Re: [ccp4bb] Ambiguous metal ion at active site.
Dear Dilip, it's difficult to exactly analyse the coordination of your metal in 2D. But, my suggestion is : Try another metal like Zn, which maybe is a contamination (from the plastic of eppendorf for example). Zn has more electron than Mg maybe that's the reason why it's still green. I suggest that you calculate anomalous difference map, maybe it could help, depending of the wavelenght used for data-collection. And you can also try this phenix.refine which has an algorithm to build solvent. Hope to help. Nicolas De : CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] de la part de Dilip Kumar [dku...@igib.in] Envoyé : jeudi 9 juillet 2015 11:35 À : CCP4BB@JISCMAIL.AC.UK Objet : [ccp4bb] Ambiguous metal ion at active site. Dear All I have solved a structure of a metal-ion dependent exonuclease enzyme. In homologous structures, two or three Manganese ions are present at catalytic center. However, I have used 2 mM MgCl2 in protein purification buffer. I tried to fit both of these metal ions at catalytic center but in both cases it still shows green density (Sigma level ~ 7) in difference map and low b-factor (10) for these metal ions. For better understanding I have attached the screenshot of metal ions with difference map on. Please suggest me the possible reasons or methods to validate the presence of any other metal ions at catalytic center. Thanks in advance. Regards Dilip Kumar Research Associate Chemical and Systems Biology Unit CSIR-Institute of Genomics Integrative Biology Delhi-110025
Re: [ccp4bb] anomalous phasing with HySS - phaser - Autobuild
Dear Almudena, I have some questions to help you, how many molecules are presents in the ASU ? If more than one, maybe you can try to find NCS in the substructure. To perform that it could be necessary to increase (more than 20) the radius of NCS research parameters. You can try to find NCS with sitcom. Or by hand. If you can use NCS to average density during the solvent flatening steps, it could help. If you haven't NCS in your substructure. After phenix and solvent flattening step, is the map interpretable ? Is phaser keeping all the sites fund by HySS ? or it find less sites ? Can you see nice density, continue, protein shape ? or is it more or less a lot of blob random ? You can try to select only the best sites found by HySS, maybe some are not well determined. HySS locate 127 but how many is it suposed to find ? One last thing, for the autobuild programm it's not the same to work with dataset at 1.8 A of res compare with one at 3 A. And you must not expect the same level of quality. And if you look the model build, are you able to continue manually, correct some clearly wrong chain? In other word be as sure as possible that the initial phases that you give to autobuild programm are not to far from the good one. Hope to help you. I am not very familiar with HySS that's why i don't go deeper for this parts. But you can try shelx which is parts of CCP4 or via HKL2map. And compare the sites obtain with these from HySS. Nicolas De : CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] de la part de Almudena Ponce Salvatierra [maps.fa...@gmail.com] Envoyé : vendredi 26 juin 2015 12:32 À : CCP4BB@JISCMAIL.AC.UK Objet : [ccp4bb] anomalous phasing with HySS - phaser - Autobuild Hi all, I have SeMet data for which HySs locates 127 Selenium atoms with a CC of 0.31, which I think is decent. Then I run Phaser to generate the first maps, and it gives a score of 25 and LLG of 190 or so... The next step would be running Autobuild, however, the first models I realize it is building have R work and R free between 50 and 55%. So I guess it is just some random stuff there. If the result from HySS and Phaser looks more or less good, why does AutoBuild show these numbers?? Any idea of what I could do to improve so? Any ideas are welcome. Best wishes, Almudena -- Almudena Ponce-Salvatierra Macromolecular crystallography and Nucleic acid chemistry Max Planck Institute for Biophysical Chemistry Am Fassberg 11 37077 Göttingen Germany
Re: [ccp4bb] CCP4 not working with OSX Yosemite version 10.10.3
Hi Saleem, in my own experience with the yosemite update, after the update, i have had to do that : 1) Xcode via apple store (itunes) you have to create or use you apple ID etc... 2) Install XQuartz : http://xquartz.macosforge.org/downloads/SL/XQuartz-2.7.7.dmg 3) Install ccp4 : http://www.ccp4.ac.uk/download/index.php#os=mac In my case it was sufficient for ccp4, ADXV and some other. Nicolas De : CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] de la part de saleem raza [mysaleemr...@hotmail.com] Envoyé : jeudi 28 mai 2015 14:33 À : CCP4BB@JISCMAIL.AC.UK Objet : [ccp4bb] CCP4 not working with OSX Yosemite version 10.10.3 Hi, I recently updated the OSX on my IMAC to OSX Yosemite version 10.10.3. Since then I am unable to open CCP4. I have tried to reinstall the CCP4 but still the problem is there. Anyone have any information how to fix this issue. I have attached the screen shot of error message. regards Saleem
Re: [ccp4bb] Hi
Dear Vijay, Have you try to use superpose in ccp4 ? In superpose it's possible to select exactly what you want (atoms, residues...) . In your case if you use only the atoms or the nucleotids that you want to be superpose, it could be ok. Hope to help. Nicolas De : CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] de la part de vijay srivastava [vijaytec...@yahoo.co.in] Envoyé : mardi 26 mai 2015 14:41 À : CCP4BB@JISCMAIL.AC.UK Objet : [ccp4bb] Hi Dear All, I want to superpose the nucleotide form one GTPase on to the nucleotide of other GTPase in order to study the interaction in the nucleotide binding pocket. I tried to superpose but it is superposing on the basis of secondary structure as a result both the nucleotides from two structutres are not properly aligned. I want to superpose both the nucleotide, so that I will get the matrix, which I want to apply on my desired strcuture and study the interacting residues. Do any one have the align program with you or any other program which can solve this problem. waiting for your kind response regards vijay
Re: [ccp4bb] Hi
I am not absolutly certain to understand the steps that you followed. But if it doesn't work with the nucleotid as reference and target, maybe if you face two GTPase, you can find some conserve residues in the binding pocket. In this cas, you can use these residues as ref. and target. To be more helpful, i think we need more information about the output message givent by superpose. I assume that it take correctly all the pdb files in count but it was not able to superpose, am i right ? One other solution is to use one of the solution descibed here : http://www.cgl.ucsf.edu/home/meng/grpmt/structalign.html or here : http://wishart.biology.ualberta.ca/superpose/ Or maybe directly in the chimera soft (tools =structure comparison) (it's not as good as lsqkab (superpose)). regards. Nicolas De : vijay srivastava [vijaytec...@yahoo.co.in] Envoyé : mardi 26 mai 2015 14:56 À : FOOS Nicolas Objet : Re: [ccp4bb] Hi Dear Nicolas, I ahd tried in superpose but it is not alligning and simultaenousli I had given only the pdb containing only the nucleotide but both of them dosen't work. regards Vijay On Tuesday, 26 May 2015 6:22 PM, FOOS Nicolas nicolas.f...@synchrotron-soleil.fr wrote: Dear Vijay, Have you try to use superpose in ccp4 ? In superpose it's possible to select exactly what you want (atoms, residues...) . In your case if you use only the atoms or the nucleotids that you want to be superpose, it could be ok. Hope to help. Nicolas De : CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UKmailto:CCP4BB@JISCMAIL.AC.UK] de la part de vijay srivastava [vijaytec...@yahoo.co.inmailto:vijaytec...@yahoo.co.in] Envoyé : mardi 26 mai 2015 14:41 À : CCP4BB@JISCMAIL.AC.UKmailto:CCP4BB@JISCMAIL.AC.UK Objet : [ccp4bb] Hi Dear All, I want to superpose the nucleotide form one GTPase on to the nucleotide of other GTPase in order to study the interaction in the nucleotide binding pocket. I tried to superpose but it is superposing on the basis of secondary structure as a result both the nucleotides from two structutres are not properly aligned. I want to superpose both the nucleotide, so that I will get the matrix, which I want to apply on my desired strcuture and study the interacting residues. Do any one have the align program with you or any other program which can solve this problem. waiting for your kind response regards vijay
Re: [ccp4bb] merge weak anomalous signal from multiple datasets
Hi Charles, I have some differents suggestions, maybe it could be a good idea to reconsider the criteria for keep or discard data : Diederichs, K., et P. A. Karplus. « Better Models by Discarding Data? ». Acta Crystallographica Section D: Biological Crystallography 69, nᵒ 7 (1 juillet 2013): 1215‑22. doi:10.1107/S0907444913001121. This paper is about the quality of the final model with or without discarded data. But in my opinion, it maybe usefull to adopt a similar point of view about the anomalous data. What i mean is that if the anomalous signal seems to be weaker, it's not necessarly worth to find the good substructure. If the signal that you measured, is well measured, with a good error estimation it maybe lower in terms of signal / noise but more accurate. You didn't precise if your datasets were collected on only one crystal or with differents crystals. In function of that, you have to tak in count differents effects. Radiation damage, non-isomorphism... One last point, depending of how you process your data, you have to keep in mind the over-scaling effect. If you use aimless directyl without options, if your data are already scaled (for example de XDS_ASCII.HKL), you may have some problem. I hope to help. In my opinion, if you have to discard data, you have to do that not only based on qualitativ criterias, but based on reliable criteria, cell parameters, B-factor, Bad correlation between data set from differents crystals. Finnaly, even if it's seems that the anomalous signal is low, you have to try to find heavy atoms sites. Nicolas De : CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] de la part de Thanh Nguyen [hongthanh1...@gmail.com] Envoyé : lundi 2 mars 2015 10:24 À : CCP4BB@JISCMAIL.AC.UK Objet : Re: [ccp4bb] merge weak anomalous signal from multiple datasets Hi Charles, Actually I also tried the method of Q. Liu et al and got one structure solved after merging 3 different datasets. The only thing different in my case is the Se-SAD phasing instead of Br. But the signal was also weak due to the disorder of incorporated Se in the protein crystals (I supposed so, because I used only 50% of Se-Met during the culture). So in my case, first I processed the data by XDS and made some resolution limitation according to the I/sigma and CCanom. I also did some limitation on the number of images because SCALA can only process not more than 10.000 images (remove the bad images according to the I/sigma and B-factor). And in fact, I did some combination between cut datasets and non-cut datasets and found that the cut datasets really gave me better merged datasets as some noises were removed and the resolution was limited, of course you also lose some information. But in our case, improving the weak anomalous signal to just enough for solving something is more important than losing some information of noise, so we need to be compromised. And any way, for facing with the weak anomalous signal issue, we usually collected such a high abundance of datasets, so losing some information of data is not the problem. Hope you will be able to solve it. Regards, Thanh Nguyen On Mon, Mar 2, 2015 at 5:19 AM, Andreas Förster docandr...@gmail.commailto:docandr...@gmail.com wrote: Hi Charles, I don't know what multiscale does. Probably the right thing. If the anomalous signal is weaker after scaling of multiple datasets, non-isomorphism might be at fault. Try Blend to scale your datasets. Blend is part of ccp4 and gives good graphical diagnostic feedback. Andreas On 01/03/2015 4:20, CPMAS Chen wrote: Dear CCP4 users, Recently, I got some datasets with weak anomalous Br signal. I tried to merge them according to Q. Liu et al Science 336, p1033 (2012). I am using the script multiscale@SSRL. The merged dataset has WEAKER anomalous signals. Liu et al used SCALA for scaling and merging while multiscale@SSRL using AIMLESS. Should this cause such a difference? The SCALA@SSRL has a limitation on the number of frames it can process. So I cannot directly check if this caused the difference. Any suggestions? Thanks! Charles -- *** Charles Chen Research Associate University of Pittsburgh School of Medicine Department of Anesthesiology ** -- Nguyen Hong Thanh, Ph.D. student Lab 20B. Macromolecular Structures Department Centro Nacional de Biotecnologia, CSIC C/ Darwin 3, Campus de Cantoblanco 28049, Madrid, Spain Genetic Engineering Laboratory Institute of BioTechnology Viet Nam Academy of Science and Technology 18 Hoang Quoc Viet, Cau Giay, Hanoi, Vietnam
Re: [ccp4bb] Protein precipitating at higher concentration!!
