Re: [ccp4bb] problem with LIBCHECK
If you have model coordinates for your CSA, I send those to the PRODRG server and let it generate a REFMAC style dictionary. You will need to make sure it is labelled as a peptide - cf the standatd residue cif files to see how to do that.. Then you need to enter the LINKR record into the pdb file connevting the CSA ands your CYS. But if you run refmac from the GUI with the REVIEW restraints option that should be done for you.. I thinjk the cif file generated should contain both the CSA entry and an apppropriately named LINKR dictionary.. Eleanor On 05/20/2011 09:59 PM, Geoffrey Feld wrote: Greetings fellow Crystallographers, I'm working on a structure at 1.8-A resolution that contains an acetone crosslink between 2 cysteines (crosslink was incorporated by adding 1,3-dichloroacetone). I figured that the easiest way to model this is to mutate one of the cysteines to S-acetonylcysteine (CSA in the PDB) and then link it to the other cys. I've seen how to do this in COOT using the mutate-by-overlap function; however, CSA is of course not in the CCP4 library that is installed on our system. I've built restraints for CSA using phenix.elbow and tried importing the residue into COOT that way, still to no avail. So I figured the way to go now is to import the cif file directly into the LIBCHECK library in our system and then I should (in theory) be able to use mutate-by-overlap to place the residue. However, this is where I'm stuck. I can't seem to figure out the notation for importing the cif file into LIBCHECK. I tried using FILE_CIF CSA.cif and I get an error "reading title of input file." What am I doing wrong? Is there another approach I should consider? Any help or advice would be greatly appreciated. Cheers, Geoff
Re: [ccp4bb] Crystal structure and NMR structure
I think calmodulin is a classic example, in all its forms (I believe that there are both NMR and crystal structures for all of these): +/- calcium, +/- peptide. Especially the no-peptide +/- calcium forms differ pretty substantially. The calcium-bound no-peptide structures are particularly interesting, as it seems that the NMR structure is much more plausible (take a look if you don't believe me--the xtal structure has a really extended alpha helix, which it seems to me would be bending all over the place in solution). And yet, the crystal structure was solved at 1.0 Ang. Now, assuming that the NMR structure is really a truer picture of the structure in solution, this contradicts a premise which always seemed reasonable to me, that as resolution increases, the model more accurately represents the protein as found in solution. I guess the bottom line is that resolution does not necessarily imply a better picture of the molecule as it functions physiologically, but simply means that the crystallography worked out better. Do others agree with this, that resolution implies little about the physiological veracity of the model? Jacob On Sat, May 21, 2011 at 4:03 AM, Chen Guttman wrote: > Here you go: > http://www.ncbi.nlm.nih.gov/pubmed/12015150 > Domain swapping of Cyanovirin. > Chen > > --- > Chen Guttman > The Zarivach laboratory for Macromolecular Crystallography > Building 39, Room 009B > Ben-Gurion University of the Negev > POBox 653 > Zip Code 84105 > Beer-Sheva > Israel > http://lifeserv.bgu.ac.il/wb/zarivach > Tel. +972-8-6479519 > Fax. +972-8-6472970 > > > On Sat, May 21, 2011 at 08:34, Vandu Murugan wrote: >> >> Dear all, >> I would like to get some information on proteins where there is >> conformation/structural change between the crystal structure and solution >> structure of the same protein. Do anybody came across such situations? >> Thanks in advance.. >> >> cheers, >> Vandu > > -- *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program cel: 773.608.9185 email: j-kell...@northwestern.edu ***
Re: [ccp4bb] How to obtain apo protein?
