Re: [ccp4bb] ligand occupancy
Hi Monica, Calculate the mean B-factor of all atoms that making interactions with each ligand in monomer A and B. Use those means values as B-factors for each ligand respectively. Adjust manually the occupancies, in order the B-factors for each ligand to stay after refinement close to the above values. In order to calculate occupancies more precisely, it would help to have the un-liganded structure and thus the location of the water molecules in each binding site. If you have the above information you could refine water molecules and ligands simultaneously in the binding site and get accurate refined occupancies. George From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Monica Mittal Sent: Friday, April 18, 2014 1:04 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] ligand occupancy Dear all I have a protein which is dimer having one ligand binding site in each monomer. I refined the crystal structure with ligand in both sites finally. I refined will full occupancy of 1 for ligands (same in both). But now i want to see is there any difference in the occupancy of both ligands in ligand binding sites of monomer A and B. Is there any way i can get any information about the occupancy of ligands in two monomers like one is binding more tightly than another so that i can get an idea about their differential binding contacts also. Thank you very much in advance. Regards Monica
Re: [ccp4bb] occupancy of Metal
What is the mean B-factor of atoms that making interactions with each metal ion? Use those means values as B-factors for each Mg2+ ion respectively. Adjust manually the occupancies, in order the B-factors for each Mg2+ to stay after refinement close to the above values. George From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Anirudha Dutta Sent: Sunday, March 23, 2014 6:38 AM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] occupancy of Metal Dear all In the structure I am working has three Mg2+ ions having B-factors of 36.6, 8.6 and 20.3 Å2 with occupancy of one. The mean B-factor and resolution are 22.65 Å2 and 2.5 Å respectively. I want to adjust the occupancy of the Mg (with 36.6 Å2) manually. Please suggest what could be the final B-factor for that Mg, based on which I can assign the occupancy. Thanks in advance Anirudha
Re: [ccp4bb] Assays for protein-ligand interaction?
Specifically for fluorescence does your ligand fluoresce? It is possible if it has indol group or some aromatic organic compound Does your protein has a tryptophan or tyrosines in the binding site? If yes may be a fluorescence titration experiment could be the solution. Also fluorescence needs very low concentration of protein (nM to microM) george From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Acoot Brett Sent: Monday, January 13, 2014 3:09 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Assays for protein-ligand interaction? FRET， CD， Fluorescence， NMR chemical shift assay, isotope-labelled ligand interaction assay, protein melting temperature assay, gel filtration retention assay, gyration radius assay by Malls, native page gel analysis, etc. Acoot On Monday, 13 January 2014 8:51 PM, HJ Lee mailto:hojunle...@gmail.com hojunle...@gmail.com wrote: Sorry for the off topic. I'm looking for a way to monitor protein-(potential) ligand interaction. The ligand is small molecule (mw~250) and we're looking for its potential interaction with couple human proteins. (We do not know this small molecule interacts with these human protein or not.) Is there any efficient way to quickly identify whether this ligand interacts with those human protein? We can buy some protein, but the amount of commercially available purified proteins is very little, making them hard to be analyzed by some good methods (e.g. ITC). That would be really great if anyone suggest any idea. Sorry for the off topic question again. Thanks!
Re: [ccp4bb] Isothermal titration calorimetry
Chris, indeed nanoITC instrument analysis software is very robust and user friendly (probable more friendly than microcal, GE). Although when you need to subtract Q (heat) values (from 2 or 3 blank experiments) from your experimental data you cannot. NanoITC software can subtract Q values only from 1 blank experiment. Also if you want to present your data in a form of heat/mol in Y (vertical) axes again you cannot. It presents data in Y axes only in form of heat/injection. If you have found a way to extract 2 or 3 blank experiments from experimental data or present data in form of heat/mol, please let me know it will be very useful. The main problem in the output files from nanoITC come with an extension .nitc, by default. Unfortunately Origin (that can do all the above) can read only, filenames with an extension .itc Cheers, George -Original Message- From: Colbert, Christopher [mailto:christopher.colb...@ndsu.edu] Sent: Saturday, March 23, 2013 5:56 PM To: George Kontopidis; CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Isothermal titration calorimetry George, would you please explain your comments? We've found the TA Instruments analysis software very robust and user friendly. We have the low volume nanoITC from TA instruments and get equivalent #'s in our comparison tests to the Microcal instrument. Cheers, Chris -- Christopher L. Colbert, Ph.D. Assistant Professor Department of Chemistry and Biochemistry North Dakota State University P.O. Box 6050 Dept. 2710 Fargo, ND 58108-6050 PH: (701) 231-7946 FAX: (701) 231-8324 On 3/23/13 8:47 AM, George Kontopidis gkontopi...@vet.uth.gr wrote: Keep in mind that output files from nanoITC, TA instrument cannot be red by Origin. At some point you will need to analyse your data further. George -Original Message- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Anastassis Perrakis Sent: Saturday, March 23, 2013 12:46 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Isothermal titration calorimetry It might be worth to consider the question more in detail. Do you want to study thermodynamics of the interaction, or a KD would do? If the former, you need ITC. If the latter, and you want to study things at the level of KD only, maybe investing on a plate reader, thermophoresis, or some biosensor technology (spr or interferometry based systems) should be considered. Then, what interactions will you study with the ITC? In general, I would agree that the lower sample volume is worth the nano options, but depending on the typical systems under study, sometimes the gain on sample quantity is not worth the money - while many times its worth it. John is if course right that for studying specific systems as the one he describes the 200 is great. A. Sent from my iPhone On 23 Mar 2013, at 11:00, John Fisher johncfishe...@gmail.com wrote: I would recommend the Microcal ITC 200, hands down. Not only is it an amazing instrument with the optional automated sample loader (which is worth every penny), but we were able to do experiments (multiple) using FULL-LENGTH p53 binding to a weak cognate protein. I believe this was the first time ITC was ever used with full length p53, as it is so labile and just loves immediately to oligomerize. Sample sizes pay for the instrument. Best, John John Fisher, M.D./PhD St. Jude Children's Research Hospital Department of Oncology Department of Structural Biology W: 901-595-6193 C: 901-409-5699 On Mar 23, 2013, at 4:45 AM, Sameh Soror shamd...@googlemail.com wrote: Dear All, I am sorry for the off topic question. I am going to buy ITC to study protein-protein protein-ligand interactions I am comparing microcal, GE and nanoITC, TA instrument.. any suggestions, recommendations, good experiences or bad experiences. is there a better system. Thank in advance for the help. Regards Sameh -- Sameh Soror Postdoc. fellow
Re: [ccp4bb] Isothermal titration calorimetry
Keep in mind that output files from nanoITC, TA instrument cannot be red by Origin. At some point you will need to analyse your data further. George -Original Message- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Anastassis Perrakis Sent: Saturday, March 23, 2013 12:46 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Isothermal titration calorimetry It might be worth to consider the question more in detail. Do you want to study thermodynamics of the interaction, or a KD would do? If the former, you need ITC. If the latter, and you want to study things at the level of KD only, maybe investing on a plate reader, thermophoresis, or some biosensor technology (spr or interferometry based systems) should be considered. Then, what interactions will you study with the ITC? In general, I would agree that the lower sample volume is worth the nano options, but depending on the typical systems under study, sometimes the gain on sample quantity is not worth the money - while many times its worth it. John is if course right that for studying specific systems as the one he describes the 200 is great. A. Sent from my iPhone On 23 Mar 2013, at 11:00, John Fisher johncfishe...