Re: [ccp4bb] Crystallizing a tough target
Hi Kavya, In addition to other excellent suggestions, I would recommend trying reductive lysine methylation, which can make the protein more hydrophobic. PMID 17098187. Best wishes, Tomas On Mon, Feb 5, 2024 at 10:27 AM kavyashreem wrote: > > Dear All, > > Has anyone worked on a protein which is highly soluble even at 80mg/ml? > > We have one such candidate, which does not precipitate even at 80mg/ml > instead forms phase separated globules in crystallization plate, which > eventually hardens over a period of 1 to 1.5 months (which is florescent > under UV microscope.) > > We tried screening at different pH, but failed to get any hits. > > Since we got few conditions in which the phase separated globules solidified, > we focused on them and expanded with 120mg/ml protein, still there were not > visible precipitates except for the phase separation. This has been a > challenging target so far. We have tried with different constructs, which > unfortunately are not soluble! > > Does POMs help in such cases? Or do you have any other suggestion. > > Thank you > > Regards > > Kavya > > > > > > CONFIDENTIAL: This email and any files transmitted with it are confidential > and intended solely for the use of the individual or group to whom they are > addressed. If you have received this email in error, please notify the sender > immediately and delete this e-mail from your system. It is NOT AUTHORISED to > copy, forward, or in any way reveal the contents of this message to anyone. > > > > To unsubscribe from the CCP4BB list, click the following link: > https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1 To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1 This message was issued to members of www.jiscmail.ac.uk/CCP4BB, a mailing list hosted by www.jiscmail.ac.uk, terms & conditions are available at https://www.jiscmail.ac.uk/policyandsecurity/
Re: [ccp4bb] RMSD per residue graph for multiple aligned PDB files
Dear All, Thank you very much to everyone who replied, both on and off the mailing list (Andy Karplus and Ensemblator from his team; Joseph Ellaway and his scripts available at https://github.com/Joseph-Ellaway/per-residue-distance). As I was analysing conformations of the same protein from the MD simulation and had corresponding trajectories from GROMACS/OpenMM, I transitioned away from PyMOL and used the GROMACS 'gmx rmsf' command. Best wishes, Tomas On Fri, Jan 5, 2024 at 10:49 AM Tomas Malinauskas wrote: > > Dear All, > > I apologize for asking a somewhat off-topic question. > > I have multiple aligned PDB files loaded in PyMOL, each representing > different conformations of the same protein. I'm interested in > creating a graph displaying RMSD per residue, similar to those shown > at > https://www.ks.uiuc.edu/Training/Tutorials/science/aars/aars_html.bak/node22.html > or > https://www.compchems.com/how-to-compute-the-rmsf-using-gromacs/#the-gmx-rmsf-command. > > I'm wondering if anyone has a script available that can calculate RMSD > per residue and write the data to a text file for graph generation. If > so, would they be able to share it with everyone? > > Thank you for your help. > > Best wishes, > Tomas To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1 This message was issued to members of www.jiscmail.ac.uk/CCP4BB, a mailing list hosted by www.jiscmail.ac.uk, terms & conditions are available at https://www.jiscmail.ac.uk/policyandsecurity/
[ccp4bb] RMSD per residue graph for multiple aligned PDB files
Dear All, I apologize for asking a somewhat off-topic question. I have multiple aligned PDB files loaded in PyMOL, each representing different conformations of the same protein. I'm interested in creating a graph displaying RMSD per residue, similar to those shown at https://www.ks.uiuc.edu/Training/Tutorials/science/aars/aars_html.bak/node22.html or https://www.compchems.com/how-to-compute-the-rmsf-using-gromacs/#the-gmx-rmsf-command. I'm wondering if anyone has a script available that can calculate RMSD per residue and write the data to a text file for graph generation. If so, would they be able to share it with everyone? Thank you for your help. Best wishes, Tomas To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1 This message was issued to members of www.jiscmail.ac.uk/CCP4BB, a mailing list hosted by www.jiscmail.ac.uk, terms & conditions are available at https://www.jiscmail.ac.uk/policyandsecurity/
Re: [ccp4bb] eLBOW aromatic restraints
Hi Henry, You could try keeping selected atoms in the plane by manually editing restraints in the CIF. Set _chem_comp_plane_atom.dist_esd to something low. E.g. loop_ _chem_comp_plane_atom.comp_id _chem_comp_plane_atom.plane_id _chem_comp_plane_atom.atom_id _chem_comp_plane_atom.dist_esd NAG-b-D plan1 N20.010 NAG-b-D plan1 C70.010 NAG-b-D plan1 O70.010 NAG-b-D plan1 C80.010 NAG-b-D plan2 C10.010 NAG-b-D plan2 C20.010 NAG-b-D plan2 C40.010 NAG-b-D plan2 C50.010 I would also check https://mmcif.wwpdb.org/dictionaries/mmcif_pdbx_v40.dic/Items/_chem_comp_plane_atom.dist_esd.html and try Grade2 https://grade.globalphasing.org/cgi-bin/grade2_server.cgi Best wishes, Tomas On Mon, Jun 12, 2023 at 11:06 AM Henry Padleywrote: > > Hi all, > > > I'm trying to generate a CIF file of an iridium piano stool complex ligand > (Cp*Ir-Phenanthroline). > > Can Phenix eLBOW generate correctly restrained aromatic ring systems which > stay flat in refinement? > > The phenanthroline bonds in the output CIF are listed as "aromatic" in the > REEL text table but they are not recognised as such in refinement. I've tried > manually editing the CIF via this text table to produce the alternating > "double/single bond" structure but it still puckers slightly in refinement. > > My eLBOW input is a PDB file of the ligand (I have also tried SDF input). I > have tried Automatic and eLBOW AM1 QM settings. Might a different Geometry > optimisation software selectable within eLBOW generate the restrained > phenanthroline? > > Any advice greatly appreciated. > > > Henry Padley, MSci > School of Pharmacy > University of Nottingham > > > This message and any attachment are intended solely for the addressee > and may contain confidential information. If you have received this > message in error, please contact the sender and delete the email and > attachment. > > Any views or opinions expressed by the author of this email do not > necessarily reflect the views of the University of Nottingham. Email > communications with the University of Nottingham may be monitored > where permitted by law. > > > > > > > To unsubscribe from the CCP4BB list, click the following link: > https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1 To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1 This message was issued to members of www.jiscmail.ac.uk/CCP4BB, a mailing list hosted by www.jiscmail.ac.uk, terms & conditions are available at https://www.jiscmail.ac.uk/policyandsecurity/
[ccp4bb] Adding hydrogens to a specific residue using Coot
Dear All, Is it possible to add hydrogens to a specific residue using Coot? Something like Calculate -> Scripting -> Python -> coot_reduce(0) but targeting one residue only. I thank you for your help. Best wishes, Tomas To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1 This message was issued to members of www.jiscmail.ac.uk/CCP4BB, a mailing list hosted by www.jiscmail.ac.uk, terms & conditions are available at https://www.jiscmail.ac.uk/policyandsecurity/
Re: [ccp4bb] Eukaryotic protein expression
Hi Dhiraj, pHLsec vector for transient and its variants for lentivirus-based expression (PMIDs 17001101 and 30455477, respectively). Best wishes, Tomas On Thu, Jun 24, 2021 at 9:42 PM Srivastava, Dhiraj wrote: > > Hi all > I am trying to express my protein in HEK293 cells for crystallization > purpose but getting poor expression. I am using cmv promoter. Which > promoter/vector people use to get good expression in eukaryotic expression > system? > > Thank you > > Dhiraj > > > > To unsubscribe from the CCP4BB list, click the following link: > https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1 To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1 This message was issued to members of www.jiscmail.ac.uk/CCP4BB, a mailing list hosted by www.jiscmail.ac.uk, terms & conditions are available at https://www.jiscmail.ac.uk/policyandsecurity/
Re: [ccp4bb] coot & other graphics programs draw too many bonds
Hi Fred, At least Schrodinder's PyMOL 2.3.2 can open Vina's PDBQR files (with multiple docked conformations) without any problems or additional CIFs in my experience. Different conformations of ligands are loaded as different states. It if is an older version you could try to convert Vina's PDBQR to PDB: 1. Split PDBQT file (vina_split comes together with Vina): vina_split --input file.pdbqt 2. Convert PDBQT to PDB: cut -c-66 file_1.pdbqt > file_1.pdb grep 'ATOM' file_1.pdb > file_1_lines_with_atoms_only.pdb Best wishes, Tomas On Thu, Apr 1, 2021 at 2:02 PM Fred Vellieux wrote: > > Hello there, > > After running autodock vina on certain small molecules, the graphics > software I am using (e.g. Pymol, Coot) draws far too many bonds on the > docked small molecule. See enclosed screen capture. > > Is there any way to prevent this from happening? This isn't very > satisfactory of you wish to produce figures for a presentation or for > publication. > > Ta, > > Fred. > > -- > MedChem, 1st F. Medicine, Charles University > BIOCEV, Vestec, Czech Republic > > > > > To unsubscribe from the CCP4BB list, click the following link: > https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1 > > This message was issued to members of www.jiscmail.ac.uk/CCP4BB, a mailing > list hosted by www.jiscmail.ac.uk, terms & conditions are available at > https://www.jiscmail.ac.uk/policyandsecurity/ To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1 This message was issued to members of www.jiscmail.ac.uk/CCP4BB, a mailing list hosted by www.jiscmail.ac.uk, terms & conditions are available at https://www.jiscmail.ac.uk/policyandsecurity/
Re: [ccp4bb] About a Protein Crystal Optimization
