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 i

Re: [ccp4bb] RMSD per residue graph for multiple aligned PDB files

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

[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/Trai

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

[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 T

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 crystalliz

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

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

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 aff

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...@j

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 w

Re: [ccp4bb] protein expression in human cells

. 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

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 re

Re: [ccp4bb] Non-specific disulfides in a secreted protein

k 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, > &

Re: [ccp4bb] Non-specific disulfides in a secreted protein

y 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 k

[ccp4bb] Non-specific disulfides in a secreted protein

fully 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 Kingd

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, >

Re: [ccp4bb] Asn/Gln - pi-stacking prevalence

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.u

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 Newslett

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 Evans wrote: > I have recently solved a novel structure which previously did not have any > structural homologues (as related by sequence identity). I was wondering i

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 ZHONG wrote: > Dear CCP4BB members, > > We

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 Khayat wrote: > Hi, > > Sorry for the non crystallography question. We're using Rh

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 J wrote: > Dear All, > > Is anyone aware of a recombinant s

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 help

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

[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 wrote: > 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 > Goo

[ccp4bb] Postdoc position in membrane protein crystallography and pharmacology

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

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 wrote: > Hi al

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 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

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 amoun

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 wrote: > Hi all, > > I was wondering if there are examples of successful MR with l

Re: [ccp4bb] pdf to text

Hi, On Mon, Sep 13, 2010 at 2:30 PM, Oganesyan, Vaheh wrote: > The molecule is polymyxin B and doesn’t exist in databases like CSD, Hic-up > or PDB. If some of you happen to have it please share. Thank you. PDB file: http://129.128.185.122/drugbank2/drugs/DB00781/pdb/download More information: