Re: [ccp4bb] clashscore question
Thank you all for the input. I think my problem got solved. The structure was determined by cryo-EM at 3.2 A. I used phenix.real_space_refine to refine the structure using the model with no hydrogen. the statistic report directly from phenix was pretty good but not from pdb validation server (especially the clashscore changes a lot). As John and Sharan mentioned here that the PDB server calculates the clashscore with the hydrogen added and that makes the difference. What I did is adding the hydrogen first and followed by one run of phenix refinement. Finally, the problem got solved, the clashscore was dropped to 2 reported from both the phenix and PDB server. I do consider the alternative conformation and symmetry. While I think adding the hydrogen is the key to my case. Thank you again for your kindly suggestion. I wish my case may serve the people who have the similar problem in the future. Xiaodi From: CCP4 bulletin board on behalf of John Berrisford Sent: Wednesday, January 16, 2019 12:06 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] clashscore question Dear Xiaodi The wwPDB validation service does indeed add hydrogens using Reduce before calculating clashes. For more details of what processes are run when generating the wwPDB validation report see the on-line help https://www.wwpdb.org/validation/2017/XrayValidationReportHelp#close_contacts If you would like us to look into this in more detail please can you email us directly on validat...@mail.wwpdb.org<mailto:validat...@mail.wwpdb.org> with details of your validation session. This email address is also listed on the wwPDB validation help page https://www.wwpdb.org/validation/validation-reports Regards John From: CCP4 bulletin board On Behalf Of Sharan Karade Sent: 16 January 2019 04:28 To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] clashscore question Dear Yu, I think pdb validation server add hydrogen to the structure and then calculate the clashscore. The other methods you mentioned calculate clashscore without adding hydrogen to the structure. Regards Sharan On Tue, Jan 15, 2019, 11:23 PM Xiaodi Yu mailto:uppsala@hotmail.com> wrote: Hi All: I have a pdb file and the clashscores reported from both phenix and MolProbity were around 5, while the clashscore from the pdb validation server is 17. I wonder what may cause the difference? Any suggestion is appreciated. Thank you. Xiaodi To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1 To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1 To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1 To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1
[ccp4bb] clashscore question
Hi All: I have a pdb file and the clashscores reported from both phenix and MolProbity were around 5, while the clashscore from the pdb validation server is 17. I wonder what may cause the difference? Any suggestion is appreciated. Thank you. Xiaodi To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1
Re: [ccp4bb] model bias
Hi Aleks: Maybe you can try CNS ( Initial refinement by simulated annealing) also. It may help to get rid of the model bias and takes short time to run. Xiaodi Date: Wed, 29 Apr 2015 13:52:53 +0200 From: frederic.velli...@ibs.fr Subject: Re: [ccp4bb] model bias To: CCP4BB@JISCMAIL.AC.UK Hello, I would certainly try the usual approaches (map coefficients that are less sensitive to model-bias, i.e. Sigmaa; OMIT maps - there are several of these which can be calculated). In addition, you may have a look at the approach of Liu and Xiong (2014, J. Mol. Biol. 426, 980-993). It is a well known technique (applying a negative temperature factor to amplitudes, for map sharpening) but this paper describes a systematic study indicating improvement whatever the situation. As usual in macromolecular crystallography, confidence is gained by the use of several approaches. HTH, Fred. On 29/04/15 13:16, Aleksandar Bijelic wrote: Dear CCP4 users, I am currently solving a structure (2.8-2.9 A resolution) of a protein complexed with a ligand using MR with the apo-form of this protein as model (resolution of the model is 2.4). After MR-phasing I performed a regular autobuild run giving me good outputs and thus I refined the best pdb leading to good values according to R-values and geometry, however, the denstiy doesn´t look well (but I think it´s due to the moderate resolution). Now I want to get sure if the side chains which are involved in the ligand binding are correctly positioned. However, the active site is suspicously similar to the active site of the model (apo-form) and so I am afraid that this could be due to model bias. My question is how to check and to