Re: [ccp4bb] protein dodecamer
Check with PISA@EBI, it has database searches where you can fetch all dodecamers in the PDB Eugene On 20 Feb 2013, at 06:29, Hui Wang wrote: Dear all, I am looking for proteins that form a dodecameric ring structure (Not two rings of hexamer Nor tetrahedral distribution of subunit trimers). I found some phage portal proteins after a quick search in PDB, but I need more. Does anyone know more protein dodecamers that has C12 symmetry? Thanks, Hui -- Scanned by iCritical.
Re: [ccp4bb] protein dodecamer
Hi You can do this type of selection from this service at PDBe. It provides you with many options that you can combine into a query to make selections of PDB-ID. http://www.ebi.ac.uk/pdbe-as/pdbeselect/PDBeSelect.jsp From this page : select from the drop down [Assembly] - Assembly Name Click [show distribution] You will need to use the max scroll slider - drag this left to until you see dodecamer (it has 0.7% occurrence) Click on the edge of the Pie to select this - it will appear on the list to the right Clicking on [how many entries] will tell you that as of today there are 1728 entries of this type. From [Assembly] - symmetry number [Show Distribution] - will show a pie chart of symmetry-number You need need to select the bar = 12 ; which might be easier if you slide the minimum scroll bar right (Note you can click and drag to select a range here - but not useful for your question) This will appear in the selection on the right with the assembly name Now click [How many entries ?] This gives 415 entries From the table on the right - click on the ID column cells to open the atlas page for any entry of interest. Go to the quaternary page on the atlas page to check that this correct. You can also [Get] a list of entries as text or XML, or put the SQL into the query browser to fine tune this. Regards Tom Oldfield Check with PISA@EBI, it has database searches where you can fetch all dodecamers in the PDB Eugene On 20 Feb 2013, at 06:29, Hui Wang wrote: Dear all, I am looking for proteins that form a dodecameric ring structure (Not two rings of hexamer Nor tetrahedral distribution of subunit trimers). I found some phage portal proteins after a quick search in PDB, but I need more. Does anyone know more protein dodecamers that has C12 symmetry? Thanks, Hui -- Tom Oldfield , PhD Team Leader Head of PDBe Databases and Services Protein Databank in Europe European Bioinformatics Institute, Hinxton, Cambridge, CB10 1SD, UK Tel : ++44 (0) 1223 492526
[ccp4bb] Problems in running refine_den in cns_solve
Dear all, I have a problem while running CNS DEN refinement. After running the CNS minimize refinement, the program was aborted and an error message was displayed as: ** Error: test set array test does not exist ** % error encountered: ABORT statement specified. (CNS is in mode: SET ABORT=NORMal END) * ABORT mode will terminate program execution. * Program will stop immediately. I couldn't find the reason. If anyone knows, it would be of great help. many thanks -- Zhubing Shi Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, 320 Yue-yang Road, Shanghai 200031, China
[ccp4bb] off topic question BIAcore problem
Dear all, Recently I have measure a set of kinetic data of a receptor ligand interaction using BIAcore 3000. To my surprise, the dissociation rate is very low (~ 10e-6). During the measurement, I use a long dissociation time (2 hours) .I repeat several time which give very similar results. So I am wondering if the BIAcore can measure such a low off-rate kinetic. What is the limitation of BIAcore? Any review about that? Thanks in advance. Xianchi Dong Research Fellow Children's Hospital Boston Harvard Medical School
[ccp4bb] Thrombin cleavage of membrane protein with fusion tag
Hi Folks, Sorry this isn't a non-ccp4 post. I am working with a membrane protein for which I am finally able to scale up expression. I am now also able to partially purify my protein from a medium-scale (12-18L) bacterial culture using a two-step tandem affinity purification protocol (Talon followed by amylose affinity steps). As the next purification step, I am about to set up a pilot thrombin cleavage experiment to separate my protein from the His.MBP fusion tag (see below). The construct that I am working with is as follows: His.MBP--ThrombinSite--Membrane Protein There is only one theoretical thrombin cleavage site in the entire fusion protein i.e., at the desired cleavage site with no theoretical secondary sites. I would like to try cleavage both at 4C and around 25C from 4h to overnight but I also have to balance the trials with the material I must generate for the endless permutations and combinations one can try. Each sensible pilot experiment is going to use up partially purified protein from 6-12L preps. FYI. All purification buffers contain DDM and I haven't yet done extensive detergent screens. Please could I ask the community to share tips/suggestions about large-scale thrombin cleavage experiments with their favorite membrane proteins. Many thanks. Raji -- Raji Edayathumangalam Instructor in Neurology, Harvard Medical School Research Associate, Brigham and Women's Hospital Visiting Research Scholar, Brandeis University
