53 PM, Tim Gruene t...@shelx.uni-ac.gwdg.de wrote: Dear Rajan kumar choudhary, could you explain why you expect the volume to depend on the conformation of the molecule? Do you have some effective volume in mind, e.g. the elution volume during size exclusion chromatography? Regards, Tim On Wed, Mar 05, 2014 at 08:42:21PM +0530, rajan kumar wrote: Dear all, sorry for asking an off topic question. My protein is composed of two domains connected by a flexible linker (15aa). after 50ns simulation i came across the fact that one domain is flexible. so my question is that how could i be able to calculate the size of molecule or radius of my molecule and which conformation i should prefer to calculate the size of molecule. your any suggestion will be very helpful. thank you all in advance with best regards -- *Rajan kumar choudhary* *Senior Research Fellow* *Department of Atomic Energy(Govt.Of India)* *ACTREC TATA Memorial Center * *Kharghar Navi-Mumbai* *Mumbai-410210* *India* -- -- Dr Tim Gruene Institut fuer anorganische Chemie Tammannstr. 4 D-37077 Goettingen GPG Key ID = A46BEE1A
[ccp4bb] Off-Topic: Hydrodynamic/Thermodynamic Workshop in Dallas, TX
Dear All, I am very pleased to announce that a workshop entitled “Methods for the Analysis of Thermodynamic and Hydrodynamic Properties of Macromolecules and their Complexes” will be held on May 19-23, 2014 in Dallas, TX, at The University of Texas Southwestern Medical Center. I post here because, in the past, we have had a very strong level of participation from the Structural Biology community. The workshop will feature lectures on the practical aspects of performing AUC, ITC, DLS, and fluorescence spectroscopy experiments. There will also be demonstrations and step-by-step instruction on how to analyze your data using SEDFIT and SEDPHAT, and lectures covering the theoretical underpinnings of the analyses. I think we can all agree that these methods and analytical techniques can add significant value to our structures. The instructors will be experts with many years of experience with these technologies and analytical methods. They include Drs. Peter Schuck, Rodolfo Ghirlando, Joy Zhao, and myself. You can find more information about the workshop and registration at the following links: http://www.utsouthwestern.edu/education/cme/symposia/macromolecules/index.html http://www.utsouthwestern.edu/education/cme/symposia/macromolecules/registration.html Best regards, Chad
[ccp4bb] Job opportunity at Constellation pharmaceuticals in Cambridge MA
Constellation Pharmaceuticals has the following job opportunity. Please use the 'care...@constellationpharma.com' email to reply. Scientist - Structural Biology Constellation Pharmaceuticals leverages insights from the rapidly expanding field of epigenetics to discover and develop small molecule therapeutics for the treatment of cancer, inflammatory/immunologic disorders and other diseases. Research here and by others has shown that abnormal epigenetic regulation of gene expression contributes to many different diseases. Our innovative product discovery engine targets both the enzymes that modify the dynamic structure of chromatin and other proteins that interact with chromatin to control gene expression. The Structural Biology Department at Constellation is looking for a Scientist level candidate to join our team and contribute to our drug discovery efforts. This position requires a passion to do high quality scientific research in a collaborative, goal-oriented and fast-paced team environment. The primary responsibilities of this role include (but are not limited to) conducting laboratory experiments related to all aspects of crystallography from crystal growth to structure solution, analyzing, and interpreting scientific data, collaborating with other scientists, and performing computer-assisted structure based drug design. The ideal candidate will have a background in chemistry and an in-depth understanding of target/ligand interactions. We require 2-5 years of post-doctoral experience, and industrial experience (in pharmaceutical or biotech) will be considered a plus. A strong commitment to frequently and articulately communicate structural information to colleagues in chemistry, enzymology and biology is expected. Constellation Pharmaceuticals is an Equal Opportunity Employer and a participant in E-Verify, and offers a comprehensive benefits package, including health, dental, 401(k), paid vacation, tuition reimbursement, and much more. If you are interested in joining Constellation's team please submit your resume to the following address: care...@constellationpharma.com This email message and any attachments are being sent by Constellation Pharmaceuticals, Inc. The information contained in this e-mail message, and any attachments, may contain information that is confidential or privileged. If you are not the intended recipient, any dissemination or copying of this communication is strictly prohibited. If you have received this communication in error, please notify the sender immediately by return e-mail and destroy all copies of this message and any attachments. Thank you. For more information about Constellation, please visit us at http://www.constellationpharma.com.
Re: [ccp4bb] Stereo monitors for use with Pymol and Coot
A rather US-centric question on passive 3D monitors... I'm just getting set up in the US, and I'm surprised on how few passive 3D monitors seem to be around - many models seem listed as 'out of stock' when looking in the usual places (Amazon, NewEgg, BestBuys, Walmart etc.) The best deal I have found is for an LG D2343PB-BN (http://www.lg.com/us/commercial/lcd-computer-monitors/lg-D2343PB-BN) at US$274 Does anyone have any experience with this model, or any suggestion about where best to buy 3D monitors in the US? many thanks in advance Shaun
Re: [ccp4bb] Peptide solubility issues
Many thanks to all the people who contacted me. I actually went rogue and decided to initially reconstitute in 10mM glycine, pH 9.5 to 2 mg/mL, sonicate for a few seconds and then immediately add an equal volume of more concentrated neutral pH buffer to minimize any possibility of alkaline hydrolysis. The peptide solubilized nicely, and the SPR sensorgrams are much more promising. My only concern is if glycine might interfere with peptide binding (I suspect not, but please let me know if you think otherwise) - a control doesn't suggest glycine likes to stick to the protein surface. A few people pointed me to links on solubilizing lyophilized peptides; however, the one I had previously been consulting is, in my opinion, the most thorough: http://www.bachem.com/service-support/faq/handling-of-peptides/ Thanks again. On Wed, Mar 5, 2014 at 6:52 AM, Toufic El Arnaout elarn...@tcd.ie wrote: Hello Mo Wong, Some points below are good, but don't underestimate custom peptides sometimes... can be much harder/expensive than recombinant proteins. If you ordered the peptides it is good to know how they synthesized them, and how the elution profiles during purification (RP HPLC..) looked like, particularly that they are very hydrophobic. Did they use formic acid, or maybe they already dissolved them in DMSO.. Did they verify the product/bonds with MALDI-TOF for example and NMR? Furthermore, you can try 0.1-0.5 % Triton X-100. My vote would be for a final concentration of 5-25 uM (containing up to 5 % solvent), from a stock of 50-100 % solvent at 1 mM.. Btw I forgot.. for SPR (depends on the system, and especially for a 12mer), some detergents and even a minor DMSO/ethanol can affect the measurements drastically. Best wishes toufic el arnaout On Wed, Mar 5, 2014 at 12:19 AM, Mo Wong mowon...@gmail.com wrote: Hi all, Slightly off topic - but I'm having trouble solubilizing some peptides for SPR and hoped someone on the BB might have some other suggestions. The peptides are intra-chain S-S cross-linked 12mers with pIs of ~3. 10 residues are hydrophobic and 2 are acidic. Peptides have been tested with and without N- and C-terminal modifications (amidation/acetylation). I have tried: ddH2O raising (and lowering) pH (tested up to 8.5) with different buffers Including DMF as a co-solvent (I'm avoiding DMSO because of a methionine present in the peptide) - peptide still visibly precipitates out at 100uM in 5% DMF (also a 1mM stock in 50% DMF shows significant amounts of ppt) Adding a trace amount of detergent (0.005% Tween 20) I'm guessing I could try other co-solvents such as ethanol or initially solubilizing peptide in dilute NaOH before bringing the pH down with addition of a buffer (though I'm concerned about alkaline hydrolysis). Anyway, I'd rather have some insight from people before I waste any further peptide. Thanks for any suggestions.