Hi Ivan, according to my experience, if you remove at the same time GuHCl and Triton, you have huge risk of precipitation if the protein is not properly folded. In my opinion, you have to do something like re-folding. It seems that your protein could be solubilized from inclusion-body. In the same case, the first thing that i will do : I start with the same protocol as you do, but after the TALON, i will dialysis gently the sample against the same buffer without the GuHCL but with Triton. Maybe you ca try to change the pH. Tris-HCl is a good strating point, but maybe you a to refine this. Phosphate buffer (7.5) helped me sometimes. What is the Pi of your protein ? Hope to help you. Nicolas De : CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] de la part de CHAVAS Leonard [ccp4hnaa...@gmail.com] Envoyé : vendredi 9 janvier 2015 01:41 À : CCP4BB@JISCMAIL.AC.UK Objet : Re: [ccp4bb] Protein precipitating at higher concentration!! Hi Ivan this will not be an answer to your question, but did you consider not concentrating 'too much' your sample? It happened few times to me that the protein was precipitating when concentrating for SEC because of the presence of other impurities. Trying the good old AS precipitation helped a bit, but wasn't really the magical solution as I was losing a bit of the protein of interest as well. The solution was to concentrate only slightly the sample, and pass it though multiple (at the time it was quite a lot actually) runs of SEC. I ended up with again a lot of pure sample to concentrate, however, this sample was pure enough and did not precipitate. Other than that, I guess playing with the salt concentration might help keeping things stable... or not. I know people also tried the addition of glycerol or EG, but I don't have personal experience in that and cannot really comment if it is working well or not. Cheers, Leo On Jan 9, 2015, at 9:00 AM, xaravich ivan xaravich.i...@gmail.com wrote: Hi Xtallographers, I have been able to purify a protein that was initially going into inclusion bodies from the excellent suggestions that I got here. So my lysis buffer has 0.5M Guanidium Hydrochoride, 2% TritonX-100, 500mM NaCl, 5% Glycerol in 20 mM Tris-HCL pH 8.0 The problem is that the protein is first purified as a SUMO-fusion protein which is further proteolysed and passed through the Talon resin to get the final SUMO-Free construct. However as I have around 250mM Imidazole (pH elution did not work) from the elution of the first round, I have to dialyse the sample to get rid of the imidazole so that I can use the proteolysed sample again on the column. All these I do in a buffer that does not have GuHCL or Triton. However I have kept the NaCl concentration same (0.5 M). I start to see white insoluble precipitate right from the dialysis step. If I spin the precipitate out, I still have a lot of protein to go to the next step of proteolysis. The problem is that when I finally want to concentrate the protein to run the SEC step, all my protein starts precipitating starting from 5mg/ml to all the way to 25-30 mg/ml. Are there some smart ways to keep the protein soluble at higher concentrations, assuming that I do not have to remove them before setting up trays? Should I keep on using Guanidium Hcl and Triton for all the steps all the way into the SEC column. Have people got any good results using 5% Acetronitrile, 50mM Arginine or DTT? (used for NMR samples) Any help in this regard will be very helpful. The protein is an engineered bacterial transcription factor. (not a membrane protein) Thanks in advance as always, ivan
Re: [ccp4bb] water at the same exactly position
Dear Lu, one simple solution is to remove the water molecules with text editor for example. It depend of how-many times you have multiply water molecules and if your model have several or more water molecules. In coot you can remove it graphically, but according to my knowledge not automatically, and it maybe time consuming. Hope to help Nicolas De : CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] de la part de luzuok [luzuo...@126.com] Envoyé : mercredi 29 octobre 2014 13:08 À : CCP4BB@JISCMAIL.AC.UK Objet : [ccp4bb] water at the same exactly position Dear all, I found that there are some water molecules in my pdb that share the same position. This maybe cause by merging molecules in coot. It seems that I have mereged water molecules into my protein for more than one time. Does anyone tell me how to fix this problem? Best regards! Lu Zuokun -- 卢作焜 南开大学新生物站A202
Re: [ccp4bb] How to separate BSA present in Thrombin
Hi Manjalu, In my opinion, one way is to use iEX column. Depending of the Pi of you protein and the one of the BSA, if they are sufficiently different, Ion exchange is on possible way. To separate your protein from BSA, you have to give us more information about your protein if it's possible. HTH. Nicolas De : CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] de la part de Manjula Ramu [manjula@gmail.com] Envoyé : mercredi 8 octobre 2014 15:27 À : CCP4BB@JISCMAIL.AC.UK Objet : [ccp4bb] How to separate BSA present in Thrombin Hi all, My protein size is nearly the same size of BSA and it has His tag. If I have to cleave the tag I have to use Thrombin which is having huge amount of BSA in it. Now how will I separate this BSA from my protein. Thanks and Regards, Manjula R Research Scholar Department of Biophysics National Institute of Mental Health and Neurosciences Bengaluru-29, Karnataka INDIA E-mail: manjula@gmail.commailto:manjula@gmail.com Mobile no:+91-9538553356 http://www.nimhans.kar.nic.in/ https://www.nimhans.kar.nic.in/
Re: [ccp4bb] calculation of the orientation angles between helices
Hi Gajanan, i never try Qhelix, but i use Chimera to do that. It's possible to mesure angle, distance etc between helix. Hope to help. Nicolas De : CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] de la part de Gajanan Arbade [gajanan_pbi...@diat.ac.in] Envoyé : mercredi 17 septembre 2014 09:31 À : CCP4BB@JISCMAIL.AC.UK Objet : [ccp4bb] calculation of the orientation angles between helices Hello, I am using Qhelix, for the calculation of the orientation angles between helices. when am running the demo its working,but when am uploading my pdb file it is not working, can someone help me to resolve the problem.suggest me the other softwares for the same. Thank you. Regards, Gajanan __ Gajanan K Arbade Research Scholar Defence Institute of Advanced Technology (DIAT) Pune Maharashtra, India Pin Code-411025 Contact No. Lab.+ 91-20-24304377 Mob: 08698198016
Re: [ccp4bb] Protein-DNA structure solution
Dear Sasha, i try to answer one by one your question : 1) Not sur to understand what you mean by validate DNA constraints, if it's about the geometry parameters liken angle bond length, you have to use Moleprobity for example. 2) Fitting DNA in density, you can use coot, it's exactly the same spirit as protein building. You have to be careful of the possible special case like hoogsteen bases pair, or fliping base, but out of that, most of the DNA building is comparable with protein. 3) The kind of parameters, is the same as for protein. But, you have to be carefull that refmac understand well that you pdb file contain DNA. I do not use Refmac, but i think with the right library for restraint (the same as ccp4 or coot) it should be OK. If it's possible, sometimes it could be good to use TLS group for DNA refinement. 4) I think it's always good to use up-to-date version of the programm. You can use the ccp4 method to install all the programm at the same time. But if it's timpossible for you, i can't rememeber which version it was, but i worked with DNA protein structure 5 years ago, and it was already more or less good. Hope to help. De : CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] de la part de Sasha Pausch [sashapau...@gmail.com] Envoyé : mercredi 30 juillet 2014 19:45 À : CCP4BB@JISCMAIL.AC.UK Objet : [ccp4bb] Protein-DNA structure solution Hello CCP4bb I am solving a 3.2A resolution crystal data for a protein DNA complex. I would like to know- 1) How to validate the DNA constraints in the crystal structure? 2) How to fit DNA into density? How different it is from protein fitting into density? 3) What kind of parameters to consider for refining DNA in Refmac for a Protein-DNA complex? 4) Which versions of refmac and COOT support DNA refining and model building? Thank you in advance, Sasha Pausch
Re: [ccp4bb] Problem in molecular replacement
You can use molrep or phaser as you prefer with DNA. To do that, you can edit you pdb file by hand with your favorit text editor (vim, emacs...) And you can cure your pdb file with Edit PDB File in ccp4 coordinate Utilities De : dusky dew [duskyde...@gmail.com] Envoyé : jeudi 24 juillet 2014 14:46 À : FOOS Nicolas Cc : CCP4BB@JISCMAIL.AC.UK Objet : Re: Problem in molecular replacement How do I use DNA as search model? Can I use it in molrep or phaser? On Thursday, July 24, 2014, FOOS Nicolas nicolas.f...@synchrotron-soleil.frmailto:nicolas.f...@synchrotron-soleil.fr wrote: Dear Yang, you should try different search model, for example : 1) DNA only, 1) DNA 2) protein 1)DNA+Prot etc... You can also try to find a partial solution and keep this solution to place the other molecules with another round of MR. Be careful if you use only DNA, if the density doesn't show clear proteic shape, you could have a false positive (a bit like with alpha helix for coil structure) One tips, sometimes the DNA could be organised in infinite Helix in the crystal, you can try to use DNA 2 times longer than you expect especially if your DNA molecules has over-hang. Nicolas De : CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UKmailto:CCP4BB@JISCMAIL.AC.UK] de la part de dusky dew [duskyde...@gmail.commailto:duskyde...@gmail.com] Envoyé : jeudi 24 juillet 2014 13:50 À : CCP4BB@JISCMAIL.AC.UKmailto:CCP4BB@JISCMAIL.AC.UK Objet : [ccp4bb] Problem in molecular replacement DEAR ALL I am trying to solve the structure of a protein DNA complex with molecular replacement. The resolution in about 2.5 angstrom. The spacegroup is P21. The search model has about 50% identity and is a dimer of a dimer. The problem is the unit cell is huge and so the number of molecules in asu becomes 4 or 3. I am not finding any solution with phaser/molrep. Please suggest. Thanks Yang zhang
Re: [ccp4bb] ITC with heterogeneous protein