Re: [ccp4bb] off topic question BIAcore problem
I assume you use CM5 chips ? I further assume you run at pH 7.5 perhaps ? What's the pI of your analyte ? 7.5 ? Do you get significant binding to your reference cell under the conditions you are running ? You might get rebinding to your negatively charged surface and the dissociation you are measuring might not really be correct (masked through rebinding) Check that first I would say. You can measure low picomolar dissociations. I recently had one with ~80 pM. 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: +1-410-614-4742 Lab: +1-410-614-4894 Fax: +1-410-955-2926 http://lupo.jhsph.edu On Feb 20, 2013, at 12:03 PM, xianchi dong wrote: Dear all, Recently I have measure a set of kinetic data of a receptor ligand interaction using BIAcore 3000. To my surprise, the dissociation rate is very low (~ 10e-6). During the measurement, I use a long dissociation time (2 hours) .I repeat several time which give very similar results. So I am wondering if the BIAcore can measure such a low off-rate kinetic. What is the limitation of BIAcore? Any review about that? Thanks in advance. Xianchi Dong Research Fellow Children's Hospital Boston Harvard Medical School
Re: [ccp4bb] Thrombin cleavage of membrane protein with fusion tag
Hi Raji, Thrombin is a rather good protease and behaves well in a large set of different detergents ( there is a paper by Michael Wiener that describes the relative efficiencies of several usual proteases, amongst those thrombin is inlcuded, used routinely for cleavage of membrane protein fusions in detergents. This is a rather extensive survey henceforth it is very informative. The variable detergent sensitivity of proteases that are utilized for recombinant protein affinity tag removal James M. Vergis 1, Michael C. Wiener in Protein Expression and Purification 78 (2011) 139–142 thrombin tends to be sensitive to reducing agents so I would stay away from DTT and TCEP , b-mercapto is acceptable in reasonable amounts. We cut in salt concentrations as high as 500 mM (NaCl) at pH 7.5-8.5 with 5-10% glycerol around no chelating agents should be present and imidazole in our hands tends to be an inhibitors (probably because it has some chelating/complexing activity). we have good cleavage in DDM , OG and somehow more difficulties in foscholines but it still cuts reasonnably well given the cost of the enzyme and the targets, the most crucial parameter is Protease/target ratio and incubation time and temp. you can do trials on small scale digests in PCR tubes at different temperatures. we usually cut at 4 or room temp. I hope this helps, Best regards, -- Pascal F. Egea, PhD Assistant Professor UCLA, David Geffen School of Medicine Department of Biological Chemistry Boyer Hall room 356 611 Charles E Young Drive East Los Angeles CA 90095 office (310)-983-3515 lab (310)-983-3516 email pe...@mednet.ucla.edu
Re: [ccp4bb] Thrombin cleavage of membrane protein with fusion tag
Hi Raji, I addition to the tips from Pascal, I would like to say that for a memb protein I worked on with a his-tag separated by a thrombin site, I used thrombin cross linked to agarose from Sigma (1 mL). The beads can be collected, washed and reequilibrated, making it ready for use so many times. In my case 1 mL of this thrombin was enough to cleave up to 5-10 mg of target protein, at 4 °C for 16 h (with slight rotation in a 15 mL tube, total volume up to 10 mL). In FC-12 there was a little better cleavage than in DDM. My opinion is that how thrombin (or most other proteases) will cleave may mostly depend on your protein/fusion type/protein-micelle complex structure/access to the site... You just have to try. Best wishes. toufic On Wed, Feb 20, 2013 at 5:15 PM, Raji Edayathumangalam r...