@gmail.com wrote: I would recommend the Microcal ITC 200, hands down. Not only is it an amazing instrument with the optional automated sample loader (which is worth every penny), but we were able to do experiments (multiple) using FULL-LENGTH p53 binding to a weak cognate protein. I believe this was the first time ITC was ever used with full length p53, as it is so labile and just loves immediately to oligomerize. Sample sizes pay for the instrument. Best, John John Fisher, M.D./PhD St. Jude Children's Research Hospital Department of Oncology Department of Structural Biology W: 901-595-6193 C: 901-409-5699 On Mar 23, 2013, at 4:45 AM, Sameh Soror shamd...@googlemail.com wrote: Dear All, I am sorry for the off topic question. I am going to buy ITC to study protein-protein protein-ligand interactions I am comparing microcal, GE and nanoITC, TA instrument.. any suggestions, recommendations, good experiences or bad experiences. is there a better system. Thank in advance for the help. Regards Sameh -- Sameh Soror Postdoc. fellow
[ccp4bb] column tube
Dear Colleagues, Apologies for the non-crystallographic question but it very likely that someone out there may have the answer. I have a chromatography column. Unfortunately the company does not provide a replacement for the plastic tube, which should not cost more than 100 €. The cost for a new column is more than 1500 €! Does anybody know if there is a company that could provide custom-made plastic tubing for chromatography columns? Thanks in advance for your answers, George Kontopidis Assοciate Professor of Biochemistry Veterinary School, University of Thessaly Karditsa 43100, Greece Tel: +30 24410 66017 Mob: +30 69 342 643 75 e-mail: mailto:gkontopi...@vet.uth.gr gkontopi...@vet.uth.gr web site: http://www.vet.uth.gr/english/
Re: [ccp4bb] How to know if ADP exists in the ATP-binding site of bacterial expressed proteins
At 280nm absorbs Trp but Tyr and Phe (much less) as well. So your protein sample even without Trp will absorb. A mass spect experiment should show a presence of molecule with MW 507 (ATP) or MW 427 (ADP) George From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Xinghua Qin Sent: Monday, June 04, 2012 7:52 AM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] How to know if ADP exists in the ATP-binding site of bacterial expressed proteins Deer CCP4ers, How to know if ADP exists in the ATP-binding site of bacterial expressed proteins? Check the UV spectrum at 280 nm is one way, but there is no Trp in my protein. Is there any other conveniet way to find out? Thanks in advance Best wishes Xinghua Qin -- Xinghua Qin State Key Laboratory of Plant Physiology and biochemistry College of Biological Sciences China Agricultural University No.2, Yuan Ming Yuan West Road Haidian District, Beijing, China 100193 Tel: +86-10-62732672 E-mail: mailto:xhqin1...@gmail.com xinghua...@126.com
Re: [ccp4bb] Kinase crystallization
if the ligand binding site is exposed to the solvent a bound ligand may help. if the protein has is flexible domains and ligand fix it in one conformation, a bound ligand will help even more. All the above assuming that the purity and the concentration of the protein are high. George From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Debreczeni, Judit Sent: Monday, January 30, 2012 3:45 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Kinase crystallization Does your kinase autophosphorylate by any chance? -- That can produce differently phosphorylated species and affect crystallisability. You can detect it by e.g. mass spec, and tackle it by dephosphorylating the protein prior to crystallisation or by coexpression with a phosphatase. _ AstraZeneca UK Limited is a company incorporated in England and Wales with registered number: 03674842 and a registered office at 2 Kingdom Street, London, W2 6BD. Confidentiality Notice: This message is private and may contain confidential, proprietary and legally privileged information. If you have received this message in error, please notify us and remove it from your system and note that you must not copy, distribute or take any action in reliance on it. Any unauthorised use or disclosure of the contents of this message is not permitted and may be unlawful. Disclaimer: Email messages may be subject to delays, interception, non-delivery and unauthorised alterations. Therefore, information expressed in this message is not given or endorsed by AstraZeneca UK Limited unless otherwise notified by an authorised representative independent of this