get rid of the bias (if present) at this stage (after several refinements). I read the publication of Terwilliger about iterative-build OMIT maps but since I am a bloody novice in this field I didn´t really understand it. I originally thought iterative-build OMIT maps are performed to compare the output map with one´s map in order to detect uncertainties, but what to do next? Or should I start from the beginning but how to proceed than, what should I do (I am using Phenix via GUI) ... Is it possible and reasonable to run autobuild with iterative omit map option? Or is it only reasonable if experimental phases are available? I didn´t run iterative-build OMIT maps yet because I am not sure how to run it correctly (what method is the best?) and at my institute the run will take more than 1 day and I don´t want to block one computer until I am not sure if it is reasonable. I hope you can give me some advice and help me. Thank you in advance. Regards, Aleks -- Fred. Vellieux (B.Sc., Ph.D., hdr) IBS / ELMA Campus EPN 71 avenue des Martyrs CS 10090 F-38044 Grenoble Cedex 9 Tel: +33 457428605 Fax: +33 476501890
Re: [ccp4bb] ITC with unfolded proteins
I have done once by using two proteins, one is disordered, the other is very well folded. The result I got is the baseline drift. The baseline goes up upon each injection. The reason I thought at that time is the heat capacity changed dramatically in the system. The disordered protein may form some degree of structure after interacting with its partner. And we did find some precipitation after the experiment. Dee Xiaodi.yu@childrens.harvard edu Sent from my iPad On Mar 14, 2014, at 9:57 AM, Anita P crystals...@gmail.com wrote: That is a very interesting question, which I would request the seniors out there to give their insights on. I was imagining that a recombinant purification of an unfolded partner would aggregate which would cause trouble in ITC. Am I correct in this theory? Would love to have more insights. thanks in advance Anita On Fri, Mar 14, 2014 at 7:18 PM, rkha...@ccny.cuny.edu wrote: Hi, I think the experiment is doable, but how would you decouple protein-protein interaction from folding of the unfolded protein due to protein interaction? Reza Reza Khayat, PhD Assistant Professor The City College of New York Department of Chemistry, MR-1135 160 Convent Avenue New York, NY 10031 Tel. (212) 650-6070 Original message Date: Fri, 14 Mar 2014 18:07:48 +0530 From: CCP4 bulletin board CCP4BB@JISCMAIL.AC.UK (on behalf of Anita P crystals...@gmail.com) Subject: [ccp4bb] ITC with unfolded proteins To: CCP4BB@JISCMAIL.AC.UK Hello everyone, I have a query for the scientists working on protein-protein interaction. It is known that some proteins exist in unfolded or molten globule state and attain structure on interaction with other folded proteins. Many a times, it is difficult to obtain the structure of these complexes. Is it possible to quantitatively determine the thermodynamics of interaction between an unfolded protein and a folded protein using ITC? Later may be perform an alascan to determine the residues of the unfolded partner involved in the interaction. Please share your ideas cheers** Anita
Re: [ccp4bb] A question on protein microheterogenity for crystalization
Hi Acoot: Can your protein form some kind of dynamic self oligomers? When you test by using the gel-filtration column, if the peak is symmetry? Sometime if the target protein can have week self interaction, you can observe a tailed peak. If the protein can have a strong self interaction, maybe you can observe isolated peaks, each peak is corresponding to different amount of the oligomersation state of the protein. You also can test your protein by using native or IEF gels. Dee Date: Sat, 14 Dec 2013 19:01:09 +0100 From: mjvanra...@cnb.csic.es Subject: Re: [ccp4bb] A question on protein microheterogenity for crystalization To: CCP4BB@JISCMAIL.AC.UK Dear Brett, We have seen this behaviour several times for different adenovirus fibre head proteins and don't really have an explanation for it. We have always set the peaks up separately when we had enough protein. For this purification, as you don't have enough protein to pool them separately, I would pool them together and do a first crystallisation screen. But I would also immediately start a larger scale purification so you in the next crystallisation trial you can set the peaks up separately. If you are lucky, by the time you have done the second prep, from the first screen you may have some conditions to optimise. If not, the first screen should at least give some ideas about which precipitants, pHs and perhaps additives are most suitable.Mark On 14 Dec 2013, at 13:16, Acoot Brett wrote:Dear All, When I purified my protein by ion exchange chromatography for crystallization, there were several peaks containing the target protein as analyzed by SDS-PAGE. All these peaks have the same MW as determined by gel filtration coupled MALLS. For crystallization purpose, can I merge the corresponding ion exchange chromatography peaks together? Otherwise the protein yield will be too low. And how to explain the heterogeneity by ion exchange chromatography in this situation? I am looking forward to getting a reply from you. Acoot