@brandeis.eduwrote: Hi Folks, Sorry this isn't a non-ccp4 post. I am working with a membrane protein for which I am finally able to scale up expression. I am now also able to partially purify my protein from a medium-scale (12-18L) bacterial culture using a two-step tandem affinity purification protocol (Talon followed by amylose affinity steps). As the next purification step, I am about to set up a pilot thrombin cleavage experiment to separate my protein from the His.MBP fusion tag (see below). The construct that I am working with is as follows: His.MBP--ThrombinSite--Membrane Protein There is only one theoretical thrombin cleavage site in the entire fusion protein i.e., at the desired cleavage site with no theoretical secondary sites. I would like to try cleavage both at 4C and around 25C from 4h to overnight but I also have to balance the trials with the material I must generate for the endless permutations and combinations one can try. Each sensible pilot experiment is going to use up partially purified protein from 6-12L preps. FYI. All purification buffers contain DDM and I haven't yet done extensive detergent screens. Please could I ask the community to share tips/suggestions about large-scale thrombin cleavage experiments with their favorite membrane proteins. Many thanks. Raji -- Raji Edayathumangalam Instructor in Neurology, Harvard Medical School Research Associate, Brigham and Women's Hospital Visiting Research Scholar, Brandeis University -- ** Toufic El Arnaout Trinity Biomedical Science Institute (TCD) 152-160 Pearse Street, Dublin 2 Tel.: +353 85 83 40 157 **
Re: [ccp4bb] off topic question BIAcore problem
Dear Xianchi, unfortunately, dissociation rate constant kd 10^-6 s^-1 was just beyond the limit of Biacore in 1999 (e.g. see Fig. 1 in http://www.ncbi.nlm.nih.gov/pubmed/10556876). I am not sure about these days. As Juergen noted, you may have a problem with rebinding (you could try to reduce amount of the ligand on the chip, see other tricks here http://users.path.ox.ac.uk/~vdmerwe/internal/spr.pdf). Regarding reviews, David Myszka writes enjoyable reviews on SPR. Hope that helps, Tomas On Wed, Feb 20, 2013 at 5:03 PM, xianchi dong dongxian...@gmail.com wrote: Dear all, Recently I have measure a set of kinetic data of a receptor ligand interaction using BIAcore 3000. To my surprise, the dissociation rate is very low (~ 10e-6). During the measurement, I use a long dissociation time (2 hours) .I repeat several time which give very similar results. So I am wondering if the BIAcore can measure such a low off-rate kinetic. What is the limitation of BIAcore? Any review about that? Thanks in advance. Xianchi Dong Research Fellow Children's Hospital Boston Harvard Medical School Dea
Re: [ccp4bb] Thrombin cleavage of membrane protein with fusion tag
Dear Pascal and Toufic, Many thanks to both of you for the pointers. Toufic, I actually poked around online and bought exactly the same kit that you are suggesting, especially because the beads are reusable. So your experience is reassuring. So I'll find out soon. I also plan to play around with the detergent type and concentration among other things. I have no plans to take the shortcut but it's nice to have at least one other human being to talk to on these matters. I appreciate your responses :) Many thanks! Raji On Wed, Feb 20, 2013 at 1:15 PM, Toufic El Arnaout elarn...@tcd.ie wrote: Hi Raji, I addition to the tips from Pascal, I would like to say that for a memb protein I worked on with a his-tag separated by a thrombin site, I used thrombin cross linked to agarose from Sigma (1 mL). The beads can be collected, washed and reequilibrated, making it ready for use so many times. In my case 1 mL of this thrombin was enough to cleave up to 5-10 mg of target protein, at 4 °C for 16 h (with slight rotation in a 15 mL tube, total volume up to 10 mL). In FC-12 there was a little better cleavage than in DDM. My opinion is that how thrombin (or most other proteases) will cleave may mostly depend on your protein/fusion type/protein-micelle complex structure/access to the site... You just have to try. Best wishes. toufic On Wed, Feb 20, 2013 at 5:15 PM, Raji Edayathumangalam r...