Hi, In addition to all great suggestions, I would try reductive lysine methylation (PMID 17098187). Good luck! Tomas On Thu, Jan 21, 2021 at 6:46 PM Jon Cooper < 488a26d62010-dmarc-requ...@jiscmail.ac.uk> wrote: > > Hello, I always recommend limited chymotrypsinolysis. It's not a miracle cure but in a proportion of cases (~1/4?) it does work. > Cheers, Jon.C. > > > Sent from ProtonMail mobile > > > > Original Message > On 21 Jan 2021, 08:26, 商国辉 < sz20183020...@cau.edu.cn> wrote: > > > Hi,Everyone! > > Glad for me to ask us a question.Recently, I have optimized a solube protein crystal, and the crystal shape is like sea urchin at first. After I try different methods-Different Protein and Kit proportion 1:1 1.2:0.8 1.4:0.6,etc; try to add different additives and detergents ; different PEGs and salts; Seeding; Try different pH and PEG Concentrations orthogonally;change primary protein sequence and so on. And the crystal is plates, but still not a mono three-dimentional crystal, so I cannot solve the structure finally by the X-ray data. > >It is strange that the protein can only crystal in low salt concentration 60 mM-100 mM NaCl SEC Buffer(Superdex 200 buffer), as the salt concentration rises to 200 mM-1000 mM, the protein does not crystal at all, and the protein with Kit mixture is clear.So I use Superdex 200 to test the protein assembly using different salt concentration 60 mM/200 mM/1000 mM, the peak is moving to right as the salt concentration improve.So maybe the protein dimer form crystal(in 60 mM salt concentration). > > Here, I really need any available ideas for my protein crystal optimization, and I will spare no effort to solve the problems. Once I make it, I promise I would share my experiments for us to help anyone in the same situation.Figures and SD200 Peaks are below. > > Greatly thanks! > > Best Wishes. > > Sincerely To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1 This message was issued to members of www.jiscmail.ac.uk/CCP4BB, a mailing list hosted by www.jiscmail.ac.uk, terms & conditions are available at https://www.jiscmail.ac.uk/policyandsecurity/
Re: [ccp4bb] dimeric tag to induce the homodimerization of protein
Hi Dhiraj, You could try a C-terminal Fc-tag if it is an extracellular protein, e.g. see pHL-FcHis vector described in the supplement of PMID 17001101. Best wishes, Tomas On Tue, Sep 22, 2020 at 6:08 PM Srivastava, Dhiraj wrote: > > Hi > I want to make my protein dimeric to increase its affinity for its > interaction partner which is a dimer. does anyone know a suitable tag/fusion > protein which can be used as C terminal fusion for this purpose? I can not > use any of the leucine zipper as I am looking for the distance between the c > terminus to be around 30-40 A. > > > Thank you > Dhiraj > > > > To unsubscribe from the CCP4BB list, click the following link: > https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1 To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1 This message was issued to members of www.jiscmail.ac.uk/CCP4BB, a mailing list hosted by www.jiscmail.ac.uk, terms & conditions are available at https://www.jiscmail.ac.uk/policyandsecurity/
Re: [ccp4bb] Ligand building
Hi Hari, I typically draw formulae using ChemDraw online, export SMILES and use them to get PDBs/CIFs from Grade web server (Global Phasing). Both ChemDraw and Grade web servers are free to use. Hope that helps, Tomas On Sat, Jun 13, 2020 at 6:01 PM Hari shankar <465d10db143e-dmarc-requ...@jiscmail.ac.uk> wrote: > > Hi all, > > I want to build a ligand that does not exist in reality (or be able to modify > an existing ligand as per choice). > 1. JLigand in the ccp4 suite seems to work only on java and gives me an > “configuration problem” as an error message on my Windows. I am new to this > and unaware of how to solve this issue. Could I get some suggestions on how > to start this? > > 2. Alternatively, I was trying to use eLBOW from phenix but it seems to only > build ligands but unable to delete atoms. Is there another program or option > for me to delete sections of the ligand and geometrical optimise them in > phenix? > > Thanks > Hari > > > > 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 This message was issued to members of www.jiscmail.ac.uk/CCP4BB, a mailing list hosted by www.jiscmail.ac.uk, terms & conditions are available at https://www.jiscmail.ac.uk/policyandsecurity/
Re: [ccp4bb] incorporating disulfide linkage
Hi Kahkashan, Indeed, homodimers formed by engineered disulfides could crystallize readily in novel crystal forms: Banatao et al., "An approach to crystallizing proteins by synthetic symmetrization”, 2006, PNAS, PMID 17050682. Best wishes, Tomas On Mon, Mar 16, 2020 at 7:58 AM Firdous Tarique wrote: > > Hi > > I have a weird question. Is it possible to incorporate or mutate amino acid > residues at the interface of two interacting surfaces in a multi subunit > protein complexes hoping it to form a disulfide bond making the over all > complex more stable for downstream structural studies ? > > Is there any sort of literature available where creating these types of > modification or kind of protein engineering helped in making a more stable > complex, eventually helping in further structural studies by x-ray or cryoem ? > > Best > > kahkashan > > > > 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
Re: [ccp4bb] protein expression in human cells
