[ccp4bb] "From Gene to Protein Crystal Structure" School - 21-24th January and 3-6th March 2020
As Organizing Committee, we are proud to invite you to the school entitled "From Gene to Protein Crystal Structure" (GeCrySchool2020) that will be held at ELETTRA synchrotron (Basovizza, TS) next year. Below, you will find some information about the school: - Aim of the School: providing the technical knowledge to get the X-ray crystal structure of a protein by starting from the genetic information; - Target: postDoc and PhD students with limited experience in the field of protein crystallography; - School organization: the school is divided in two sessions: * session I "From Gene to Crystal" (from 21 to 24 January 2020, max 12 participants). topics: expression, purification, and crystallization of proteins; * session II "From Crystal to Structure" (from 3 to 6 March 2020, max 15 participants) topics: diffraction experiment, obtaining the crystalline structure,validation and Protein Data Bank deposition. Each session includes short theoretical lessons, technical lessons, and lab sessions (at least 4h per day). - registration: each participant can choose to which School session apply, according to his background. Deadline: 10th December 2019 for both sessions. Please, remember that lessons will be held in Italian languages mainly. You can find more information on our website (http://www.gecryschool-edition-1.it) or you can contact us to our e-mail address gecryschool@gmailcom. We are waiting for you at the ELETTRA synchrotron!!! Dr. Caliandro Rosanna University of Bolzano, Facoltà di Scienze e Tecnologie Dr. Pozzi Cecilia University of Siena, Dip. di Biotecnologie, Chimica e Farmacia Dr. Tassone Giusy University of Siena, Dip. di Biotecnologie, Chimica e Farmacia Dr. Belviso Benny Danilo CNR, Istituto di Cristallografia -- Dr. Belviso Benny Danilo, PhD Istituto di Cristallografia (IC) Consiglio Nazionale delle Ricerche (CNR) via Amendola 122/o, 70126 Bari - Italy Tel ++39 080 5929273 Fax ++39 080 5929170 mail: danilo.belv...@ic.cnr.it web: users.ba.cnr.it/ic/crisdb00 To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1
Re: [ccp4bb] ligand mismathces in pdb deposit
Dear Bing Wang, To solve your problem, you may try to take a eme ligand directly from a PDB that contains it, e.g. 1HRC. You should download the PDB file and to copy and paste the ligand coordinates in a new file and then use this new pdb file to refine your structure. It could be a good solution. Danilo Il 2014-09-13 00:28 Wang, Bing ha scritto: Hi guys, A quick question about pdb deposition! My protein has a common ligand 'heme' which mismatches with the ligand in pdb CCD (_Chemical Component Dictionary_). However i didn't find any differences in it. Is that because of the positions of double bonds or hydrogen atoms, since my model don't have hydrogen atoms. Actually i replace the heme with exact one from CCD and run refmac which give me a new pdb file. Unfortunately, CCD still didn’t recognize the heme. Solutions? Thank you!! Bing Wang
Re: [ccp4bb] Refinement with new ligands to PDB
Dear Meisam, In the past I had similar problems, because of jligand and other cif generators can make errors when generate cif files, as it has been written before. I have solved these problems by using sketcher (ccp4) and PRODRG server (http://davapc1.bioch.dundee.ac.uk/cgi-bin/prodrg). I use the first one to create the pdb file (it is very easy to use) and the second to generate the restrains file both for refmac and for phenix.refine. I hope I've helped you. Danilo Il 2014-04-01 01:21 Meisam Nosrati ha scritto: Dear CCP4ers I have crystallized a protein with a series of ligands that are not in the PDB. I have made the pdb and the torsion libraries in the jligand and given them the new three letter codes, and then I try the refinement in the PHENIX or CCP4, but since these ligands are not in the library the refinement aborts. Even when I open the ligand files in COOT and I import the cif dictionary, still it does not recognize it, and does not let me rotate around the bonds. I need to know how to fix this problem, and how can I choose the three letter codes for my ligands that are not already chosen by other people. Thanks in advance for your help Meisam
Re: [ccp4bb] error xds