@brandeis.eduwrote: Hi Folks, Sorry this isn't a non-ccp4 post. I am working with a membrane protein for which I am finally able to scale up expression. I am now also able to partially purify my protein from a medium-scale (12-18L) bacterial culture using a two-step tandem affinity purification protocol (Talon followed by amylose affinity steps). As the next purification step, I am about to set up a pilot thrombin cleavage experiment to separate my protein from the His.MBP fusion tag (see below). The construct that I am working with is as follows: His.MBP--ThrombinSite--Membrane Protein There is only one theoretical thrombin cleavage site in the entire fusion protein i.e., at the desired cleavage site with no theoretical secondary sites. I would like to try cleavage both at 4C and around 25C from 4h to overnight but I also have to balance the trials with the material I must generate for the endless permutations and combinations one can try. Each sensible pilot experiment is going to use up partially purified protein from 6-12L preps. FYI. All purification buffers contain DDM and I haven't yet done extensive detergent screens. Please could I ask the community to share tips/suggestions about large-scale thrombin cleavage experiments with their favorite membrane proteins. Many thanks. Raji -- Raji Edayathumangalam Instructor in Neurology, Harvard Medical School Research Associate, Brigham and Women's Hospital Visiting Research Scholar, Brandeis University -- ** Toufic El Arnaout Trinity Biomedical Science Institute (TCD) 152-160 Pearse Street, Dublin 2 Tel.: +353 85 83 40 157 ** -- Raji Edayathumangalam Instructor in Neurology, Harvard Medical School Research Associate, Brigham and Women's Hospital Visiting Research Scholar, Brandeis University
Re: [ccp4bb] off topic question BIAcore problem
Hi Xianchi, First of all: a very slow dissociation rate can also be an artifact: the analyte can be simply precipitating on the surface. You do need to rule this out by proper controls. But there is no rule saying that 10e-6 s-1 off rate is not realistic. Even with protein-small molecule binding, one can get extremely slow dissociation if the interaction is very strong. For example, the dissociation of biotin from streptavidin has a rate constant of the 10e-6 s(-1) order. A very slow dissociation rate is often (if not always) correlated with very tight binding (for example KD in picomolar range or even smaller). What is your calculated KD? The binding phase of the BIAcore curves should also reflect the fact that the off rate is low (the Kon-obs has an off rate term). We have measured some pico molar bindings with BIAcore in the past. It is doable, but difficult. You may also want to consider in-solution methods (so that you do not need to worry about artifacts caused by the surface), such as ITC (by competition method), or fluorescence-based methods. For BIAcore, when working with very strong bindings (KD 100pM), there are a few things to consider: 1) You may find great difficulties regenerating the chip - probably the biggest concern with BIAcore (considering the ridiculous price of chips). 2) How much RU should you conjugate to the chip? With strong interactions, as low as possible amount of your ligand should be labeled on the chip, for 4 purposes: a) to make regeneration easier; b) to reduce rebinding effect; c) to reduce mass transfer effect; d) to make sure you do not take away significant amount of analyte from the solution (discussed in 3)). 3) If you plan to span the KD range with the analyte, then if the KD is in picomolar molar range, you are supplying the surface with extremely dilute analyte solutions. In such case, you need to calculate if your flow rate is high enough, to compensate the loss of solute due to binding to surface, otherwise the real concentration of the analyte in the mobile phase will be much lower than the assumed analyte concentration. 4) The association phase of the BIAcore experiment is also affected by the dissociation rate. The observed binding rate Kon-obs contains a Koff term. When you are loading the analyte at near KD concentrations, the binding will take similar amount of time as the dissociation phase to reach plateau. The Kon-obs also contains a concentration terml, so the time required for reaching plateau will be shorter and shorter when you load with higher concentrations of the analytes. 5) The only proper way of labeling chip for slow dissociations is by covalent means. HTH, Zhijie From: xianchi dong Sent: Wednesday, February 20, 2013 12:03 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] off topic question BIAcore problem Dear all, Recently I have measure a set of kinetic data of a receptor ligand interaction using BIAcore 3000. To my surprise, the dissociation rate is very low (~ 10e-6). During the measurement, I use a long dissociation time (2 hours) .I repeat several time which give very similar results. So I am wondering if the BIAcore can measure such a low off-rate kinetic. What is the limitation of BIAcore? Any review about that? Thanks in advance. Xianchi Dong Research Fellow Children's Hospital Boston Harvard Medical School