Hi Gloria, two key papers describing expression (transient and lentivirus-based) of proteins in HEK293 cells we use to make milligrams of proteins for crystallization and cryo-EM: Acta Crystallogr D Biol Crystallogr. 2006 Oct;62(Pt 10):1243-50. Epub 2006 Sep 19. A time- and cost-efficient system for high-level protein production in mammalian cells. Aricescu AR, Lu W, Jones EY. PMID: 17001101 Nat Protoc. 2018 Dec;13(12):2991-3017. doi: 10.1038/s41596-018-0075-9. Lentiviral transduction of mammalian cells for fast, scalable and high-level production of soluble and membrane proteins. Elegheert J, Behiels E, Bishop B, Scott S, Woolley RE, Griffiths SC, Byrne EFX, Chang VT, Stuart DI, Jones EY, Siebold C, Aricescu AR. PMID: 30455477 Hope that helps, Tomas Dr. Tomas Malinauskas University of Oxford Wellcome Centre for Human Genetics Division of Structural Biology Roosevelt Drive Oxford OX3 7BN United Kingdom to...@strubi.ox.ac.uk tomas.malinaus...@gmail.com On Fri, Jan 24, 2020 at 11:50 PM Gloria Borgstahl wrote: > > Hello CCP4-ers, > I was wondering what people have found to be the best human cell line > expression system for making a large quantity of purified recombinant protein. > Any information and protocols would be greatly appreciated. > Happy 2020, Gloria > > > > 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
Re: [ccp4bb] Problematic on-line reference
Dear Gerard, you can find a copy of the page here: https://web.archive.org/web/20110624033607/http://ccp4wiki.org/~ccp4wiki/wiki/index.php?title=Scaling_experimental_intensities_with_Scala Hope that helps, Tomas On Mon, Apr 15, 2019 at 4:19 PM Gerard Bricogne wrote: > > Dear all, > > In a recently completed piece of writing I used as a reference > the following on-line material: > > http://ccp4wiki.org/~ccp4wiki/wiki/index.php?title=Scaling_experimental_intensities_with_Scala#Corner_corrections > > that I had found in 2017. > > Now that I have to quote, at the proof-checking stage, an access > date for this reference, I find that it is no longer available. > > Would anyone know of the whereabouts of this material? Or of > other, more recent and/or more permanent material on the same subject > (i.e. corner effects in 3x3 CCD detectors)? > > Any help will be greatly appreciated. > > > With best wishes, > > Gerard. > > -- > === > * * > * Gerard Bricogne g...@globalphasing.com * > * * > * Global Phasing Ltd. * > * Sheraton House, Castle Park Tel: +44-(0)1223-353033 * > * Cambridge CB3 0AX, UK Fax: +44-(0)1223-366889 * > * * > === > > > > 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
Re: [ccp4bb] Non-specific disulfides in a secreted protein
Dear Zhijie et al, thanks a lot for your thoughtful suggestions. a) we do get a fraction with a "proper" monomer but it would be nice to minimise losses because of non-specific disulfides; b) yes, it has 1 Asn-linked glycan which gets nicely trimmed upon treatment with Endo F1; d) yes, we do get some monomer on SEC without DTT. We thought ~0.5 mM DTT (which degraded relatively fast anyway) might be OK to reduce some non-specific disulfides but not sufficiently high to destroy the whole folded domain, we used it out of desperation/curiosity; e) we will try longer tags and adding more residues at termini. Best wishes, Tomas On Fri, Dec 14, 2018 at 6:57 PM Zhijie Li wrote: > > Hi Tomas, > Some thoughts: > a) I guess the thermodynamic drive for all part of this small ectodomain to > fold into a single lowest energy conformation is not very strong. The cells > can’t know that the little artificial domain is supposed to be a monomer when > the oligomers are also sufficiently hydrophilic. We also occasionally see > aggregates coming out of human cell lines. Sometimes barely anything good. > > (One wild thought: the disulfide shuffling system in eukaryotic cells have > something to do with the N-glycans. Does the natural form of this protein > have N-glycans, while the ecotodomain is somehow deprived of that?) > > b) Is there absolutely no monomer on the Western? If this is the case, since > the bacterial preps can be refolded, can you try incubating the protein with > Cysteine or GSH or even add PDI or DsbC to try to increase the population of > properly folded species? > > c) If you have some monomers or can manage to generate some by disulfide > shuffling: for a small domain with such high disulfide content a further > reverse-phase C18 HPLC step may give you the properly folded species without > irreversibly denaturing them. Or maybe a Superdex75 gel filtration or a > silica SEC on HPLC, without reducing reagents, see below. > > d) Have you ever run the gel filtration without DTT? When putting the DTT, > because of its strong disulfide cleaving power, you might be sending the > misfoled junk to the monomer peak. They look like monomers when DTT is > present but they are misfolded still. If you have a small fraction of > monomers that had passed the cell’s QC, they are more likely to be correctly > folded therefore active. Then the right thing to do I think is to try to > separate the monomers from the oligomers, rather than forcing everything into > “monomers”. > > e) Try a larger tag? Add more natural sequence back to the domain? Try > periplasmic expression in E coli? The NEB pMal-p vector is my favourite for > disulfide forming proteins, when not using mammalian cells. > > Zhijie > > > On Dec 14, 2018, at 11:57 AM, Tomas Malinauskas > > wrote: > > > > Dear All, > > > > we are purifying a small secreted protein from conditioned media and > > have a rather unusual problem. > > > > It is a small ectodomain (~11 kDa, pI ~6, His-tagged) of the type 1 > > transmembrane receptor, crystal structures are known (of the protein > > that was produced in E.coli and