Dear Almudena, You can try to run XDS with 300 frames. At the end, you have to open the file called INTEGRATE.LP and copy the refined value of REFLECTING_RANGE= REFLECTING_RANGE_E.S.D.= BEAM_DIVERGENCE=BEAM_DIVERGENCE_E.S.D.= at the end of your XDS.INP. Now, you can re-run XDS with all your frames. However, if xds fails, it means that your data are not good. This is only a strategy to overcome the error. Danilo Il 2014-04-01 17:28 Almudena Ponce Salvatierra ha scritto: sorry, I missed this part of the error message: !!! ERROR !!! AUTOMATIC DETERMINATION OF SPOT SIZE PARAMETERS HAS FAILED. YOU MAY RESTART THIS STEP AFTER SPECIFYING VALUES IN XDS.INP FOR: REFLECTING_RANGE= REFLECTING_RANGE_E.S.D.= BEAM_DIVERGENCE= BEAM_DIVERGENCE_E.S.D.= 2014-04-01 17:27 GMT+02:00 Almudena Ponce Salvatierra maps.fa...@gmail.com: Dear CCP4 users, while I am running CORRECT within XDS, the program suddenly stops, and gives the following message: forrtl: severe (24): end-of-file during read, unit 2, file /data/almudena/160314SLS/dts2/bin1_01.tmp I actually don't know what it means. I could run it with no problems with 300 frames, but not with 600 or with 500. I get this message when I do so. Any suggestions are welcome. I'm looking forward to hearing from you. Best wishes, Almudena -- Almudena Ponce-Salvatierra Macromolecular crystallography and Nucleic acid chemistry Max Planck Institute for Biophysical Chemistry Am Fassberg 11 37077 Göttingen Germany -- Almudena Ponce-Salvatierra Macromolecular crystallography and Nucleic acid chemistry Max Planck Institute for Biophysical Chemistry Am Fassberg 11 37077 Göttingen Germany
Re: [ccp4bb] density near Histidine
Hello, it is not the first time that I see such electron densities near to His residue. However, in my structures (3N30, 3N32), I was sure that Zn, Pt or other metals were present in the crystallization condition. You have tried to refine with Zn rather than Mg that seems to be present in the crystallization condition, but is there some reason or indication that led you to follow this way? Maybe it's trivial to point out this, but Zn is much more heavier than Mg and this can affect occupancy/B-thermal factor of the refined atom. Strange values for occupancy and B-thermal factor of your refined atoms (Zn and water) can be a good indication about the correctness of your refining procedure. The distance between N of His and water or metal are compatible with the range expected for metals coordinated to Neps of Ndelta of His residue? Have you try to refine with a more complex ligand, such as metal coordinated to water molecules? Danilo Il 2014-03-08 06:28 SD Y ha scritto: Hi, During refinement, I see a strong density next to His and I have tried metal ions and water but it seems to be different. Known components of crystallization are Tris, NaCl, BME, MgOAc, PEG3350. Its only seen in one chain of AU. Are there any suggestions? Thanks in advance SDY
Re: [ccp4bb] program for protein - RNA docking
Dear Rongjin, I think that HADDOCK (http://www.nmr.chem.uu.nl/haddock/) is the best solution for your requests. Danilo On Tue, 7 Jan 2014 16:36:43 -0500, rjguan rjg...@gmail.com wrote: Dear All, We have a RNA binding protein with structure known. We want to dock a RNA molecule to the protein structure, and we roughly know where RNA binds to the protein. Which program does a better job (generally speaking) in the following cases: 1. RNA is rigid; 2. RNA is allowed to change conformation. Please share your experience. Thanks Rongjin Guan
Re: [ccp4bb] Unidentified blob
Dear Afshan, Maybe what I suggest you have already tried: have you tried to fit a citrate molecule? The blob seems to be branched, thus compatible with such organic molecule. Danilo On Thu, 12 Dec 2013 03:34:07 -0800, Afshan Begum afshan...@yahoo.com wrote: Dear Experts, I collected a data set (1.12A) of one of my target proteins, and built a model by molecular replacement. I then exam the model by Coot and found one unidentified blob. I am unable to to identified a blob which appeared on the surface of the molecule its close to residues 115 SER the protein purified in 10 mM Na-phosphate buffer, 100mM KCl and crystallized in 1.6 M sodium citrate, 3.5% _v/v _glycerol I have attached the picture of the blob. I would be thankful for your kind suggestions. Best Regards AFSHAN
[ccp4bb] Ligandfit - problem with ligand_start