[ccp4bb] Dell linux box and stereo monitor
Hi All, I am about to order some linux boxes for protein crystallography use from DELL. Could you guys share the experience which model would be the best for this purpose? Also I know the stereo monitor topic has been discussed previously, but I would like to get the opinion for the current best stereo monitor available for model building in protein crystallography. Thanks so much in advance for sharing your experience. Jinyu
[ccp4bb] Opening for a Post Doctoral Fellow in Crystallography at Genentech
Dear CCP4bb readers, We have an opening for a talented Post Doc in X-ray crystallography in the Genentech Department of Structural Biology. If interested, please apply online at http://www.gene.com/careers/detail/00411397/Postdoctoral-Research-Fellow-Xray-Crystallography . Postdoctoral Research Fellow - Xray Crystallography South San Francisco, California JOB ID: 00411397 The Position Crystallography Post Doctoral Fellow - Hymowitz Lab A position is available for a postdoctoral fellow with expertise in crystallography to join the Department of Structural Biology at Genentech, Inc. Active areas of research include the structure and function of intracellular signaling cascades involving ubiquitin as well as the structure and function of protein complexes relevant to human disease. The successful candidate will be involved in all aspects of crystallography, including expression and purification of proteins, crystallization, data collection, structure determination and analysis of protein structures. Who You Are Highly motivated candidates are sought who are capable of independent work in a collaborative environment. Candidates must have a Ph.D. in structural biology, biophysics, biochemistry or related discipline. Candidates should have a strong background in protein crystallography and structure determination as well as demonstrated by successful purification, characterization and crystallization of challenging proteins as evidenced by a first author paper published or accepted in a peer-reviewed journal. Familiarity with biophysical techniques is a plus. Who We Are At the Roche Group, about 80,000 people across 150 countries are pushing back the frontiers of healthcare. Working together, we've become one of the world's leading research-focused healthcare groups. A member of the Roche Group, Genentech has been at the forefront of the biotechnology industry for more than 30 years, using human genetic information to develop novel medicines for serious and life- threatening diseases. The headquarters for Roche pharmaceutical operations in the United States, Genentech has multiple therapies on the market for cancer and other serious illnesses. Please take this opportunity to learn about Genentech, where we believe that our employees are our most important asset and are dedicated to remaining a great place to work. Sarah G. Hymowitz Director Department of Structural Biology Genentech, Inc. 1 DNA Way, South San Francisco, CA 94080
[ccp4bb] Improving Homology Models
Dear Crystallographers, it has been my experience that homology modelling programs get folds pretty well, but sometimes the details are pretty obviously bad, like too-close contacts. One might think that the modelling software would put in a sort of polishing step, but they don't seem to. Is there any way to trick the CCP4 or other software to fix these things, such as by simulated annealing or otherwise, I guess without any weight on the [non-existent] structure factors? Thanks, Jacob -- *** Jacob Pearson Keller, PhD Postdoctoral Associate HHMI Janelia Farms Research Campus email: j-kell...@northwestern.edu ***
Re: [ccp4bb] Dell linux box and stereo monitor
