Re: [ccp4bb] Per-residue RMSD for multiple structures?+ CCP4BB Gestapo
Dear all, I have just subscribed to the CCP4BB. The description for this BB is "The CCP4BB mailing list is for discussions on the use of the CCP4 suite, and macromolecular crystallography in general. " So, please, give us all possible solutions to crystallographic problems, including protein expression, purification etc. For many of us the CCP4BB is our school of crystallography. As this is probably the biggest crystallographic forum, any relevant information is wellcome plus job postings. Maia - Original Message - From: "Gerard DVD Kleywegt" To: Sent: Tuesday, February 23, 2010 7:57 AM Subject: Re: [ccp4bb] Per-residue RMSD for multiple structures? Thanks Stephen! I was going to suggest that, but I was afraid of the self-appointed CCP4BB Gestapo that has been seen goose-stepping in this neighbourhood recently (Tassos recently accused me of becoming mellow and diplomatic in my dotage, so I hope I've set the record straight now). However, since this solution is neither CCP4 nor Phenix, we may get away with this heinous act of bulletin-board heresy... On the other hand, I've learned that it is often more expedient to beg for forgiveness than to ask for permission. I would add that: - I assume that the sequences and numbering are identical - you should put the structures in one big PDB file and read it into LSQMAN - since LSQMAN doesn't do true multiple-structure alignment, you could pre-align them, e.g. with SSM/PDBeFold - if you didn't, you could indeed use the MCentral and MAlign commands to align them - my favourite plot would be the "CD plot" (but then again, it would, wouldn't it?) - see for instance: http://xray.bmc.uu.se/cgi-bin/gerard/image_page.pl?image=usf/pics/cdplot_1ldn.gif - which is also produced with the MPlot command - http://xray.bmc.uu.se/usf/lsqman_man.html#S82 - a normal MPlot would look like this: http://xray.bmc.uu.se/cgi-bin/gerard/image_page.pl?image=usf/pics/mplot_1ldn.gif - the output file of the normal MPlot command is in a form that can be quickly converted into an O datablock for those handy with an editor and familiar with O datablocks, and could then be used to ramp a model inside O - you may also want to consider showing how the (main-chain or side-chain) torsion angles differ between the structures, e.g. by plotting the circular variance of phi and psi - see for instance http://xray.bmc.uu.se/cgi-bin/gerard/image_page.pl?image=usf/pics/vmain_1ldn.gif - as described here: http://xray.bmc.uu.se/usf/lsqman_man.html#S83 - or a multiple-model Ramachandran plot like this http://xray.bmc.uu.se/cgi-bin/gerard/image_page.pl?image=usf/pics/mrama_1ldn.gif (with the MRama command). The advantage is that no superposition is required at all and that any domain movements won't debeautify your results --dvd On Tue, 23 Feb 2010, Stephen Graham wrote: > I am pretty sure you can do this using LSQMAN from Gerard (BluRay?) Kleywegt. > > The pertinent commands are MCENTRAL to determine the 'most > representative structure' (i.e. the one to align upon and show in the > figure), MALIGN to do the alignment and then MPLOT to calculate a > 'multi-RMSD' for each residue (see manual for details - set the > 'cut-off for printing' to 0 to get all values). > > Regards depiction, I think pymol can also represent structures as > sausages based on their B values: > cartoon putty > show cartoon > > HTH, > > Stephen > > On 23 February 2010 01:31, Ethan Merritt wrote: >> Hi all, >> >> I am comparing 4 very similar (<1.5A rmsd) large (750 residues) structures, >> but struggling to find a way to generate a figure that conveys where they >> are most alike and where they diverge. >> >> Simply drawing a superimposed set of backbone traces results in what looks >> like colored spaghetti. I don't think that's going to work. >> >> So I had the idea of drawing a single backbone trace, or ribbon diagram, >> and coloring by the RMSD of the four C-alphas at each residue position. >> But I can't find a program that will output this as a table of numbers >> I can use. All of the multiple structure superposition programs must >> have this information internally. After all, that's what they are minimizing. >> But do any of the programs provide an option to write it out? >> >> I can get pairwise per-residue deviations by doing SSM superposition in Coot, >> but that doesn't get me to an RMSD for all four structures jointly. >> >> Ethan >> >> >> -- >> Ethan A Merritt >> Biomolecular Structure Center >> University of Washington, Seattle 98195-7742 >> > > > > -- > Dr Stephen Graham > 1851 Research Fellow > Cambridge Institute for Medical Research > Wellcome Trust/MRC Building > Addenbrooke's Hospital, Hills Road > Cambridge, CB2 0XY, UK > Phone: +44 1223 762 638 > Best wishes, --Gerard ** Gerard J. Kleywegt Dept. of Cell & Molecular Biology University of Uppsala
Re: [ccp4bb] Rsym problems...maybe???
There are Rpim and Rrim, Rpim is sqrt(1/(N-1)) and is usually small and Rrim (or Rmeas)=sqrt(N/(N-1)) and is large. I usually go with I/sigma cutoff. Maia - Original Message - From: "Edward A. Berry" To: Sent: Thursday, April 22, 2010 11:59 AM Subject: Re: [ccp4bb] Rsym problems...maybe??? There are plenty of structures in the database with R-sym=0.99. But something is odd here. If I understand R-pim, it should always be bigger than Rsym, because this factor of sqrt(N/(N-1)) is always >1 Are you saying Rpim is .30 and Rsym is 1.00? Last time I deposited a structure, Rsym and Rmerge in the last shell are optional. I would leave it out and rely on the excellent I/sigI in the last shell, and use all the data (provided after refinement R-free in the last shell is < .4). Ed Daniel Bonsor wrote: Hello again. At first I was not worry but maybe now I am. I have completed a structure and submitted to the PDB. They queried my Rsym value in the highest resolution bin, 2.5-2.37A (may I dare say it 100%). I was not worried at the time as I had: 99.4% completeness Mean(I/sdI) of 2.5 and a redundancy of 11 (which would explain the high Rsym) Space group I422 My Rpim in this shell is 30%. Should I reduce the resolution and start from scratch again or is everything fine and dandy and I should stop worrying?
Re: [ccp4bb] question - GFP fusion - cleavage sites
Why do you need a fusion protein? Can you use His-tag directly? If you have to have a fusion protein, use one that has no hydrophobic patches on the surface, because otherwise they will be glued to each other. For separation of your mixture that you have now try dilute it as much as possible ( but keep it detectable) and hopefully the complex might dissociate. I also think that adding some organic solvent might help to dissociate it. Then you apply to your column. Maia - Original Message - From: Celina R. To: CCP4BB@JISCMAIL.AC.UK Sent: Monday, May 24, 2010 6:05 AM Subject: [ccp4bb] question - GFP fusion - cleavage sites Dear CCP4er's, Sorry for the non-crystallography related question and was hoping someone on the bulletin board might have some suggestions to overcome my peculiar protein purification problem. I am working on several membrane proteins (for crystallization trials) that have a C-Terminal eGFP fusion partner followed by a His-tag. The membrane protein and the GFP-His tag are separated by a TEV protease site. After purifying the fusion protein by IMAC, I add TEV protease to cleave the linkage between the membrane protein and the GFP-His tag.The cleavage reaction is also dialyzed to get rid of the imidazole. This cleavage seems to go to completion as judged by SDS-PAGE. However, when I try to separate the membrane protein from the GFP-His tag by passing through a IMAC column twice (excess nickel resin), a significant amount (about 1 mg) of the GFP-His tag doesn't bind the IMAC column and flows through along with my protein. In addition, other methods such as centricons (30, 50 or 100 kDa M.W.C.O.), Gel Filtration and Ion-Exchange are also not able to separate them. All my buffers have 5 mM reducing agent and 500 mM NaCl to try and prevent any non-specific interaction between my protein and the GFP-His tag. It appears that the GFP-His tag is somehow stuck to my protein and co-elutes on any chromatographic column that i use. Has anyone encountered such a problem and managed to overcome it? Any suggestions/tricks would be helpful. I also have a question: Which is better to use for the cleavage of His-tags, in case I want to clone the membrane protein without GFP: TEV protease or thrombin? Thanks in advance. C.
Re: [ccp4bb] monomeric coiled coil
The multimeric state depends on a protein concentration. You can get any multimer to dissociate if you dilute it to low enough concentration. If your complex is a homodimer, then Kdiss=[complex]/[monomer]^2. Let's say your Kdiss~10^(-3)M, and your protein concentration is ~10^(-4)M, then [complex]=Kdiss/[monomer]^2=10^(-3)/10^(-4)^2=10^(-5), that means, the dimer concentration is approximately ~10 times less then the monomer concentration at this particular protein concentration. Let's say, the mol weight is 50 kDa, then at 5mg/ml you will have only about ~10% of the dimer. Of course, if your Kdiss~10^(-4)M, then you will have approximately similar concentrations of monomers and dimers at 10^(-4). Because this is a dynamic equlibrium between multimers and monomers, some methods are not good for the determination of a multimeric state. Some reviewers demand to prove the multimeric state by size-exclusion chromatography (SEC) or analytical centrifugation. The analytical ultracentrifugation method will not work, as the characteristic time of the dissociation/association is much lower than the centrifugation time (`24 hours). The separated monomer will start association and the separated dimer will start dissociation according to Kdiss and the bands will be smeared. SEC is faster, like half an hour, it gives you a better chance. The methods without separation are the best Like light scattering), just make protein concentration high. Here comes the other question. What is the physiological concentration. You want to be close to it. I read some literature on this and it looks like it is between 10^-(4) to 10^-(6) for majority of proteins. - Original Message - From: "aidong" To: Sent: Saturday, July 03, 2010 6:26 AM Subject: [ccp4bb] monomeric coiled coil Sorry for this ccp4 unrelated question. We recently have a protein that a multicoil program (http://groups.csail.mit.edu/cb/multicoil/cgi-bin/multicoil.cgi/cgi-bin/multicoil ) predicts to have very high probability for dimer and trimer. Their scores are close to 0.4 and 0.6 for lengths of more than 60 amino acids. However, two constructs that cover this region have demonstrated monomers in solutions by Multiangle light scattering?!For the same question, we could not get any response from this program manager therefore we turn to ccp4 for help. We wonder whether some of you might have similar experience. Thank you in advance. Sincerely, Aidong
Re: [ccp4bb] monomeric coiled coil--updated
It's an interesting discussion. 1.Usually it's not possible to use mass-spec for non-covalent complexes. 2. Most methods depend on macromolecule shape and concentration. 3. SAXS method looks limited to me. It uses diluted monodisperse solutions. That excludes complexes that can associate/dissociate. How can you calculate Kd from it? 4. All methods for determination of multimeric state using separation technique depend on three different cases: time of equilibrium (teq)>> time of separation (tsep), or teq << tsep or teq ~ tsep. Even without equilibrium, you cannot have only one component. For a successful separation, you would want teq >> tsep, which is less likely in AUC method. 5. There are papers on capillary electrophoresis methods where they study exactly these effects (time of equilibrium vs time of separation). Maia - Original Message - From: "aidong" To: Sent: Sunday, July 04, 2010 3:01 AM Subject: Re: [ccp4bb] monomeric coiled coil--updated > In light of several wonderful responses,I would like to provide an > update for this question: > > 1. I would agree that SEC might not be able to identify monomer vs > multimer forms for this likely rod-shaped protein. > > 2. It is extremely low kd for dimer. AUC and SAXS experiments have > measured its kd at ~0.1 mM. > > 3. MALS might not be able to pick up dimer form since it might be only > a few percent when the concentration is low. We might overcome > concentration effect by direct injection to dawn heleos and refraction > index. > > 4. Mass spec has found both monomer and dimer forms although the > abundance of each one is not known. > > 5. Intramolecular coiled coil is quite possible since intermolecular > dimer is unstable. We hope our structure might provide an answer. > > Many thanks for your time and ideas > > Cheers > > Aidong > > > On Jul 4, 2010, at 1:09 AM, Anastassis Perrakis wrote: > >> A few thoughts on these, since I do not fully agree. >> >> 1. Detection by light scattering is a method that can be used either >> without separation, or while separating. >> If you have a scattering detector, you can stick in a cuvette, or >> stick it to the end of a column, your choice. >> >> 2. Sec is not a good method to show if especially a coiled coil is >> monomer-multimer. A long coil, will >> have a hydrodynamic radius bigger than its MW, thus any prediction >> based on SEC will be misleading, >> especially for this class of proteins. >> >> 3. In AUC (although I am not an expert at it at all) I cant see the >> connection between the disassociation time >> and the run time. In sedimentation or equilibrium runs, depending on >> what you want to see, I think you can look >> at monomer-multimer equilibrium over a wide range of kD and >> combinations of k(on) and k(off). >> >> 4. The physiological concentration is a bit misleading. First, its >> clear now that cells have microenvironments, >> and 'physiological' concentrations are hard to define. Also, in a >> cell, I think (and I think others tend to agree) >> that kD plays little role at the end. kD is a combination of k(on) - >> which is concentration dependent but in a cell >> very likely diffusion limited - and of k(off) which I think is what >> matters most in the cell. >> >> Going to Aidong's question, I think that MALLS was a good >> experiment. The fact that these constructs do no associate, >> can mean that >> >> a. the prediction is wrong - likely with these scores, but not >> necessary >> b. the kD in solution is indeed higher that the concentration you >> used for MALLS >> c. The constructs are not well chosen for some reason >> >> You could use AUC to detect kD as high as ~100uM, depending on the >> concentration of the start sample of course. >> The next question will anyway be if that kD has any sort of >> physiological significance - which you cannot tell by magnitude - >> so you are back at the drawing board for mutants. Three years later >> the referees will still not believe it ... sorry, now it gets >> personal, >> so I stop here. >> >> My two cents. >> >> A. >> >> >> On 3 Jul 2010, at 18:10, chern wrote: >> >>> The multimeric state depends on a protein concentration. You can >>> get any >>> multimer to dissociate if you dilute it to low enough >>> concentration. If >>> your complex is a homodimer, then Kdiss=[complex]/[monomer]^2. >>> Let's say >>> you
Re: [ccp4bb] MR with polysaccharide
I fitted manually the chondroitin sulfate into my complex (3c9e.pdb and 3h7d) using program coot. The chondroitin-sulfate model was used from the PDB. I used CNS for the refinement of the first structure, and phenix for the second structure. Phenix was more convenient with this ligand as it was a polymer chain running through many unit cells. What sulfated sugar did you use? How big it is? Maia - Original Message - From: Vandu Murugan To: CCP4BB@JISCMAIL.AC.UK Sent: Friday, July 30, 2010 5:28 AM Subject: [ccp4bb] MR with polysaccharide Dear all, I am seeing some bulk extra denisty near my protein molecule, in a 2.7 angstrom map. The crystallization condition condition has a sulfated polysaccharide. If I want to perform a MR with this polysaccharide, in presence of the refined protein model in the cell, which program should I use?. In the phaser, there are options only for proteins and nucleic acids. Is there, any automated ligand identifying/fitting program that can work at this resolution? All suggestions are wellcome. Thanks in advance. warm regards, Murugan
Re: [ccp4bb] Query regarding GST fusion protein purification
I worked with several viral cystene proteases. I do not recommend using basic pH for them dring purification or storage or crystallization. Keep it below 7, because the active site cysteine oxydazes very easily. The higher the pH, the easier the oxydation. PH 10 can also cause hydrolysis of some peptide bonds. Try His tag, then you don't need to use proteases for cleavage, you can use Ni column. Maia - Original Message - From: "Ashok Ranjan Nayak" To: Sent: Sunday, August 29, 2010 5:28 AM Subject: [ccp4bb] Query regarding GST fusion protein purification Hello one and all !! I have been working on cloning and purification aspects on a Leishmanial cysteine peptidase (pI= 8.2) for quite some time. I had cloned it in pGEX expression vector. Expression seemed quite okay when induced with 0.5 and 1mM IPTG, so did the solubility in 4 buffers at different pH conditions. i. e. Tris (6.8), HEPES (7.5), Bicine (pH = 9) and Glycine (pH=10). The problem is when I purify it using glutathione sepharose column I get only GST (size wise estimation; no western tried ) i.e. a prominent 25 KD band. At the same time I get the fusion protein in the load, equilibration and wash fractions. When I increased Nacl concentration upto 400 mM I only could exclude the fusion protein band from wash. I had tried protease inhibitors like PMSF, sigma cocktail, and DTT in the lysate before sonication. I had also tried reduced glutathione upto 40 mM in the elution buffer with two different pH at 8 and 9. I also read from literature that similar intracellular cysteine proteases behave same even after mutating the conserved cysteine residue at its active site. They all say that its not because of autocatalytic property of the enzyme its because of some proteases from E.coli. Should I try ion exchange or affinity chromatography using any inhibitor of this enzyme?? Can anyone suggest me some tip?? Guys help me out. i am kind of struck here Ashok Ranjan Nayak Research Scholar Molecular and Structural Biology Division Central Drug Research Institute, Lucknow