Dear all, I am working on a membrane protein covalently bound to a molecular antanna: it is known that this molecule binds to lysine residue but I do not know how many and which lysine residues it binds. 20 diffraction datasets of this protein-ligand complex have been obtained and now, I would quickly localize the ligand using the Fo-Fc map of each data set and using the information on the covalent bound protein-ligand. Ligandfit tool (PHENIX) seems to be indicated to do this; to use the information on the covalent bound, I am using the ligand_start keyword with a pdb containing a ghost atom (however present in ligand model) perfectly superposed to the lysine atom that should bind the ligand. The command used is: phenix.ligandfit data=prot.mtz model=prot.pdb ligand=lig.pdb ligand_start=lig_start.pdb input_labels=FOFCWT PHFOFCWT \ refine_ligand=True \ nproc=32 \ cif_def_file_list=lig.cif description: - prot.mtz (data) - prot.pdb (protein without ligand) - lig.pdb(ligand containing ghost atom) - lig_start.pdb (ghost atom superpose to NZ of a lysine) - lig.cif(restrain of lig.pdb) Strangely, no ligand is found at the end of the process even reducing ligand_cc_min to 0.01. I have run the same command by using an other protein where an other ligand has been correctly fitted but, also in this case, no ligand has been detected. Conversely, without the use of ligand_start, ligandfit properly localizes the ligand. I'm doing some mistake in the use of ligand_start? Do you know an other tool to perform a ligand fitting in these conditions? Thanks for your answers. Danilo
Re: [ccp4bb] crystals with large solvent content -dehydratation
Dear Andre, you could try with the protocol described in the following paper Acta Crystallogr D Biol Crystallogr. 2013 69,920-3. Using high-throughput in situ plate screening to evaluate the effect of dehydration on protein crystals. Douangamath A, Aller P, Lukacik P, Sanchez-Weatherby J, Moraes I, Brandao-Neto J. It gives very good results with membrane proteins where the water content is high. Danilo On Tue, 29 Oct 2013 08:18:21 -0700, Andre Godoy andre_go...@yahoo.com.br wrote: Dear all I'm trying to solve a beautiful large crystal that, unfortunately, doesn't go further than 5 A resolution. I believe that in this case, the lack of resolution is due the high solvent content (about 66%). Therefore, my next strategy should be the dehydratation. Yet, I never (sucessfully) did that. I read different approachs, were people equilibrate crystals in dehydratation solution for days, or do more than 20 steps, or add solvents. Since i never had sucess in my trials, I was thinking that someone can suggest a protocol (should I remove all salt?, should I keep the additive concentration?, how much precipitant should I add? how many steps?). crystal condition: 23% PEG 3350, 0.2M NaCl, 0.1M Tris pH 8.5, 3% galactose (orthorhombic crystals, with about 0.6 x 0.6 mm) all the best, Andre Godoy
Re: [ccp4bb] changes in small sections of secondary structure
Dear Mahesh, In addition to RMSD plots, you could try to quantitatively analyse the protein residue flexibility in order to detect backbone conformational transitions by means of the method proposed in: Local Fluctuations and Conformational Transitions in Proteins Rocco Caliandro, Giulia Rossetti, and Paolo Carloni J. Chem. Theory Comput. 2012, 8, 4775−4785 By starting from a set of several models of the same protein, the method allows to detect the hinge points of the protein and the residues where transitions between two different conformational states occur. However, by inspecting your picture, also I am not convinced that they are alterations of the protein, but rather inconsistency in assignment of secondary structure. Danilo
[ccp4bb] R-factor for radiation damage (R_d)
Dear all, I am using xds (with graphical interface xdsgui) to process several diffraction data of a membrane protein that I have crystallized. At the end, I run XDSSTAT in order to check the statistic parameters of the process and my attention is captured by the R_d plot: R_d drops during the firsts 10-15 frames and then reaches a maximum (20-30 frames). Then, its value remains quite stable (I suppose due to the radiation damage correction performed by xds). The trend seems the same that is shown in ActaCryst. (2006). D62, 96–101 where Some aspects of quantitative analysis and correction of radiation damage Fig.1(a)(b) where R_d (decay R-factor) was introduced. The question is: By considering that radiation damage increases during the data collection due to the progressive dose of radiation to which the crystal is subjected, why does the R_d drop during the first period of data collection? Thank you in advance for your answers. Danilo
Re: [ccp4bb] Staining Crystals with comassie