Re: [ccp4bb] Peptide solubility issues
For that purpose we’ve developed this expression system that may or may not be useful to your specific problem: http://www.ncbi.nlm.nih.gov/pubmed/23996492 J Mol Recognit.http://www.ncbi.nlm.nih.gov/pubmed/23996492# 2013 Oct;26(10):496-500. doi: 10.1002/jmr.2292. Development of a multifunctional tool for drug screening against plasmodial protein-protein interactions via surface plasmon resonance. Boucher LEhttp://www.ncbi.nlm.nih.gov/pubmed?term=Boucher%20LE%5BAuthor%5Dcauthor=truecauthor_uid=239964921, Bosch Jhttp://www.ncbi.nlm.nih.gov/pubmed?term=Bosch%20J%5BAuthor%5Dcauthor=truecauthor_uid=23996492. Sorry, for the self advertisement (at least it’s not attached), but perhaps it is a useful tool for others as well. Jürgen .. 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: +1-410-614-4742tel:%2B1-410-614-4742 Lab: +1-410-614-4894tel:%2B1-410-614-4894 Fax: +1-410-955-2926tel:%2B1-410-955-2926 http://lupo.jhsph.edu On Mar 5, 2014, at 8:15 PM, Mo Wong mowon...@gmail.commailto:mowon...@gmail.com wrote: Many thanks to all the people who contacted me. I actually went rogue and decided to initially reconstitute in 10mM glycine, pH 9.5 to 2 mg/mL, sonicate for a few seconds and then immediately add an equal volume of more concentrated neutral pH buffer to minimize any possibility of alkaline hydrolysis. The peptide solubilized nicely, and the SPR sensorgrams are much more promising. My only concern is if glycine might interfere with peptide binding (I suspect not, but please let me know if you think otherwise) - a control doesn't suggest glycine likes to stick to the protein surface. A few people pointed me to links on solubilizing lyophilized peptides; however, the one I had previously been consulting is, in my opinion, the most thorough: http://www.bachem.com/service-support/faq/handling-of-peptides/ Thanks again. On Wed, Mar 5, 2014 at 6:52 AM, Toufic El Arnaout elarn...@tcd.iemailto:elarn...@tcd.ie wrote: Hello Mo Wong, Some points below are good, but don't underestimate custom peptides sometimes... can be much harder/expensive than recombinant proteins. If you ordered the peptides it is good to know how they synthesized them, and how the elution profiles during purification (RP HPLC..) looked like, particularly that they are very hydrophobic. Did they use formic acid, or maybe they already dissolved them in DMSO.. Did they verify the product/bonds with MALDI-TOF for example and NMR? Furthermore, you can try 0.1-0.5 % Triton X-100. My vote would be for a final concentration of 5-25 uM (containing up to 5 % solvent), from a stock of 50-100 % solvent at 1 mM.. Btw I forgot.. for SPR (depends on the system, and especially for a 12mer), some detergents and even a minor DMSO/ethanol can affect the measurements drastically. Best wishes toufic el arnaout On Wed, Mar 5, 2014 at 12:19 AM, Mo Wong mowon...@gmail.commailto:mowon...@gmail.com wrote: Hi all, Slightly off topic - but I'm having trouble solubilizing some peptides for SPR and hoped someone on the BB might have some other suggestions. The peptides are intra-chain S-S cross-linked 12mers with pIs of ~3. 10 residues are hydrophobic and 2 are acidic. Peptides have been tested with and without N- and C-terminal modifications (amidation/acetylation). I have tried: ddH2O raising (and lowering) pH (tested up to 8.5) with different buffers Including DMF as a co-solvent (I'm avoiding DMSO because of a methionine present in the peptide) - peptide still visibly precipitates out at 100uM in 5% DMF (also a 1mM stock in 50% DMF shows significant amounts of ppt) Adding a trace amount of detergent (0.005% Tween 20) I'm guessing I could try other co-solvents such as ethanol or initially solubilizing peptide in dilute NaOH before bringing the pH down with addition of a buffer (though I'm concerned about alkaline hydrolysis). Anyway, I'd rather have some insight from people before I waste any further peptide. Thanks for any suggestions.
[ccp4bb] PhD position to explore the molecular mechanisms of ubiquitin signaling
Dear colleagues, We invite applications for a PhD position in the research group of Dr. Sonja Lorenz at the Rudolf Virchow Center for Experimental Biomedicine, University of Würzburg.The successful applicantwill have the opportunity to be part of the 'Graduate School of Life Sciences' in Würzburg- a highly-regarded international graduate program funded by the German Excellence Initiative. The PhD position isfunded by the Emmy Noether Program of the German Research Foundation (DFG) to explore the molecular mechanisms of ubiquitin signaling. Available projects aim to uncover the catalytic mechanisms, specificities, and regulation of ubiquitination enzymes as well as the crosstalk of ubiquitination with other types of posttranslational modifications in the cell. The lab combines structural biology (X-ray crystallography, NMR spectroscopy) with complementary biophysical, biochemical and cell biological analyses. Depending on individual qualifications and interests, the successful applicant will have the opportunity to gain experience in various aspects of this multidisciplinary approach. The Lorenz lab has access to state-of-the-art instrumentation in X-ray crystallography, high-field solution NMR spectroscopy, biophysics and an extensive range of modern biochemical, and cell biological equipment. The Rudolf Virchow Center is integrated into a stimulating, collaborative research environment that includes the Biocenter, the Research Center for Infectious Diseases, various institutes of the university hospital, and the Max Planck Research Group for Systems Biology in Würzburg. Applicants should have a M.Sc./diploma in biochemistry, biophysics, molecular biology or a related discipline and a strong interest in structure-function studies on proteins. Experience in either protein expression and purification, the characterization of protein interactions in vitro or in cells, enzymology, NMR or X-ray crystallography would be beneficial. Good English language skills are essential; knowledge of German is not required. The positionwill start on 1 April 2014 or later, upon mutual agreement, and is initially available for 3 years. Salary will be according to German TVL scale. In case of equivalent qualifications, disabled applicants will be preferentially considered. Applications and informal queries about the positions or ongoing projects should be sent to Sonja Lorenz by email (*sonja.lor...@virchow.uni-wuerzburg.de sonja.lor...@virchow.uni-wuerzburg.de*). Applications are expected to include a letter of motivation detailing research interests and expertise, a CV, and the contact information for at least two references. Review of applications will begin immediately. Lab homepage: *http://www.rudolf-virchow-zentrum.de/en/research/research-groups/lorenz-group/research.html http://www.rudolf-virchow-zentrum.de/en/research/research-groups/lorenz-group/research.html* Website of the Graduate School of Life Sciences: *http://www.graduateschools.uni-wuerzburg.de/life_sciences http://www.graduateschools.uni-wuerzburg.de/life_sciences* *Sonja Lorenz, PhD* *Rudolf Virchow Center for Experimental Biomedicine* *University of W**ü**rzburg* *Josef-Schneider-Strasse 2, Haus D15* *97080**W**ü**rzburg*
[ccp4bb] Efforts been made to solve protein (soluble) aggregation
Dear CCP4 friends, I have been searching around the CCP4 mails for the discussion of soluble protein aggregations, as I have the same problem recently. Many efforts I have tried but without success. Here, I would like to summarise what I have done and maybe any of you come across some ideas. 1. My protein (not membrane protein) is around 35kDa with a pI of 8.6. It was expressed in E.coli as a fusion protein where its N-ter was fused by MBP-tag with a flexible linker (Factor Xa cleavage site available). The whole fusion protein is ~77kDa (pI 6.1). 2. After MBPTrap affinity purification, the protein was run through gel-filtration column it came out at void volume. The aggregation was also confirmed by DLS. The buffer condition is: 20 mM HEPES, pH7.4, 125 mM KCl, 20% glycerol, 40 mM maltose, 5 mM DTT. 3. I made several truncations and only one gave me a monomer (The other truncations were all soluble aggregates). However, this monomeric truncation is small (~5 kDa) and not important for the function (not interesting fragment). At least, this monomeric truncation suggests that the aggregation is not due to the aggregation of MBP. The truncations were designed in both two versions: long linker (including TEV cleavage site) and short linker (-Ser-Ser-Ser-). 4. Hampton detergent screens and high salt (1-2 M KCl or NaCl) were tried on both truncations and full-length protein, but none of them work (DLS confirm). Divalents and other ions should not bind to this protein. 5. The full-length aggregated protein is still functional in Gel-shift assay, which indicates the protein is corrected folding. I guess soluble aggregation is a common problem for tough proteins. If anyone has experience on it and can share with us, please drop an email. Kind regards, Wenhe