refolded; we are secreting the same > > protein using mammalian cells) so we can design reasonable constructs. > > The protein is expressed and secreted by transiently transfected > > HEK293T cells that work very well for other ectodomains and > > extracellular proteins in our hands (PMID 17001101). The target > > protein has 10 cysteines that form 5 disulfides in the crystal > > structure (of E.coli-expressed and refolded protein), there should be > > no free cysteines and no non-specific disulfides. Unfortunately, once > > the protein is secreted, it forms non-specific dimers and higher-order > > oligomers in the media (standard DMEM/2% FBS) before purification > > (confirmed by Western blotting under non-reducing conditions). Using > > 0.5 mM DTT during SEC gives a nice monomeric peak (however, the > > protein suffers as suggested by weaker interactions with its binding > > partners). We don't understand how a secreted protein (which passes > > trafficking quality control in the cell) with a known disulfide > > pattern forms non-specific disulfide linked oligomers in the > > extracellular media. We tried expressing it at 37 C and 30 C, and have > > sequenced our constructs (plasmids) multiple times. > > > > If anyone has seen this kind of problem and successfully solved it > > (purified homogeneous crystallisation quality protein), please let us > > know if possible. I thank you for your help. > > > > Best wishes, > > Tomas > > > > > > Dr. Tomas Malinauskas > > University
Re: [ccp4bb] Non-specific disulfides in a secreted protein
Thanks! We will try a DNA dilution trick. Yes, a fraction (~30-70%) is a monomer we want. On Fri, Dec 14, 2018 at 6:12 PM wrote: > > Hi Tomas, > > I have seen something similar in the past, see PMID: 26998761. The problem > could be alleviated by tuning down expression levels, through plasmid > dilution. I guess that cellular QC mechanisms are overwhelmed if you push too > hard for overexpression. I'd test a range of 1:10 to 1:1000, or higher, > dilutions in empty (pLEXm or any irrelevant plasmid) to keep total DNA amounts > constant. Is there no hint of monomer whatsoever in your prep? > > Best wishes, > > Radu > > -- > Radu Aricescu > MRC Laboratory of Molecular Biology > Francis Crick Avenue > Cambridge Biomedical Campus > Cambridge CB2 0QH, U.K. > tel: +44-(0)1223-267049 > fax: +44-(0)1223-268305 > www: http://www2.mrc-lmb.cam.ac.uk/group-leaders/a-to-g/radu-aricescu > > > Dear All, > > > > we are purifying a small secreted protein from conditioned media and > > have a rather unusual problem. > > > > It is a small ectodomain (~11 kDa, pI ~6, His-tagged) of the type 1 > > transmembrane receptor, crystal structures are known (of the protein > > that was produced in E.coli and refolded; we are secreting the same > > protein using mammalian cells) so we can design reasonable constructs. > > The protein is expressed and secreted by transiently transfected > > HEK293T cells that work very well for other ectodomains and > > extracellular proteins in our hands (PMID 17001101). The target > > protein has 10 cysteines that form 5 disulfides in the crystal > > structure (of E.coli-expressed and refolded protein), there should be > > no free cysteines and no non-specific disulfides. Unfortunately, once > > the protein is secreted, it forms non-specific dimers and higher-order > > oligomers in the media (standard DMEM/2% FBS) before purification > > (confirmed by Western blotting under non-reducing conditions). Using > > 0.5 mM DTT during SEC gives a nice monomeric peak (however, the > > protein suffers as suggested by weaker interactions with its binding > > partners). We don't understand how a secreted protein (which passes > > trafficking quality control in the cell) with a known disulfide > > pattern forms non-specific disulfide linked oligomers in the > > extracellular media. We tried expressing it at 37 C and 30 C, and have > > sequenced our constructs (plasmids) multiple times. > > > > If anyone has seen this kind of problem and successfully solved it > > (purified homogeneous crystallisation quality protein), please let us > > know if possible. I thank you for your help. > > > > Best wishes, > > Tomas > > > > > > Dr. Tomas Malinauskas > > University of Oxford > > Wellcome Centre for Human Genetics > > Division of Structural Biology > > Roosevelt Drive > > Oxford OX3 7BN > > United Kingdom > > to...@strubi.ox.ac.uk > > tomas.malinaus...@gmail.com > > > > > > > > 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] Non-specific disulfides in a secreted protein
Dear All, we are purifying a small secreted protein from conditioned media and have a rather unusual problem. It is a small ectodomain (~11 kDa, pI ~6, His-tagged) of the type 1 transmembrane receptor, crystal structures are known (of the protein that was produced in E.coli and refolded; we are secreting the same protein using mammalian cells) so we can design reasonable constructs. The protein is expressed and secreted by transiently transfected HEK293T cells that work very well for other ectodomains and extracellular proteins in our hands (PMID 17001101). The target protein has 10 cysteines that form 5 disulfides in the crystal structure (of E.coli-expressed and refolded protein), there should be no free cysteines and no non-specific disulfides. Unfortunately, once the protein is secreted, it forms non-specific dimers and higher-order oligomers in the media (standard DMEM/2% FBS) before purification (confirmed by Western blotting under non-reducing conditions). Using 0.5 mM DTT during SEC gives a nice monomeric peak (however, the protein suffers as suggested by weaker interactions with its binding partners). We don't understand how a secreted protein (which passes trafficking quality control in the cell) with a known disulfide pattern forms non-specific disulfide linked oligomers in the extracellular media. We tried expressing it at 37 C and 30 C, and have sequenced our constructs (plasmids) multiple times. If anyone has seen this kind of problem and successfully solved it (purified homogeneous crystallisation quality protein), please let us know if possible. I thank you for your help. Best wishes, Tomas Dr. Tomas Malinauskas University of Oxford Wellcome Centre for Human Genetics Division of Structural Biology Roosevelt Drive Oxford OX3 7BN United Kingdom to...@strubi.ox.ac.uk tomas.malinaus...@gmail.com To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1
Re: [ccp4bb] CHAPS provider
Hi Sebastiano, we often buy chemicals (including CHAPS) from Melford: https://www.melford.co.uk/products/biochemicals/detergents-surfactants/chaps-1-g.html It is often cheaper than Sigma-Aldrich. Hope that helps, Tomas On Fri, Nov 30, 2018 at 2:42 PM Sebastiano Pasqualato wrote: > > > Dear all, > we are considering whether to buy the columns and reagents to purify our own > Wnt3 protein or to buy it. > Given that the purification requires large amount of column eluents with 1% > CHAPS, we are looking for the cheapest CHAPS provider to understand if the > in-house purification is worth the cost. > Could anybody share their experience with low-cost chemical providers, please? > Thank you very much, > have a nice weekend, > Sebastiano > > > -- > Sebastiano Pasqualato, PhD > Biochemistry and Structural Biology Unit > Department of Experimental Oncology > European Institute of Oncology > IFOM-IEO Campus > via Adamello, 16 > 20139 - Milano > Italy > > tel +39 02 9437 5167 > fax +39 02 9437 5990 > > > > > > 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
Re: [ccp4bb] Asn/Gln - pi-stacking prevalence
Dear Michael, On Sat, Nov 10, 2018 at 9:56 AM Michael Jarva wrote: > > Dear ccp4 community, > > > I have recently been working with a structure that has an Asparagine that > makes a planar stacking connection with a Tryptophan ring > (pep_ASN-TRP_v2.png), that seem to be a true pi-stacking interaction and I'd > like to find more examples of this. You could check the following paper: https://elifesciences.org/articles/31486 Pi-Pi contacts are an overlooked protein feature relevant to phase separation They have a list of examples (including Asn-Trp pi-pi stacking) in PDB (Figure 1, data source): Pi-Pi contact annotations for the full PDB set. Text file listing the pi-pi contacts observed across our non-redundant PDB set, with contact types shown by residue annotations where single amino acid names refer to sidechains and pairs of amino acids refer to the backbone peptide bond between residue i and residue i + 1. https://doi.org/10.7554/eLife.31486.005 Best wishes, Tomas Dr. Tomas Malinauskas University of Oxford Wellcome Centre for Human Genetics Division of Structural Biology Roosevelt Drive Oxford OX3 7BN United Kingdom to...@strubi.ox.ac.uk tomas.malinaus...@gmail.com To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1
Re: [ccp4bb] Joint CCP4 and ESF-EACBM Newsletter number 31
Hi Pedro, it seems PostScript files (one can easily convert to PDFs) with the contents are here: ftp://ftp.ccp4.ac.uk/ccp4/newsletter/nov_94/ Best wishes, Tomas On Wed, Jul 11, 2018 at 9:01 AM Pedro Matias wrote: > Dear CCP4ers, > > Is there a PDF version of the Joint CCP4 and ESF-EACBM Newsletter number > 31 (November 1994)? > > I can find the summary on the web but not the contents. > > Thanks in advance, > > Pedro > > -- > > Industry and Medicine Applied Crystallography > Macromolecular Crystallography Unit > ___ > Phones : (351-21) 446-9100 Ext. 1669 > (351-21) 446-9669 (direct) > Fax : (351-21) 441-1277 or 443-3644 > > email : mat...@itqb.unl.pt > > > http://www.itqb.unl.pt/research/biological-chemistry/industry-and-medicine-applied-crystallography > http://www.itqb.unl.pt/labs/macromolecular-crystallography-unit > > Mailing address : > Instituto de Tecnologia Quimica e Biologica António Xavier > Universidade Nova de Lisboa > Av. da República > 2780-157 Oeiras > PORTUGAL > > ITQB NOVA, a great choice for your PhD > https://youtu.be/de6j-aaTWNQ > > Master Programme in Biochemistry for Health > https://youtu.be/UKstDCFjYI8 > > > > 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
Re: [ccp4bb] 3D Structure Search
Dear Nicola, PDBeFold is great too: http://www.ebi.ac.uk/msd-srv/ssm/ Best wishes, Tomas On Thu, Feb 15, 2018 at 11:16 PM, Nicola Evanswrote: > I have recently solved a novel structure which previously did not have any > structural homologues (as related by sequence identity). I was wondering if > anyone could recommend a 3D structural search engine? I used the Dali server > which has been recommended to me in the past (and with past success) but the > top few hits this time aren't similar structurally (although I haven't > exhausted the list yet). I just want to confirm if the folds are truly novel > or not. > > Thanks in advance for your suggestions, > > Nicola