Dear All, izit dye is a solution containing methylene blue that you could prepare in your lab. I usually prepare a solution of 0.05%w/v of dye in water and then I add a volume of dye solution equals to 10% of the volume of the drop containing the crystal to test. I prefer to add the dye solution in small portions (if the volume permits) every 2-3h in order to limit the shock due to the new solution on the crystal. You should remember that this test is not definitive: the dye is a cationic dye, that needs of anion counter part to bind the protein. Therefore, the dye is not able to colour all protein crystals: in addition, colouration is affect by pH of the crystallization condition, since low pH could increase the positive charge on the protein reducing its ability to bind the dye. You could try also glutaraldehyde as alternative. In order to perform this test, you should put the crystal into a low ionic strength buffered solution containing up to 2% glutaraldehyde. In this condition, formation of Schiff bases with the lysines and N-term residues occurs and the crystal become a yellow gel, while salt crystals dissolve and should not be coloured. To perform comassie crystal staining you should prepare a solution of 1% comassie in 40% MeOH and 10% Glacial Acetic Acid and add this solution as in methylene blue test. However, I rarely use this test because the use of MeOH and Glacial Acetic Acid causes the crystal dissolution. Only a final tip: obviously these tests enable you to distinguish between protein and salt, however they do not differentiate between protein in the crystal and protein in solution. For these reason, in some cases could be difficult to see the crystal colouration due to the low contrast with the colouration of the solution. Hence, I prefer to put the crystals to test in a new solution with the same formulation of the drop where the crystals have grown but without protein and perform here the dye test that I have chosen. In this way, you can easily see the colouration of the crystal without background effect. I hope I've helped you. Danilo On Tue, 15 Oct 2013 13:29:20 +0530, Swastik Phulera swastik.phul...@gmail.com wrote: Dear All, I am looking for a method to quickly differentiate between salt and protein crystals. I have been told thats its a popular alternative to the commercially available izit dye. I would appreciate if some one would share their comassie crystal staining protocol. Swastik
Re: [ccp4bb] Staining Crystals with comassie
Dear Swastik, izit dye is a solution containing methylene blue that you could prepare in your lab. I usually prepare a solution of 0.05%w/v of dye in water and then I add a volume of dye solution equals to 10% of the volume of the drop containing the crystal to test. I prefer to add the dye solution in small portions (if the volume permits) every 2-3h in order to limit the shock due to the new solution on the crystal. You should remember that this test is not definitive: the dye is a cationic dye, that needs of anion counter part to bind the protein. Therefore, the dye is not able to colour all protein crystals: in addition, colouration is affect by pH of the crystallization condition, since low pH could increase the positive charge on the protein reducing its ability to bind the dye. You could try also glutaraldehyde as alternative. In order to perform this test, you should put the crystal into a low ionic strength buffered solution containing up to 2% glutaraldehyde. In this condition, formation of Schiff bases with the lysines and N-term residues occurs and the crystal become a yellow gel, while salt crystals dissolve and should not be coloured. To perform comassie crystal staining you should prepare a solution of 1% comassie in 40% MeOH and 10% Glacial Acetic Acid and add this solution as in methylene blue test. However, I rarely use this test because the use of MeOH and Glacial Acetic Acid causes the crystal dissolution. Only a final tip: obviously these tests enable you to distinguish between protein and salt, however they do not differentiate between protein in the crystal and protein in solution. For these reason, in some cases could be difficult to see the crystal colouration due to the low contrast with the colouration of the solution. Hence, I prefer to put the crystals to test in a new solution with the same formulation of the drop where the crystals have grown but without protein and perform here the dye test that I have chosen. In this way, you can easily see the colouration of the crystal without background effect. I hope I've helped you. Danilo On Tue, 15 Oct 2013 13:29:20 +0530, Swastik Phulera swastik.phul...@gmail.com wrote: Dear All, I am looking for a method to quickly differentiate between salt and protein crystals. I have been told thats its a popular alternative to the commercially available izit dye. I would appreciate if some one would share their comassie crystal staining protocol. Swastik