Dear Sajid, one first problem in your study is how-to adress if the deltaH mesured is caused by the ligand interaction, or by the modification of dimer-monomer equilibrium. You have to well caracterise your system dimer-monomer. One other problem is about the accessibility of the interaction site. If it's different between monomer and dimer, you have to know the ratio between these two state, to managed the proportion of active protein. Is it possible to separate the dimer from monomer ? Is this equilibrium concentration dependant ? In this case, you can try to execute your ITC experiment in low protein concentration. Or try to find condition in wich you can assume that you have large majority of dimer in your sample. To my mind it's difficult to start without more information. You can also try to titrate the protein with itself. If dimer/monomer is concentration dependent, you can try to titrate protein with itself. This can give you some information about the thermodynamics of the dimer/monomer formation. Hope to help. Nicolas De : CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] de la part de sajid akthar [b_sajid_...@yahoo.co.in] Envoyé : vendredi 18 juillet 2014 11:24 À : CCP4BB@JISCMAIL.AC.UK Objet : [ccp4bb] ITC with heterogeneous protein Dear All, This is an off-topic question. I have protein solution of heterogeneous (contains both monomer and dimer). I want to perform ITC with this protein. I doubt whether this heterogeneity will interfere the binding study. Any advice please. Thank you Sajid
Re: [ccp4bb] ITC with heterogeneous protein
it depend of what you expected as information : In this case your measure is resulting from ligand binding AND dimerization (except if all the protein is already dimerized). I am not sur to understand, do you know how many binding sites exists on one monomer ? You should be able to determine the stoechiometry ligand/protein, but it's not necessarily the same between monomer and dimer. Is the ligand trigger for dimerization ? Or the dimerization appear in all case upon the concentration is sufficient ? De : CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] de la part de Ramesh V [ramesh.c...@gmail.com] Envoyé : vendredi 18 juillet 2014 14:42 À : CCP4BB@JISCMAIL.AC.UK Objet : Re: [ccp4bb] ITC with heterogeneous protein I have a similar case, where in there are multiple binding sites on the protein for the ligand and ligand induces dimerization.So it is not helpful even if I separate the monomer and dimer. If I titrate the dimer with ligand, the stoichiometry will completely change? Any suggestions will be helpful. On Fri, Jul 18, 2014 at 12:46 PM, David Briggs drdavidcbri...@gmail.commailto:drdavidcbri...@gmail.com wrote: Hi Sajid, *Assuming* you have one site per monomer (rather than, say, one site per dimer), and *assuming* each binding event is completely independent ( I.e no co-operativity), you might just get away with running the experiment with the heterogeneous material. However, you might not be able to confidently make these assumptions, so imho it would be preferable to separate the monomer and dimer by SEC prior to ITC. If this is not possible, then pay close attention to the fit when you run the heterogeneous experiment. Poor fit to a one site model may indicate that these assumptions are invalid. Can you obtain stoichiometry information from a different technique? This might be very helpful. Hth, Dave Dr David C Briggs PhD http://about.me/david_briggs On 18 Jul 2014 10:25, sajid akthar b_sajid_...@yahoo.co.inmailto:b_sajid_...@yahoo.co.in wrote: Dear All, This is an off-topic question. I have protein solution of heterogeneous (contains both monomer and dimer). I want to perform ITC with this protein. I doubt whether this heterogeneity will interfere the binding study. Any advice please. Thank you Sajid
Re: [ccp4bb] Naccess
Hello, i cited this program with : Hubbard, S.J., and Thornton, J.M. (1993). NACCESS (Computer Program, Department of Biochemistry and Molecular Biology, University College London.). I am not absolutly certain that's th best way. Nicolas De : CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] de la part de Armando Albert [xalb...@iqfr.csic.es] Envoyé : mercredi 9 juillet 2014 17:02 À : CCP4BB@JISCMAIL.AC.UK Objet : [ccp4bb] Naccess Dear all, Does anyone know how to properly cite Naccess for calculation of solvent accessible area (http://www.bioinf.manchester.ac.uk/naccess/)? Armando
Re: [ccp4bb] Buried surface area calculation..
Dear Gajana, you can use . PISA ((Krissinel and Henrick, 2007) (EMBL server) and NACCESS (Hubbard and Thornton, 1993). (not for windows i think). Hope to help Nicolas De : CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] de la part de Gajanan Arbade [gajanan_pbi...@diat.ac.in] Envoyé : mardi 1 juillet 2014 20:49 À : CCP4BB@JISCMAIL.AC.UK Objet : [ccp4bb] Buried surface area calculation.. Hello, Am working with some DNA binding proteins. If I want to calculate the buried surface area, which software tools should i use? Suggest me some windows based programs to solve my purpose. Thank you Regards, Gajanan __ Gajanan K Arbade Research Scholar Defence Institute of Advanced Technology (DIAT) Pune Maharashtra, India Pin Code-411025 Contact No. Lab.+ 91-20-24304377 Mob: 08698198016
Re: [ccp4bb] How to transfer non-frozen crystals with less disturbance?