Re: [ccp4bb] Co-purified ligand present in protein crystal
Dear Wenhe, we had a similar case and extraction using CHCl3 plus CH3OH (2:1 ratio, v/v) (65 °C, 30 min) before mass spectrometry worked very well: https://www.ncbi.nlm.nih.gov/pubmed/21743455 Hope that helps, Tomas On Mon, Aug 21, 2017 at 4:41 AM, WENHE ZHONGwrote: > Dear CCP4BB members, > > We would like to identify a ligand that is present in crystal structure > (according to strong positive densities at active site) but absent in > crystallization condition. We already have some candidates in mind based on > our knowledges on this protein but we need to validate further. The general > method we are using now is to use methanal to precipitate protein and extract > ligand from the precipitated protein. Then we analyse the methanol extraction > sample on LC-MS. One problem of this method is that the methanol extraction > will not be 100% efficient which means there is only a small portion of > bound-ligand can be extracted from the protein— particularly if the ligand > binds very tightly to the protein. So I would like to know whether anyone has > experience to efficiently extract tighly-bound ligands from protein for > downstream analysis. One method is to digest protein with protease such as > trypsin. Or use urea to denature the protein. However, these methods require > relatively long processing time which is not optimal when the ligand that we > want to analyse is unstable (degrade overtime). Anyone has more suggestions? > > Thank you! > > Kind regards, > Wenhe
Re: [ccp4bb] Rhodamine in FPLC
Dear Reza, I used hydrogen peroxide (~0.1-2%?) to remove rhodamine stuck on Superdex columns. "Pink" columns turned "white" after this kind of treatment. Hope that helps, Tomas On Fri, Jun 9, 2017 at 10:43 PM, Reza Khayatwrote: > Hi, > > Sorry for the non crystallography question. We're using Rhodamine for > labeling some material that we need to run through our FPLC. It seems that > the Rhodamine is getting stuck to the PEEK tubing. Anyone has experience with > successfully removing the Rhodamine from the tubing? Thanks. > > Best wishes, > Reza > > Reza Khayat, PhD > Assistant Professor > City College of New York > Department of Chemistry > New York, NY 10031
Re: [ccp4bb] anti-hexahistidine antibody
Dear Fernandez et al., maybe this anti-His-tag antibody could be useful: PDB ID 1KTR, its sequence is in the paper https://www.ncbi.nlm.nih.gov/pubmed/?term=12054774 Hope that helps, Tomas On Sun, Jan 8, 2017 at 9:24 PM, Fernandez, Elias Jwrote: > Dear All, > > Is anyone aware of a recombinant system that produces an N-terminal, > hexa-histidine-directed Fv antibody fragment that can be used for structural > studies (crystallography, SAXS, cryo-EM)? > > Best regards, > > Elias > > >
Re: [ccp4bb] Heavy-atom derivatives
Hi Giulliana, here is a nice and simple method on how to select heavy atoms for phasing using native PAGE (please see Fig. 1): http://www.ncbi.nlm.nih.gov/pubmed/10903954 Structure. 2000 Jul 15;8(7):R143-9. Screening for phasing atoms in protein crystallography. Boggon TJ, Shapiro L. Hope that helps, Tomas On Mon, Jun 29, 2015 at 8:47 AM, Giulliana Rangel giulliana.ran...@gmail.com wrote: Dear all, I'm looking for a method to solve the phase problem. Thus, I would like some help about heavy-atom derivatives. What is the best heavy-atom to make soaking? I tried Iodine (because my protein have a lot of tyrosines), but no results... I read some papers about quick-soak, its a good method? Using platine, Hg, Br... Any suggestions? Thank you so much. Best wishes, -- Giulliana Rangel M.S. Biotechnology Program - UNIFESP Structural Biology Laboratory Phone: +55 (12) 3309-9698 330 Talim St ZIP CODE 12231-280 São José dos Campos - SP/ BR
Re: [ccp4bb] B-factor blurring
Hi Mike, my favourite summary on B factor sharpening/blurring is here: Acta Crystallogr D Biol Crystallogr. 2006 Aug;62(Pt 8):923-32. Epub 2006 Jul 18. Considerations for the refinement of low-resolution crystal structures. DeLaBarre B, Brunger AT. Hope that helps, Tomas On Mon, Nov 17, 2014 at 8:01 PM, Mike Lawrence lawre...@wehi.edu.au wrote: Dear all can anyone help me with literatures references to B-factor blurring as a technique to reveal low resolution features in an electron density map? I have seen the poster from Andrea Thorn at http://shelx.uni-ac.gwdg.de/~athorn/pdf/thorn_iucr2014_poster.pdf but was wondering if there were any alternative references? with many thanks! Mike Mike Lawrence, PhD Associate Professor and WEHI Fellow Structural Biology Division Walter and Eliza Hall Institute of Medical Research 1G Royal Parade, Parkville Victoria 3052, AUSTRALIA Tel. 61-3-9345-2693 Fax 61-3-9345-2686 Email: lawre...@wehi.edu.au __ The information in this email is confidential and intended solely for the addressee. You must not disclose, forward, print or use it without the permission of the sender. __
[ccp4bb] Electron density of penicillin (Re: [ccp4bb] Google Gets it Right)
Dear Gregg et al., On Mon, May 12, 2014 at 4:28 PM, Gregg Crichlow gregg.crich...@gmail.comwrote: Actually, it was noticing penG that made me mouse over it myself. After spending many years completing a thesis on beta-lactamases, I was very surprised - and excited - to see that on something as main-stream as Google. But then when I paid attention more closely, I saw that they even have a good representation of the electron density in three orthogonal planes surrounding the molecule! Dr. James Knox (my thesis advisor) just informed me that the density maps are on exhibit at the British Museum of Science. The electron density map of penicillin is deposited at the Museum of the History of Science in Oxford too, a photo I took few years ago is here: https://drive.google.com/file/d/0B4TVGcERcQ7veGZXS1lEMXBqck0/edit?usp=sharing Best wishes, Tomas
[ccp4bb] Postdoc position in membrane protein crystallography and pharmacology
Dear All, The laboratory of Hiro Furukawa at Cold Spring Harbor Laboratory (CSHL) is seeking a postdoc with dynamic interests in solving problems in neurological disorders and diseases using biochemical and biophysical techniques. We conduct x-ray crystallography on transmembrane proteins to understand basic mechanisms of ion and substrate transport across the membrane and on fragments of water soluble domains to answer specific pharmacological questions. We validate structure-based functional hypotheses by a combination of techniques such as electrophysiology and calcium imaging. We have excellent setups to conduct membrane protein biochemistry and crystallography such as Mosquito LCP and have frequent accesses to synchrotron light sources including NSLS and APS. CSHL offers a unique scientific environment to interact with scientists within the institute as well as from outside at courses and meetings. A qualified candidate should hold or soon expected to hold (within ~1yr) Ph.D. in the area of crystallography and biochemistry and have experiences in basic molecular biology, biochemistry, and protein expression. Experience in x-ray crystallography or electron microscopy is desired but not necessary. Please see the lab website (http://www.cshl.edu/public/SCIENCE/furukawa.html) for further information and send CV with a brief research statement as well as 2-3 contacts for references to furuk...@cshl.edu Best wishes, Tomas Dr. Tomas Malinauskas Cold Spring Harbor Laboratory One Bungtown Road Cold Spring Harbor New York 11724 United States of America Email: tmalinaus...@cshl.edu tomas.malinaus...@gmail.com Tel: (516) 367-6824
Re: [ccp4bb] distinguish ligand binding sites within a protein
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] structural search for homologs in pdb?
Dear Gloria, HHpred. Best wishes, Tomas On Thu, Aug 22, 2013 at 4:14 PM, Gloria Borgstahl gborgst...@gmail.com wrote: We have a protein sequence that probably contains OB folds. What is the best way to search for the top structural homologs to this sequence in the pdb? G
Re: [ccp4bb] Xtal formed during purification
Dear Zhizhi, we had a case like that. I would switch to slightly different buffer (e.g. different pH) so crystals do not appear overnight, and then do crystallisation screening. I bet you will have many hits in different conditions, likely with bigger crystals. Good luck! Best wishes, Tomas On Fri, Aug 9, 2013 at 8:31 PM, ZHIZHI WANG zzw...@u.washington.edu wrote: Hi all, After I purified my target protein by ion exchange, I left the fractions with high protein concentrations overnight @4C. Now I saw a lot of small needle crystals inside the EP tubes this morning. I wonder whether there is any technique or method to get bigger crystals from this? ZZ
Re: [ccp4bb] off topic question BIAcore problem
Dear Xianchi, unfortunately, dissociation rate constant kd 10^-6 s^-1 was just beyond the limit of Biacore in 1999 (e.g. see Fig. 1 in http://www.ncbi.nlm.nih.gov/pubmed/10556876). I am not sure about these days. As Juergen noted, you may have a problem with rebinding (you could try to reduce amount of the ligand on the chip, see other tricks here http://users.path.ox.ac.uk/~vdmerwe/internal/spr.pdf). Regarding reviews, David Myszka writes enjoyable reviews on SPR. Hope that helps, Tomas On Wed, Feb 20, 2013 at 5:03 PM, xianchi dong dongxian...@gmail.com wrote: Dear all, Recently I have measure a set of kinetic data of a receptor ligand interaction using BIAcore 3000. To my surprise, the dissociation rate is very low (~ 10e-6). During the measurement, I use a long dissociation time (2 hours) .I repeat several time which give very similar results. So I am wondering if the BIAcore can measure such a low off-rate kinetic. What is the limitation of BIAcore? Any review about that? Thanks in advance. Xianchi Dong Research Fellow Children's Hospital Boston Harvard Medical School Dea
Re: [ccp4bb] low resolution molecular replacement with partial model
Dear Daniel, here is an example of MR at 7 A: http://www.pdb.org/pdb/explore/explore.do?structureId=4GZA http://www.ncbi.nlm.nih.gov/pubmed/23104057 Best wishes, Tomas On Tue, Nov 6, 2012 at 11:06 AM, Panne Daniel pa...@embl.fr wrote: Hi all, I was wondering if there are examples of successful MR with low resolution data (~7A). Our protein crystallized in the P21 (a= 111; b= 211, c=126; beta=99.7). We only have a partial search model (267 amino acids) that accounts for ~6% of the AU (however at 100% identity). Has anybody been successful in such situations? Thanks, Daniel