[ccp4bb] Fwd: [ccp4bb] move ligand to fit the density in coot
Dear Wei, It seems that coot detects some discrepancies between cif file and your ligand. It is not uncommon that the program used to generate your ligand pdb produces atom definitions that are not correctly read by programs used to generate restrains, such as PRODRG. In order to overcome the problem, you could try to use the pdb file of your ligand created by PRODRG to fit the density that you believe related to your ligand. I hope this information will will be of help for you. Danilo Original Message Subject: [ccp4bb] move ligand to fit the density in coot Date: Mon, 7 Oct 2013 19:43:07 -0400 From: Wei Shi wei.shi...@gmail.com To: CCP4BB@JISCMAIL.AC.UK Hi all, I was solving a protein-ligand complex structure and got a molecular replacement solution with the protein as the search model and now trying to fit the ligand to the electron density in coot. It seems that the ligand needs to change conformation to fit the density well. So, I created the dictionary using PRODRG2 (the following link), and downloaded CIF topology (general) from the results file page. Is this the CIF library which I should include in order to move the ligand to fit the density in coot? http://www.ucl.ac.uk/~rmhasek/dictionary.html [1] And in coot, I loaded the pdb file, mtz file and the click import CIF dictionary to load the DRGMAC.LIB file, but when I was trying to move the ligand, coot gave the error message as follows: No restraints found! No existent or minimal description of restrained residues. Are you sure that you read a non-minimal mmCIF dictionary for this monomer. Are you sure the PDB reside name matches the dictionary residue name? If not, try File - Import CIF Dictionary Alternatively, did you check that the atom names of the PDB file match those of the restraints. I am wondering whether any of you have any suggestions about how to the fix this. Thank you so much! Best, Wei Links: -- [1] http://www.ucl.ac.uk/%7Ermhasek/dictionary.html
[ccp4bb] CC calculation by OVERLAPMAP and PHENIX.GET_CC_MTZ_PDB
Hello everybody, I am working with a protein that should be covalently bound to an organic ligand and I would calculate the correlation coefficient of this ligand both against 2Fo-Fc and Fo-Fc map arising from refining (map calculated in the presence of ligand). To do this, I am using OVERLAPMAP (in CCP4, graphic interface) by setting: List a correlation by residue First map calculated from input MTZ F1 FOFCWT or 2FOFCWT PHI PHFOFCWT or PH2FOFCWT sigma unassigned weightunassigned Second mapcalculated from input coordinates (this input contains only the ligand molecule) In addition, I am using PHENIX.GET_CC_MTZ_PDB (in phenix, command line) to compare the previous results. In order to do the same kind of calculation performed by OVERLAPMAP, I run: phenix.get_cc_mtz_pdb *.pdb *.mtz \ labin=FP=FOFCWT PHIB=PHFOFCWT \ atom_selection=chain a phenix.get_cc_mtz_pdb *.pdb *.mtz \ labin=FP=2FOFCWT PHIB=PH2FOFCWT \ atom_selection=chain a (chain a contains only the ligand) My questions are: 1) OVERLAPMAP: Are the setting correct to perform CC calculations of a model against Fo-Fc and 2Fo-Fc maps? 2) OVERLAPMAP: By considering that is not possible to define main-chain and side-chain for the ligand molecule, is Total correlation coefficient, at the end of the output, the proper CC value of the whole ligand? 3) PHENIX.GET_CC_MTZ_PDB: Is CC value, at the end of the output, the equivalent of Total correlation coefficient arising from OVERLAPMAP? 3) Are the two procedures (ccp4 and phenix) similar or they perform different calculations? Thank for your suggestions.
[ccp4bb] restrain range for ligand
Hi! Does anybody know which is the range is used by REFMAC to vary bond distances, angles, and torsions of a ligand molecule during a refinement process? Is it possible to control and choose this range? In which way? Where are these information in cif dictionary obtained by sketcher in CCP4? thank you!! Danilo
[ccp4bb] calculation of cavities within crystal protein
Dear all, I am Dr. Danilo Belviso and I am working on a platinum-based inhibitor for matrix-metallo proteasis. I have obtained the crystal structure of the adduct Pt/protein and, for me, would be very interesting to know the cavities of the protein within the crystal, namely by considering symmetry-related protein molecules around the asymmetric unit. Do you know a software (or server) that carries out this calculation? Best regards Dr. Danilo Belviso