Re: [ccp4bb] distinguish ligand binding sites within a protein
Hi Wei: Based on the structure, you can calculate the binding surface between the protein and the ligand. Maybe the two binding pockets will give you two different numbers. And the larger one usually can have the higher binding affinity. You also can analyse how the ligand interacts with the protein though hydrophobic or electrostatic interaction , etc? the last, you may also compare the b factors of the ligand or the protein binding pocket regions after you refining the structure. These things may give you some hints about which binding site is more strong. Dee Date: Mon, 18 Nov 2013 22:45:58 -0500 From: wei.shi...@gmail.com Subject: Re: [ccp4bb] distinguish ligand binding sites within a protein To: CCP4BB@JISCMAIL.AC.UK Thank you so much for the suggestions, Tomas! Yes, my ligand is a small molecule. I have the crystal structure of the ligands bound to the protein, do I still need to computationally dock the ligand to the two pockets, can I calculate the parameters of binding directly using the crystal structure? Best, Wei On Mon, Nov 18, 2013 at 9:03 PM, Tomas Malinauskas tomas.malinaus...@gmail.com wrote: Dear Wei Shi, is your ligand a small molecule? If it is a small molecule, I would try to computationally dock the small molecule to two pockets separately using AutoDock, and look at the estimated free energies of binding. Best wishes, Tomas On Mon, Nov 18, 2013 at 8:55 PM, Wei Shi wei.shi...@gmail.com wrote: Hi all, I got the crystal structure of a transcription factor, and every monomer binds two molecules of the same ligand in different binding pockets. And I also did the ITC experiment, titrating the ligand into the protein, and got a U-shaped curve. The binding affinity for the first binding site is higher than the second binding site. I am wondering whether I could computationally determine from the protein-ligand complex structure that which binding site has higher affinity for the ligand and correlate the binding sites with the parameters I got from ITC experiment. Thank you so much! Best, Wei
Re: [ccp4bb] Biacore/SPR
Another option is BLItz which is cheaper and uses interferometry. Dee Date: Mon, 28 Oct 2013 11:05:33 -0400 From: jubo...@jhsph.edu Subject: Re: [ccp4bb] Biacore/SPR To: CCP4BB@JISCMAIL.AC.UK BiaCore 3000 or if you can afford the T200.Proteon XPR36 would be a cheaper Option and should work as well.Jürgen On Oct 28, 2013, at 10:54 AM, Gang Dong wrote:We mainly measure protein-protein interactions (sometimes protein-small molecules). Thanks! _Gang From: Bosch, Juergen [mailto:jubo...@jhsph.edu] Sent: Monday, October 28, 2013 3:17 PM To: Gang Dong Cc: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Biacore/SPR Protein-protein interaction?protein-small molecule interactions ?Epitope mapping of mABs ?Could you specify what you would like to do, as different models are good for different things.Jürgen On Oct 28, 2013, at 10:08 AM, Gang Dong wrote: Dear all, Could anyone tell me your experience with Biacore (any models) or a similar SPR-based technology for measuring interaction kinetics? I also want to know the price ranges to discuss with our department/facility managers. Thanks,Gang .. 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 .. 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
[ccp4bb] The binding between disordered and ordered proteins
Dear All: I have a general question about protein- protein interactions. I have two proteins, A and B. A is a disordered protein while B is a well folded protein. The binding between A and B has been approved by GST-pull down assay previously. The strange thing is I cannot get them bind if protein A were just freshly prepared. However, if I kept these two proteins separately for one or two days at 4 degree and then did the GST-pull down assay again, I can observe very strong interaction between A and B. Protein A doesn't contain any cys residue. I have already test certain chemicals which might affect the interactions, for example, DTT and EDTA. These chemicals seems to have no effect on the binding. Although A is a disordered protein, does it need such long time to find its proper conformation? Do any people have similar experience? Any suggestions are greatly appreciated. Thanks, Dee