Dear Meisam, i don't know exactly what you need to do after the transfert, but if you want collect without frozen, maybe you can try to use capillar. Maybe can you mount your crystall in capillar before the travel ? It's simple suggestion, i never did that. Nicolas De : CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] de la part de Meisam Nosrati [meisam.nosr...@gmail.com] Envoyé : mercredi 2 juillet 2014 22:17 À : CCP4BB@JISCMAIL.AC.UK Objet : [ccp4bb] How to transfer non-frozen crystals with less disturbance? Dear CCP4ers I need to transfer some crystals mainly in sitting drops to the site of data collection without freezing them. I do not know what is the best solution to secure the boxes in their place to minimize the disturbance. I am using the 24 well VDX plates with 10-80 microliter sitting drops. I have one or two hanging drop boxes as well with 10 microliter size drops. If you have any experience about this matter, I greatly appreciate, if you share it with me. Thanks Meisam Nosrati
Re: [ccp4bb] solvent flattening with resolve - phenix
Dear Almudena, i think your problems com from the Flag in your input.mtz . You have to check if your mtz has the correct column name. And you have to designate the one which correspond to PHIB. It depend from which programm com your input.mtz . Hope to help. Nicolas De : CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] de la part de Almudena Ponce Salvatierra [maps.fa...@gmail.com] Envoyé : jeudi 5 juin 2014 11:43 À : CCP4BB@JISCMAIL.AC.UK Objet : [ccp4bb] solvent flattening with resolve - phenix Dear all, I am trying to get a sharper map with RESOLVE in Phenix and I provide the .mtz or high resolution data as well as the mtz corresponding to experimental data. However I get the following message, and I don't really know how to fix it even though it looks quite silly. Might be that today I am not inspired, :-P Please identify the column 'PHIB' in your input data file. You can specify all columns to use in 'input_labels' like this: FP SIGFP PHIB FOM HLA HLB HLC HLD where you have to put something in each of these 8 spots (use None for missing ones). You currently have: FP SIGFP None FOM hltofom.ABCD.A hltofom.ABCD.B hltofom.ABCD.C hltofom.ABCD.D Possibilities are... FreeRflag FP SIGFP FC PHIC FC_ALL PHIC_ALL FWT PHWT DELFWT PHDELWT FOM FC_ALL_LS PHIC_ALL_LS hltofom.ABCD.A hltofom.ABCD.B hltofom.ABCD.C hltofom.ABCD.D None Any suggestions? Thank you very much in advance. Best wishes, from sunny Göttingen! Almudena -- Almudena Ponce-Salvatierra Macromolecular crystallography and Nucleic acid chemistry Max Planck Institute for Biophysical Chemistry Am Fassberg 11 37077 Göttingen Germany
Re: [ccp4bb] Xia2 / XDS issues
Dear Antony, Have you try to use the option - parralel 1, to see what hapen. Maybe it's problem with the parallelism option. If you try this option you force to use only one core and i think you decrease your need in memory. If this solution help, you probably have to check the xdsInput parameters controled by xia2 if it's possible (i have not try at this time) . Hope to help. Nicolas De : CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] de la part de Antony Oliver [antony.oli...@sussex.ac.uk] Envoyé : lundi 19 mai 2014 14:37 À : CCP4BB@JISCMAIL.AC.UK Objet : [ccp4bb] Xia2 / XDS issues Dear CCP4-ers. I am (trying) to use Xia2 (svn/Build 4599) to process some diffraction data. However, I am coming across the following issue, which I don’t seem to be able to solve... Integrating SWEEP1 Status: error [XDS] cannot allocate memory” I have checked XDS, and I have the correct (64-bit version installed: VERSION January 10, 2014 BUILT=20140307). I also have set the parameter KMP_STACKSIZE to 8m, in my .bash_profile as indicated in the XDS setup instructions. I am running OS X Mavericks (10.9.3) / XQuartz 2.7.6. I also have 16 GB of RAM installed. Any help / suggestions would be very much appreciated. Tony. - - - - - - - - - - - - - - - - - - Dr Antony W Oliver Senior Research Fellow CR-UK DNA Repair Enzymes Group Genome Damage and Stability Centre Science Park Road University of Sussex Falmer, Brighton, BN1 9RQ - - - - - - - - - - - - - - - - - - email: antony.oli...@sussex.ac.uk tel (office): +44 (0)1273 678349 tel (lab): +44 (0)1273 677512 http://www.sussex.ac.uk/lifesci/oliverlab http://tinyurl.com/aw-oliver - - - - - - - - - - - - - - - - - -
Re: [ccp4bb] data processing with XDS
Hi Almudena, you can also try mosflm as said Harry. And you can try different setting in the XDS.INP, you can try to reduce the STRONG_PIXEL (because you said spots are weak) number or/and MINIMUM_NUMBER_OF_PIXELS_IN_A_SPOT (if the spot are small). Nicolas De : CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] de la part de Almudena Ponce Salvatierra [maps.fa...@gmail.com] Envoyé : lundi 19 mai 2014 18:18 À : CCP4BB@JISCMAIL.AC.UK Objet : [ccp4bb] data processing with XDS Dear all, I am looking for some suggestions here. I have lately collected some datasets but the spots are very very weak... it is very difficult to process them. At times it looks like XDS is stalled or at times it just says that it can not interpret the lattice parameters... also while running integrate I have such a message after each range of images: !!! WARNING !!! REFINEMENT DID NOT CONVERGE LAST CORRECTION SHIFT WAS 9.1E-03 (should be 1.0E-03) I guess this is not good at all. And I wonder what to do? Is there any info that I can get out from these data at all? or rather not? I wonder if the problem is to find the spots, I have tried with HKL2000 and it can't even find enough of them for indexing. Any ideas are welcome. Best wishes, Almudena. -- Almudena Ponce-Salvatierra Macromolecular crystallography and Nucleic acid chemistry Max Planck Institute for Biophysical Chemistry Am Fassberg 11 37077 Göttingen Germany
Re: [ccp4bb] Add an atom in Coot
Hi Remi, you place the pointer (pink cube) at the place where you want put the atom. After you click on Place atom at pointer (yellow square with blue cross). HTH Nicolas De : CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] de la part de Remie Fawaz-Touma [remiefa...@gmail.com] Envoyé : mardi 18 mars 2014 16:14 À : CCP4BB@JISCMAIL.AC.UK Objet : [ccp4bb] Add an atom in Coot Can anyone give directions on how to add an atom in coot?? Thank you very much, Remie
Re: [ccp4bb] Add an atom in Coot
You can place the pointer by clicking the central-button of the PC-PS/2 mousse. And you can also turn your model with the left button for that, you place the pointer for example at one place according X-Y plan, you turn by 90 along Y axe, you replace the pointer at the right place and again 90 along Y, you replace and it should be good. Finnaly, you have to turn your model in different position and replace the pointer at the right place. De : Edward A. Berry [ber...@upstate.edu] Envoyé : mardi 18 mars 2014 17:50 À : FOOS Nicolas; CCP4BB@JISCMAIL.AC.UK Objet : Re: [ccp4bb] Add an atom in Coot Neat! How do you control the depth (front of slab to back of slab)? FOOS Nicolas wrote: Hi Remi, you place the pointer (pink cube) at the place where you want put the atom. After you click on Place atom at pointer (yellow square with blue cross). HTH Nicolas De : CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] de la part de Remie Fawaz-Touma [remiefa...@gmail.com] Envoyé : mardi 18 mars 2014 16:14 À : CCP4BB@JISCMAIL.AC.UK Objet : [ccp4bb] Add an atom in Coot Can anyone give directions on how to add an atom in coot?? Thank you very much, Remie
Re: [ccp4bb] Determining concentration of membrane protein
Hi Raji, i the kit i used for this purpose was from Pierce it's call Protein BCA RAC assay. BCA : for the colorimetric parts, en ad the RAC is for Reducing agent compatibility. This RAC is also efficient with detergent. (according my remember) But you if you use at the same time detergent and Reducing Agent, it's inefficient. http://www.piercenet.com/product/bca-protein-assay-reducing-agent-compatible If you use this one, it could be costless to buy the microplate oriented kit. (you can use also without microplate and you have more experiment in the same box) Nicolas De : CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] de la part de Ho Leung Ng [h...@hawaii.edu] Envoyé : vendredi 14 février 2014 01:58 À : CCP4BB@JISCMAIL.AC.UK Objet : Re: [ccp4bb] Determining concentration of membrane protein Hi Raji, There are also some proprietary stains such as the 660 nm (can't they think of a better product name?) stain from Pierce that are detergent compatible. I used this briefly with success when comparing against Abs 280 nm. Ho Ho Leung Ng University of Hawaii at Manoa Assistant Professor, Department of Chemistry h...@hawaii.edumailto:h...@hawaii.edu Date:Thu, 13 Feb 2014 10:06:12 -0500 From:Raji Edayathumangalam r...@brandeis.edumailto:r...@brandeis.edu Subject: Determining concentration of membrane protein Dear CC4BBers, I am trying to figure out what is the best way to determine the protein concentration of my membrane protein. My purified membrane protein is in 20mM Tris pH 7, 150mM NaCl and 0.02% DDM (CMC of DDM=0.0076%). After reading the friendly manuals and searching online, I've learned that detergents interferes with assays like Bradford but can't find good descriptions of what works best. For now, I am trying to estimate concentration from absorbance at 280nm and using molar extinction coefficients based on aromatic amino acids, but again suspect detergent interference. I would like to know what other folks working on membrane proteins are doing. Thanks very much. Raji
Re: [ccp4bb] Determining concentration of membrane protein
The problem is that if you put detergent or reducing agent in Bradford or BCA, the reaction is complet also without protein. You can't determine the color gradient because every tubes are blue or purple. De : CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] de la part de Niks [nik...@gmail.com] Envoyé : vendredi 14 février 2014 07:45 À : CCP4BB@JISCMAIL.AC.UK Objet : Re: [ccp4bb] Determining concentration of membrane protein Dear All, May be a stupid question. But if we take buffer with detergent as control (Blank), would not the difference in ODs using any of the methods used e.g. Bradford assay, gives protein concentration? Regards Nishant On Thu, Feb 13, 2014 at 9:09 PM, Roger Rowlett rrowl...@colgate.edumailto:rrowl...@colgate.edu wrote: Your basic choices for protein assays are: 1. Alkaline copper methods (e.g., Biuret and micro-biuret) 2. alkaline copper + molybdate methods (e.g., Lowry, BCA assays) 3. Hydrophobic dye methods (e.g. Bradford) 4. UV methods (e.g., A280, A230, A210, etc.) Method 1 is least sensitive to amino acid composition, but is also has highest detection limits. Thiols interfere. Method 2 is very idiosyncratic with amino acid composition, and also subject to interference by thiols. Method 3 is not usable in detergent solutions. Method 4 has many inteferences as most everything absorbs in the far UV region. If you have some special protein cofactors, metals, chromophores, etc. these can be exploited for better measurements. For ecample metalloproteins are easy to quantify by ICP-OES or TXRF if they are reasonably pure. Cheers, ___ Roger S. Rowlett Gordon Dorothy Kline Professor Department of Chemistry Colgate University 13 Oak Drive Hamilton, NY 13346 tel: (315)-228-7245 ofc: (315)-228-7395 fax: (315)-228-7935 email: rrowl...@colgate.edumailto:rrowl...@colgate.edu On 2/13/2014 10:06 AM, Raji Edayathumangalam wrote: Dear CC4BBers, I am trying to figure out what is the best way to determine the protein concentration of my membrane protein. My purified membrane protein is in 20mM Tris pH 7, 150mM NaCl and 0.02% DDM (CMC of DDM=0.0076%). After reading the friendly manuals and searching online, I've learned that detergents interferes with assays like Bradford but can't find good descriptions of what works best. For now, I am trying to estimate concentration from absorbance at 280nm and using molar extinction coefficients based on aromatic amino acids, but again suspect detergent interference. I would like to know what other folks working on membrane proteins are doing. Thanks very much. Raji -- Raji Edayathumangalam Instructor in Neurology, Harvard Medical School Research Associate, Brigham and Women's Hospital Visiting Research Scholar, Brandeis University -- The most difficult phase of life is not when No one understands you;It is when you don't understand yourself
Re: [ccp4bb] KD of dimerization, off topic
I agree with Dave, and i suggest one more method to estimate Kd, The intrinsic fluorescence of proteins thanks to the aromatic chain side. Maybe it's also possible to have an estimation with native gels if you use prot A concentration as fixed and B protein concentration as variable. I am not sure. De : CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] de la part de David Briggs [drdavidcbri...@gmail.com] Envoyé : vendredi 14 février 2014 09:13 À : CCP4BB@JISCMAIL.AC.UK Objet : Re: [ccp4bb] KD of dimerization, off topic Hi Careina, I'm not sure you can assume that the ratio of monomer and dimer will stay constant through the column - as you say, the protein is diluted during the run, the ratio will change, unless you have a super tight dimer - which clearly you do not. Also, as the mass and the molar extinction coefficient will both double in the dimer, the relationship between absorbance and concentration will be unchanged. Typically, such these sorts of questions are answered (at least me) by equilibrium analytical centrifugation. Hth, Dave On 14 Feb 2014 08:03, Careina Edgooms careinaedgo...@yahoo.commailto:careinaedgo...@yahoo.com wrote: Dear CCP4 board I have a protein that exists in equilibrium between monomer and dimer and I'm trying to calculate KD using size exclusion. The problem is that the column dilutes my sample so that if I put 20 uM on to the column, I only recover 0.5 uM in dimer fraction and 2 uM in monomer fraction. I am getting confused as to how to plot my KDs. Do I not regard my initial concentrations at all and work only with the final concentrations that come off the column? I would plot [monomer] squared vs [dimer] and I will assume that the ratio of monomer to dimer will stay constant as the protein passes through the column. (also I would calculate [dimer] using 2x monomer extinction coefficient) Does this seem a reasonable way to calculate KDs and reasonable argument? Also I am looking for good references for calculating Kds when dealing with dimerization Thanks and sorry for off topic question Careina
Re: [ccp4bb] Protein Purification Problem
hello Andy, i have different questions about your problem : Is the hetero-dimer co-expressed ? Or the two partners are express separately ? Have you controlled where are you proteins after the lysis, because if you have many of your proteins in the pellet, it's not a good sign for the folding state. If this is the case, you have different solution : change the growing conditions, change the buffer for the lysis step... If you have a low amount of protein in the first step of your purification process, you have to control at each step which one is the more limiting. Because, it's maybe not really necessary to do two affinity column. You can try different condition for the purification and increase the separation. This may allow you to skip one step and go directly to the size exclusion column. Sometimes the problems with the desalting column is the high speed of the variation. This high speed is potential source of osmotic stress for the protein. Need you really to reduce the amount of salt ? If it's absolutely required, you can try to do by dialysis, but by multiple steps. Another question, as soon as you can, control the good state of your protein ( for example with Circular dichroisme or DLS). Hope to help you. Nicolas De : CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] de la part de Anindito Sen [andysen.to...@gmail.com] Envoyé : jeudi 30 janvier 2014 14:17 À : CCP4BB@JISCMAIL.AC.UK Objet : [ccp4bb] Protein Purification Problem Dear All, This may be slightly off-the-track question but your feedback will be very much appreciated. The situation is- I obtain a very low amount of the protein of my interest (a hetro-dimer) from the construct I am using (only 8% of the total amount of protein obtained is the protein of my interest). After 2 column purifications (Ni-NTA and St) the concentration of the protein is around 0.24 mg/ml (volume- ~1.0 ml) from a litre of bacterial culture and in ~300 mM NaCl present in the elution buffer. To reduce the high amount of salt I have I use a desalting column which, further lowers the protein concentration significantly. I need atleast 1.0mg/ml of protein concentration and to the amount of ~200 microlts for further experiments. As the last resort I try to use high amount of bacterial culture (~6lts) to scale up the yield and use centricon to concentrate the protein at various stages. I am partially successful to obtain 0.56mg/ml of protein concentration and up to 50 microlts of it. Another problem is that the protein is notoriously prone to aggregation ( 1.5 mg/ml), so dialysis for ~ 2 hrs or so to reduce the high salt concentration has failed miserably. Please do send your feedback. Thanks and Best Wishes Andy Dr. Anindito Sen (Ph.D) Department of Cell Biology Anatomy Graduate School of Medicine University of Tokyo Tel fax: +81-3-5841-3339
Re: [ccp4bb] Docking model
Hi Thomas, maybe you can try to use AMBER programs. http://ambermd.org/ I think these programs allow you to use different forcefield to minimise the energy of your model. Hope to Help Nicolas De : CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] de la part de Thomas RORET [thomas.ro...@univ-lorraine.fr] Envoyé : jeudi 16 janvier 2014 15:32 À : CCP4BB@JISCMAIL.AC.UK Objet : [ccp4bb] Docking model Hi, I made one docking model of a protein complex by NMR and another one by modeling. I wanted to knowwhich software to useto minimize the energy (close contacts, H bonds, ...) best regards, Thomas. -- Thomas RORET BioMod Team Tel. 00 333 83 68 47 89 CRM2 UMR CNRS-UL 7036 Faculté des Sciences et Technologies BP 70239 54506 Vandoeuvre-les-Nancy