message. No contractual relationship is created by this message by any person unless specifically indicated by agreement in writing other than email. Monitoring: AstraZeneca UK Limited may monitor email traffic data and content for the purposes of the prevention and detection of crime, ensuring the security of our computer systems and checking compliance with our Code of Conduct and policies. From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of CHAVES SANJUAN, ANTONIO Sent: 30 January 2012 10:07 To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Kinase crystallization Dear all, I am trying to crystallize a protein kinase without any success. I suspect about its characteristic catalytic loop. I have already prepared different constructs, different expression vectors, and different mutant proteins (pseudo-phosphorylated, active, inactive?). I have also tested some ligands as ANPpnp (non hidrolizable nucleotide), ADP (product) and manganese (cofactor). I am thinking in trying to cocrystallize it with a general kinase substrate (a peptide, a small molecule...). Does any one have any experience or suggestion? Thanks in advance. Sincerely, Antonio
Re: [ccp4bb] Kd's in Crystals
Determination of Kd in crystal using only crystallographic data look at The First Direct Determination of a Ligand Binding Constant in Protein Crystals Wu SY, Dornan J., Kontopidis G., Taylor P., Walkinshaw M.D. Angew Chem Int Ed Engl, 2001, 40, 582-586. George --- George Kontopidis Associate Professor of Biochemistry Head of Biochemistry Veterinary School, University of Thessaly Trikalon 224, Karditsa 43100, Greece Tel: +30 24410 66017 Mob: 69 342 643 75 Fax: +30 24410 66041 e-mail: gkontopi...@vet.uth.gr web site: http://www.vet.uth.gr/english/departments_biochemistry.html --- -Original Message- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Steven Herron Sent: Monday, June 27, 2011 9:33 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Kd's in Crystals I had success using crystallography to measure the Ca2+ affinity (in the mM range) for a Ca2+ dependent enzyme. See: Characterization and implications of Ca2+ binding to pectate lyase C. Herron SR, Scavetta RD, Garrett M, Legner M, Jurnak F. J Biol Chem. 2003 Apr 4;278(14):12271-7. We measured the occupancy of the Ca2+ ion using three different pH's and 3-4 different Ca2+ concentrations. The presence of the Ca2+ ion altered the conformation of two residues in the binding pocket. In several of the Ca2+ soak experiment the occupancy was between 35% and 70%, where both orientations of the side chains could be modeled separately and their occupancy values refined (see attached picture). We confirmed our crystallographic Kd approach using tryptophan fluorescence. Since it was difficult to measure mM binding affinities using dialysis or titration calorimetry, we turned to crystallography (since we had lots of crystals and beam time). Steve Jacob Keller wrote: Dear Crystallographers, what is the dogma with regard to affinities in crystals? For example, if I soak three crystals in 1pM, 1nM, and 1uM compound X, and they all show equivalent density, does that mean that the affinity is really better than 1pM, or is the crystal of such a high local concentration (~600mg/mL) that it will be fully occupied at nearly any concentration, provided external ligand concentration does not change due to binding in the crystal? I guess there is also the problem that the crystallization solutions are very non-physiological, but neglecting that, is there any straightforward way to think of this, or is there a good reference? Jacob Keller
Re: [ccp4bb] Putting ligand in the protein structure
You need protein structure, a different e. density map (1Fo-1Fc) and your ligand structure. Then from COOT menu select Other Modelling Tools and click Find Ligand. You can also move manually the ligand into the density map. George -Original Message- From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] On Behalf Of Anshul Awasthi Sent: Tuesday, September 30, 2008 10:57 AM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Putting ligand in the protein structure Hi all the crystallographers, I am trying to solve a structure of a protein with some inhibitor. I want to know how I can put in my inhibitor in the density map of the data i got. I can see some density in the active site where the inhibitor should be. I generated the topoly file of the inhibitor (in both pdba nd refmac5 top formats) from the Dundee PRODRG server. Now do i need to incorporate the structure of the inhibitor in ccp4 or can i do in coot?? I am not sure of how to do it. ANy sugegstion will be very valuable for me. No virus found in this incoming message. Checked by AVG - http://www.avg.com Version: 8.0.173 / Virus Database: 270.7.5/1697 - Release Date: 29/9/2008 7:25 ìì