[ccp4bb] Intra-molecular interactions
Dear All: I have a quick question: how common it is that electrostatic interactions are involved in intra-molecular interactions, particularly in intrinsically disordered proteins? Is this interaction specific and any example? Thanks, Dee Xiaodi Yu, Ph.D. Boston Children's Hospital Dana-Farber Cancer Institute Harvard Medical School 3 Blackfan Boston, MA 02115
Re: [ccp4bb] no expression
Plus check the insoluble part or the whole cell. Date: Sat, 5 May 2012 16:30:06 -0400 From: jubo...@jhsph.edu Subject: Re: [ccp4bb] no expression To: CCP4BB@JISCMAIL.AC.UK Some very stupid questions from my side:- it is an expression vector and you are in frame with your gene of interest ?- you are using E. coli strains suitable for expression ?- have you tried adding glucose to the media to avoid leaky expression in case your protein is toxic to E. coli ?- the wild type expresses well ? In the same vector strain ?- how do you determine if it was expressed ? Western blot of tag ? Jürgen Sent from my iPad On May 5, 2012, at 16:17, Jahan Alikhajeh ja...@graduate.org wrote: Dear friends, I am sorry for off-topic though it may be related indirectly! I mutated a 60kDa protein (changing from X--Pro). After doing all the steps, I sequenced the expression vector. It seems everything is fine, even with promoter, but, I can't express it. I tried different Tempratures, IPTG (e with new Stock), media, but they didnot make any difference. Any suggestion is highly appreciated. Best, Jahan
Re: [ccp4bb] Desalting columns
Hi Sangeetha: If you just want to check which buffer is good for your protein, maybe you can try to set up a crystallization screen, keeping your protein concentration just 3 mg/ml. You can observe (after several days) which conditions give you a clear drop, and maybe you can find a clue which buffer is better for your protein. If you want to speed up the whole processes, you can also add glycerol into the drops to grasp the water molecules. Yu Xiaodi Date: Mon, 27 Feb 2012 11:01:33 -0500 From: sangeetha...@gmail.com Subject: [ccp4bb] Desalting columns To: CCP4BB@JISCMAIL.AC.UK Dear bb users, I am trying to crystallize a ~320 kDa protein that crashes out if concentrated past about 3 mg/mL. I would like to try to exchange it into various buffer-salt-additive combinations to see which buffer works. For a starting point, I'd like to use desalting colums. Does anyone have suggestions for good buffer exchange and sample recovery? I woud like to load about 250 uL onto each column. Thanks a lot! Best regards, Sangeetha.
Re: [ccp4bb] about point mutation
Hello Arun: Actually, I am not sure about i couldn't get my desire point mutation. You mean you didn't get the pcr product or you could get the pcr product but there is no mutation. If you didn't get the PCR,Just lower the annealing temperature to 55 degree. And try extension temperature 72 or 68 degree. You can get it. After PCR, using DpnI to treat your PCR product to get rid of the template plasmid. Yu Xiaodi Date: Fri, 24 Feb 2012 09:05:40 + From: arungreenlo...@gmail.com Subject: [ccp4bb] about point mutation To: CCP4BB@JISCMAIL.AC.UK can any one help me in suggesting that what mistake i have did in my mutagenic pcr . actually my problem is my primer annealing temperature is 81degree. im using phusion pol enzyme. i have made many trial, i.e., made annealing at 68 and then followed 2 step pcr method and then added 1micro lit of dmso to 50micro lit of pcr mix etc.. but till now i couldn't get my desire point mutation. my primer length is about 33 and the mutation id at the centre of the primer. can anyone help me what i can improve to get result or what mistake i had did.. thank you all the members in advance, cheers, Arun
Re: [ccp4bb] protein degradation
Hi Sivasankar: Are you sure it is due to the protein degradation? Maybe you can try to do a western blot or others to check if it is the product of degradation. By the way, where you put the 6 histag, N- or C-terminal? If it is at the N terminal, maybe it is the truncation version of your protein. After looking at the gel, it seems your sample was over-load or had lots of unspecific binding to the column. Maybe you can add salt (250 mM NaCl, final concentration) and small amount of imidazle in the sample before you load onto the column (for example, 20 mM Imidazole final concentration). One small trick you can try is wash the cell with the buffer containing PMSF once before lysising the cell. Yu Xiaodi Date: Wed, 15 Feb 2012 18:39:19 +0530 From: sivasankarpu...