My 0.02 on this as always. For monitors, I was worried about this at first. I replaced all my SGI units (with what was good graphics and CRT displays), and I worried about getting a new monitor and losing information with different stereo options. I bought zalman monitors, and they work fine. They have little quirks, but overall for the price it was a great deal (a one time price for the monitor, no issues with software or hardware as you can just pick up free glasses at your local 3D movie showing). Now the surprise. Going from a dim CRT display to any standard LCD display is really all you need to do. I find I don't use stereo a lot. The nice crispness of the LCD displays is really good enough, and the software is quick enough that one can do quick rotations and get a good feel for dimensions. I find students today don't really use stereo, and I've sort of gone away from it. But I do like the Zalmans, as they do on occasion let me use stereo if that is something I need to display As far as computers for linux - I just build them. Buy a motherboard, memory, hard drive, DVD drive, and some sort of video card and you are good to go. Costs around $300-$400 or so per computer if you do it right. I have boxes that are over 10 years old that will run a typical cns job in 10 minutes. While that is slow compared to most things now, I feel I need a 10 minute break after modeling an entire molecule (I used to get a day break, how times have changed). All modern flavors of linux are very user friendly when it comes to installing (some more than others, that's a whole different matter; I still use fedora). They don't mind hardware, and in fact they do well with almost any newer hardware Good luck Dave On 2/20/2013 2:58 PM, jlliu liu wrote: Hi All, I am about to order some linux boxes for protein crystallography use from DELL. Could you guys share the experience which model would be the best for this purpose? Also I know the stereo monitor topic has been discussed previously, but I would like to get the opinion for the current best stereo monitor available for model building in protein crystallography. Thanks so much in advance for sharing your experience. Jinyu
Re: [ccp4bb] Improving Homology Models
On Wed, Feb 20, 2013 at 12:39 PM, Jacob Keller j-kell...@fsm.northwestern.edu wrote: it has been my experience that homology modelling programs get folds pretty well, but sometimes the details are pretty obviously bad, like too-close contacts. One might think that the modelling software would put in a sort of polishing step, but they don't seem to. Is there any way to trick the CCP4 or other software to fix these things, such as by simulated annealing or otherwise, I guess without any weight on the [non-existent] structure factors? What software were you using? There must be dozens of papers (at least) on this subject, and assessment of refinement and model quality is a major part of the CASP competition. The Rosetta relax protocol is one of the best known, but there are many other approaches (including MD), some of which are definitely integrated into modeling pipelines. I'd start here: https://www.rosettacommons.org/manuals/archive/rosetta3.4_user_guide/d6/d41/relax_commands.html and also: https://www.rosettacommons.org/manuals/archive/rosetta3.4_user_guide/d5/d4e/comparative_modeling.html Of course if the model is too awful, there isn't much that can be done to relieve gross errors without completely rebuilding. I don't know what the radius of convergence of the various protocols is; Rosetta relax certainly can't fix some of the truly awful models in the PDB (but it's by no means the only option). -Nat
[ccp4bb] Off Topic GST-tag Protein Purification
I am attempting to purify a protein complex of two proteins. The Protein A and Protein B have been co-expressed in media containing N15-Cys. Protein A has a 6-histadine tag. I am able to purify this complex via a Ni-NTA column with good results. I now have the Protein A-labled/B-labled complex with some excess Protein A. The A/B complex has a molecular weight of ~60 kDa. I want to exchange the labeled Protein B in the A/B complex for un-labled Protein B. I have also expressed GST-tagged Protien B in media without N15-Cys. With GST-tagged Protien B (unlabled) bound to a GSH column, I applied the A-labled/B-labled complex to the GSH column. However, almost all of the protein remained on the column after on column thrombin cleavage, 1 hour at room temperature (thrombin cleavage site at C-term of GST). This was my preferred method since it would elimnate the GST. If I use glutathione elution on the GSH column, then I will have to do thrombin cleavage in solution, which will yield more efficient cleavage. I will then have a mixture of my target Protein A-labled/B-un-labled, monomeric GST, and dimeric GST. I can separate monomeric GST from A-labled/B-un-labled by size exclusion chromotography. Here is my question: How do I elimnate the GST dimer when the molecular weight is so close to the A-labled/B-un-labled dimer? (GST ~52 kDa, A-labled/B-un-labled ~60 kDa) Caveats: We do not have monomer only GST. The pI's are too close to use ion-exchange. Protein A must be co-expressed as it does not express well alone. Many Thanks, Sarah Presley Ph.D. Candidate University of Tennessee Department of Biochemistry, Molecular, and Cellular Biology
Re: [ccp4bb] Off Topic GST-tag Protein Purification
Protein A must be co-expressed as it does not express well alone. Have you tried separating A+B on the Nickel column by flowing 2M urea to help tickle off protein B. The amount will vary depending on the affinity. In my case (~50nM) requires about 1000-1700ml of 20mM Tris, 2M urea pH 7.5. Then you can add unlabelled purified protein B to labelled protein A. Dan
Re: [ccp4bb] off topic question BIAcore problem
Thanks a lot for the kind reply. I used CM5 chip at pH 7.4. I am not quite sure about the PI of my protein but I have different truncations of my protein which behaved similarly. I can fit my data quite good with the Chi2 around 1 and Rmax 150. The kon is about 10e4 and the KD is around 0.2 nM. The concentration of analytes I used was from 100 nM to 5 nM. I used a control buffer which can disrupt the binding. In that buffer, no binding was observed. I also used fluorescence anisotropy to measure the Ki of the interaction which showed a 1 nM binding compared with the 0.2 nM binding in the SPR assay. On Wed, Feb 20, 2013 at 12:03 PM, xianchi dong dongxian...@gmail.comwrote: Dear all, Recently I have measure a set of kinetic data of a receptor ligand interaction using BIAcore 3000. To my surprise, the dissociation rate is very low (~ 10e-6). During the measurement, I use a long dissociation time (2 hours) .I repeat several time which give very similar results. So I am wondering if the BIAcore can measure such a low off-rate kinetic. What is the limitation of BIAcore? Any review about that? Thanks in advance. Xianchi Dong Research Fellow Children's Hospital Boston Harvard Medical School
Re: [ccp4bb] Improving Homology Models
Hi Jacob, In our experience using MODELLERhttp://salilab.org/modeller/ for the homology modeling part, followed by refinement with CHARMMhttp://www.charmm.org/ leads to structures with very reasonable high quality statistics as reported by MolProbity. Both programs are integrated in our commercial suite Discovery Studiohttp://accelrys.com/products/discovery-studio/, but you can also access them directly from their respective labs. Kind regards, Francisco Francisco Hernandez-Guzman, PhD, MBA Sr. Product Manager Accelrys Software, Inc. From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Jacob Keller Sent: Wednesday, February 20, 2013 12:40 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Improving Homology Models Dear Crystallographers, it has been my experience that homology modelling programs get folds pretty well, but sometimes the details are pretty obviously bad, like too-close contacts. One might think that the modelling software would put in a sort of polishing step, but they don't seem to. Is there any way to trick the CCP4 or other software to fix these things, such as by simulated annealing or otherwise, I guess without any weight on the [non-existent] structure factors? Thanks, Jacob -- *** Jacob Pearson Keller, PhD Postdoctoral Associate HHMI Janelia Farms Research Campus email: j-kell...@northwestern.edumailto:j-kell...@northwestern.edu ***
Re: [ccp4bb] Improving Homology Models
I used proteinmodelportal.org to generate homology models. It worked well for us. Huiming Sent from my iPhone On Feb 20, 2013, at 12:39 PM, Jacob Keller j-kell...@fsm.northwestern.edu wrote: Dear Crystallographers, it has been my experience that homology modelling programs get folds pretty well, but sometimes the details are pretty obviously bad, like too-close contacts. One might think that the modelling software would put in a sort of polishing step, but they don't seem to. Is there any way to trick the CCP4 or other software to fix these things, such as by simulated annealing or otherwise, I guess without any weight on the [non-existent] structure factors? Thanks, Jacob -- *** Jacob Pearson Keller, PhD Postdoctoral Associate HHMI Janelia Farms Research Campus email: j-kell...@northwestern.edu ***
Re: [ccp4bb] off topic question BIAcore problem
Well that looks pretty real then. You might have wrong concentrations in one or the other experiment perhaps hence the difference. Jürgen On Feb 20, 2013, at 5:45 PM, xianchi dong wrote: Thanks a lot for the kind reply. I used CM5 chip at pH 7.4. I am not quite sure about the PI of my protein but I have different truncations of my protein which behaved similarly. I can fit my data quite good with the Chi2 around 1 and Rmax 150. The kon is about 10e4 and the KD is around 0.2 nM. The concentration of analytes I used was from 100 nM to 5 nM. I used a control buffer which can disrupt the binding. In that buffer, no binding was observed. I also used fluorescence anisotropy to measure the Ki of the interaction which showed a 1 nM binding compared with the 0.2 nM binding in the SPR assay. On Wed, Feb 20, 2013 at 12:03 PM, xianchi dong dongxian...@gmail.commailto:dongxian...@gmail.com wrote: Dear all, Recently I have measure a set of kinetic data of a receptor ligand interaction using BIAcore 3000. To my surprise, the dissociation rate is very low (~ 10e-6). During the measurement, I use a long dissociation time (2 hours) .I repeat several time which give very similar results. So I am wondering if the BIAcore can measure such a low off-rate kinetic. What is the limitation of BIAcore? Any review about that? Thanks in advance. Xianchi Dong Research Fellow Children's Hospital Boston Harvard Medical School .. 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: +1-410-614-4742 Lab: +1-410-614-4894 Fax: +1-410-955-2926 http://lupo.jhsph.edu
Re: [ccp4bb] off topic question BIAcore problem
Hi Xianchi, How did you treat the control flow cell? And what is the composition of the control buffer that you use to disrupt binding? Is it denaturing or a mild condition? If the Rmax=150 and Koff=1e-6, then at 7200 s (2 hr), the signal will only drop 1RU from 150RU (assuming you can reach the Rmax), is that what you see on the dissociation curve? In reality, when loading analyte at 100nM, assuming you have Kon=1e4, Koff=1e-6, binding for 30min would only let you reach ~125 RU, and at the lower concentrations will be much worse. Consequently the total dissociation after 7200s would be less than 1 RU on most curves - I am not sure if the Biacore 3000 will have a stable enough baseline for you to confidently measure that. If you try to fit the dissociation phase alone to get the Koff, the reported Koff won't be too accurate due to the low S/N ratio. On the other hand the 5-100nM spanning would generate significant changes on the binding phase, thus the global fitting should be able to get a relatively accurate KD. 0.2nM from SPR and 1nM from in-solution experiment also sounds reasonable. Zhijie From: xianchi dong Sent: Wednesday, February 20, 2013 5:45 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] off topic question BIAcore problem Thanks a lot for the kind reply. I used CM5 chip at pH 7.4. I am not quite sure about the PI of my protein but I have different truncations of my protein which behaved similarly. I can fit my data quite good with the Chi2 around 1 and Rmax 150. The kon is about 10e4 and the KD is around 0.2 nM. The concentration of analytes I used was from 100 nM to 5 nM. I used a control buffer which can disrupt the binding. In that buffer, no binding was observed. I also used fluorescence anisotropy to measure the Ki of the interaction which showed a 1 nM binding compared with the 0.2 nM binding in the SPR assay. On Wed, Feb 20, 2013 at 12:03 PM, xianchi dong dongxian...@gmail.com wrote: Dear all, Recently I have measure a set of kinetic data of a receptor ligand interaction using BIAcore 3000. To my surprise, the dissociation rate is very low (~ 10e-6). During the measurement, I use a long dissociation time (2 hours) .I repeat several time which give very similar results. So I am wondering if the BIAcore can measure such a low off-rate kinetic. What is the limitation of BIAcore? Any review about that? Thanks in advance. Xianchi Dong Research Fellow Children's Hospital Boston Harvard Medical School
Re: [ccp4bb] Problems in running refine_den in cns_solve
many thanks. I'll try again. best, On Wed, Feb 20, 2013 at 10:11 PM, Mecy Shi mecy...@gmail.com wrote: Dear all, I have a problem while running CNS DEN refinement. After running the CNS minimize refinement, the program was aborted and an error message was displayed as: ** Error: test set array test does not exist ** % error encountered: ABORT statement specified. (CNS is in mode: SET ABORT=NORMal END) * ABORT mode will terminate program execution. * Program will stop immediately. I couldn't find the reason. If anyone knows, it would be of great help. many thanks -- Zhubing Shi Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, 320 Yue-yang Road, Shanghai 200031, China -- Zhubing Shi Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, 320 Yue-yang Road, Shanghai 200031, China