@iisertvm.ac.in Subject: [ccp4bb] protein degradation To: CCP4BB@JISCMAIL.AC.UK Dear All, Can anybody suggest the tricks and trades of stabilizing a 133 kDa (multi domain) DNA binding protein, that we are expressing at 18 degree Centigrade in E. Coli. The protein appears to degrade during purification; we have protease inhibitor cocktail (in the lysis buffer) as well as 2 mM PMSF, 1 mM EDTA and 1mM DTT throughout during purification ( right from lysis stage). We handle the protein at 4 degree Centigrade. Can you please suggest what precautions we can try to avoid such degradation ? Please find the attached gel picture regarding protein Sivasankar Putta
Re: [ccp4bb] pH optimisation for crystallisation
Hello Sreetama: I think for crystallization, everything is hard to say. But if you find your crystal is sensitive to the pH, you certainly can optimize the pH value but it is better not to deviate a lot. For example you can make 0.2 unit interval (for example: pH value 4.5, 4.7, 4.9...etc which are closed to your original pH value ). For the buffer, you can change or not. Another thing is that, you can also incorporate bis-tris in your last purification, since you find your crystal in this buffer. When you do additive screen, the drops which is clear, also can give you important information. You might find a compont which can inhibit crystal formation. You can use it to slow down the crystal formation to get a big or single crystal.However, you see, sometimes, this optimization is time consuming. I suggest you to try seeding. It can give you a big surprise, sometimes. Yu Xiaodi Date: Wed, 8 Feb 2012 11:56:30 +0530 From: somon_...@yahoo.co.in Subject: [ccp4bb] pH optimisation for crystallisation To: CCP4BB@JISCMAIL.AC.UK Dear all, I have a 17 KDa protein that gives crystals in a condition that has 0.1M bis-tris pH 6.5. The crystals are thin needle clusters and do not diffract. I have tried additives, but they haven't improved the crystals. I intend to vary the pH of the condition. My questions are-1. should the buffer be kept the same or can it also be changed (as long as the desired pH is within the range of both the buffers)?2. in case of a different buffer, should its molarity be the same as that of the original one in the crystallization condition? regards,sreetama
Re: [ccp4bb] Dye for protein affinity measurement
Hello Jiahong: If I understand correctly that you want to test protein-protein interaction or inhibition study in solution, maybe you can try something like ELISA to test protein-protein interaction. Or if your B protein has 6 histag, you can use Ni-NTA agrose beads to test inhibition or binding depending on your purpose. And another option (a little dangerous ), is using radio active to label one of your protein. Yu Xiaodi Date: Wed, 8 Feb 2012 14:17:47 + From: patr...@douglas.co.uk Subject: Re: [ccp4bb] Dye for protein affinity measurement To: CCP4BB@JISCMAIL.AC.UK Jiahong Thermo sells a series of kits called DyLight Fluor for fluorescent labelling of antibodies or other proteins. They have everything you need and they're very convenient and easy to use. You can pick the excitation and emission wavelength. If you label both A and B (or C) with different colors you will be able to see if both are in your crystals (assuming crystallization is part of your approach). You need only label a small percentage of your protein or peptide to see whether the protein is present in a crystal. Patrick http://en.wikipedia.org/wiki/DyLight_Fluor Forsythe, E.L., Achari, A., and Pusey, Marc L. (2006), Trace Fluorescent Labeling for High Throughput Crystallography, Acta Cryst. D62, 339-346. We used DyLight 350 NHS Ester to check we had protein crystals - see methods section of Cryst. Growth Des., 2011, 11 (8), pp 3432–3441 2012/2/8 Jiang Jiahong jiang_jiah...@126.com Dear all, I am looking for some kind of dye for protein affinity comparison, but do not know which to choose. I know protein A can contact B to form a complex,now I hope to find something simiar with A to act as an inhibitor to block the process of A-B complex formation. Maybe a short peptide, a segment of protein A or even some organic molecule. Because here is a poor access to ITC nor Biacore, I can only rely on some dye to check the competence between A and inhibitor candidates. If any one can offer any suggestions. That would be so grateful! Any way,thank kind-hearted people in advance! Regards Jiahong -- patr...@douglas.co.ukDouglas Instruments Ltd. Douglas House, East Garston, Hungerford, Berkshire, RG17 7HD, UK Directors: Peter Baldock, Patrick Shaw Stewart http://www.douglas.co.uk Tel: 44 (0) 148-864-9090US toll-free 1-877-225-2034 Regd. England 2177994, VAT Reg. GB 480 7371 36
Re: [ccp4bb] [RESUME] [OFF-TOPIC] Site-Directed Mutagenesis [OFF-TOPIC]
Hi Fred: For the mutated plasmids, it generates the nicked dna, so the transform efficiency will be lower compared to the parental plasmids. And that is the reason why people usually use super competent cell to transform these nicked plasmid. It seems ok to me to get 2 or 14 colonies from the nicked plasmid. You just need ONE. And usually for these mutagenesis PCR, the success rate is pretty high. I suggest you to sequence these colonies. And you can get it. Yu Xiaodi Date: Tue, 7 Feb 2012 10:17:43 -0200 From: ccp4bb.l...@gmail.com Subject: Re: [ccp4bb] [RESUME] [OFF-TOPIC] Site-Directed Mutagenesis [OFF-TOPIC] To: CCP4BB@JISCMAIL.AC.UK Hi CCP4 list, Thank you very much for additional messages and references. Here goes the image of the PCR product before digested and after digested and cleaned. http://ompldr.org/vY29jbA The results of the transformation of 3 microL (90 ng) of non-mutated/paretal plasmid gave hundreds of colonies; 3 microL of Dpn1 digested sample gave two colonies only; and transformation of 3 microL(90 ng) of cleaned product gave 14 colonies. So, if the amplification is not abundant, chances are that home made competent cell will not be transformed with the digested product. Don't want start another discussion but, is there any reason for differences in the transformation efficiency between the parental and the mutated (cleaned) plasmids? All the Best, Fred Em 03-02-2012 16:14, Fred escreveu: Hi CCP4 list, Thanks everyone who have answered my post concerning to mutagenesis. From quick reading most of the answers, the following seems to be a consensus: 1) Do not concentrate your PCR product; 2) Too much DNA and/or impurities like salts or whatever can inhibits transformation; 3) Purify your PCR product before transformation if possible or use 3 of 4 microL of it. This is more or less the amount of DNA showed in the uploaded image. Kind regards, Fred P.S.: I'll let you know the results.
Re: [ccp4bb] On pKa of Aspartic acid
Hi Deepak: I think it is common for the residues which participate catalysis to have a Pka deviated from the reality pKa value especially for acid/base catalysis (acid base titration assay can help you to figure out the way of catalysis). Usually the pKa values of these kind of critical residues are affected by their local environment and this character is related to the enzyme's working mechanism. I am sorry that I am not professional in enzyme, I cannot answer your questions for each questions. Yu Xiaodi Date: Tue, 7 Feb 2012 19:48:26 +0800 From: deepos...@gmail.com Subject: [ccp4bb] On pKa of Aspartic acid To: CCP4BB@JISCMAIL.AC.UK Dear colleagues, We have solved the crystal structure of a human enzyme. The pKa of a catalytically critical aspartic acid has increased to 6.44. It is hydrogen bonded (2.8 Angstroms) to a water molecule that is supposed to donate a proton during the catalysis. Can anybody help me a) interpret the significance of this increase in pKa of the aspartic acid from 3.8 to 6.44 in context with the catalysis? Is this advantageous or detrimental? b) How is pKa related to an amino acids’ ability to force a water molecule to donate a proton? c) At pH 7.4, the aspartic acid would be de-protonated irrespective of whether the pKa is 3.8 or 6.44; isn’t that true? d) Have similar increase in pKa values observed for aspartic acids before? I would be grateful if anybody could explain or comment on the above queries. Deepak Oswal
Re: [ccp4bb] Off topic: His-tag purification
Hi Theresa: If you can make sure that your target protein is expressed. You can first use 6 M urea to denature the protein and then try to bind it to the column. If the denatured protein can bind to the column, it seems the histag is hided inside of the protein. It is not exposed enough to interact with the column. In this case, you can design a new construct, for example, put the tag to the other end of the sequence, or introduce a flexible linker between the tag and the protein. Another thing you can try is using Cu ion instead of Ni ion. It will fasten the binding. Good luck Yu Xiaodi Date: Sun, 15 Jan 2012 18:23:33 + From: theresah...@live.com Subject: [ccp4bb] Off topic: His-tag purification To: CCP4BB@JISCMAIL.AC.UK Hi all I have a His-tagged soluble protein (8 His residues added to 90 kDa protein) that do not bind to IMAC column based on flowthrough showing up with Western blott. Do you have suggestions to improve the binding? Binding condition is 50 mM Tris-HCl 8.0, 300 mM NaCl, 10 mM imidazole pH to 8.0. Thank you. Theresa