Any idea of the affinity of the complex? If weak enough, you could just dialyse, dialyse, dialyse... Or, you could bind your protein to a column, and wash with a slow-flow of appropriate buffer. Or you could also try to compete it out with adenosine, for example, which presumably has a lower affinity, but at high concentrations would still compete it out, and then dialyse or rinse while bound to a column. Jacob On Fri, May 20, 2011 at 10:18 PM, jlliu liu wrote: > Hi All, > I am working with a methyltransferse protein and its later determined > crystal structure indicates its substrate SAM was bound during protein > expression or purification since no SAM was added during protein > crystallization. Now I want to obtain the apo form protein for further > characterization. Here are the strategies I can think of: > 1. Mutate residues that are possibly critical for SAM binding. The > shortcoming of this strategy is the protein is modified, also mutating one > residue may or may not be able to get rid of SAM binding. > 2. partially denature protein with denaturing reagent such as guanidine. > This strategy is hoping the partial denaturing would loose SAM binding, then > dialysis out SAM, reconcentrate protein and re-run sizing column to get rid > of aggregated protein. This strategy may only work with well-behaved > protein. The harsh denaturing reagent treatment could lead to total protein > denaturing. > I am asking more experienced biochemists if there are other better strategis > for obtaining apo protein that I am not aware of. Thanks very much for > sharing your knowledge. > Eric -- *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program cel: 773.608.9185 email: j-kell...@northwestern.edu ***
Re: [ccp4bb] crystallization of a weird protein
try to change ur buffer instead of PBS u can use Tris or HEPES. In PBS buffer u will get salt crystals in many condition becoz of phopshaphate try to use nonionic detergents in ur soluton .. On Fri, May 20, 2011 at 8:29 PM, Nian Huang wrote: > It might be SDS precipitation. Although 0.1% SDS is generally considered > not high enough to precipitate at 4 degree, it might interact with other > components in your solution to form even less soluble material. > > Nian > > > On Fri, May 20, 2011 at 9:36 AM, WEI MIN wrote: > >> Dear All >> >> I have a difficulty to crystallize my membrane protein(his tagged). I got >> the salt crystals from many different screening conditions. The protein is >> in PBS buffer and 130mM salt with 0.1% SDS. I store It in the 4 degree >> although it forms the milk solution. >> The solution is getting clear in the room temperature within 5 mins when I >> set up the trays. >> >> I would like to take any advice. I would appreciate your input. >> >> Best >> >> Min >> > > -- Vandana Kukshal Postdoc Fellow structure biology group international Center For Genetic Engineering and Biotechnolgy India
Re: [ccp4bb] Crystal structure and NMR structure
2 observations: 'collapsed' forms of calmodulin ligand also exist that indicate the 'function' of wrapping itself around a target. Second, the real question is what the 'function' to be elucidated means - if it involves a binding site detail, resolution (i.e. coordinate precision) sure is 'biologically' relevant. In the case of Cam, a large domain movement as key part of the function will be subject to all the well known crystal packing caveats and may require multiple structures etc. I.e., to get the big picture, small detail is less relevant - as many valuable low resolution structures (open-closed channels, etc) demonstrate. BR -Original Message- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Jacob Keller Sent: Sunday, May 22, 2011 7:07 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Crystal structure and NMR structure I think calmodulin is a classic example, in all its forms (I believe that there are both NMR and crystal structures for all of these): +/- calcium, +/- peptide. Especially the no-peptide +/- calcium forms differ pretty substantially. The calcium-bound no-peptide structures are particularly interesting, as it seems that the NMR structure is much more plausible (take a look if you don't believe me--the xtal structure has a really extended alpha helix, which it seems to me would be bending all over the place in solution). And yet, the crystal structure was solved at 1.0 Ang. Now, assuming that the NMR structure is really a truer picture of the structure in solution, this contradicts a premise which always seemed reasonable to me, that as resolution increases, the model more accurately represents the protein as found in solution. I guess the bottom line is that resolution does not necessarily imply a better picture of the molecule as it functions physiologically, but simply means that the crystallography worked out better. Do others agree with this, that resolution implies little about the physiological veracity of the model? Jacob On Sat, May 21, 2011 at 4:03 AM, Chen Guttman wrote: > Here you go: > http://www.ncbi.nlm.nih.gov/pubmed/12015150 > Domain swapping of Cyanovirin. > Chen > > --- > Chen Guttman > The Zarivach laboratory for Macromolecular Crystallography > Building 39, Room 009B > Ben-Gurion University of the Negev > POBox 653 > Zip Code 84105 > Beer-Sheva > Israel > http://lifeserv.bgu.ac.il/wb/zarivach > Tel. +972-8-6479519 > Fax. +972-8-6472970 > > > On Sat, May 21, 2011 at 08:34, Vandu Murugan wrote: >> >> Dear all, >> I would like to get some information on proteins where there is >> conformation/structural change between the crystal structure and solution >> structure of the same protein. Do anybody came across such situations? >> Thanks in advance.. >> >> cheers, >> Vandu > > -- *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program cel: 773.608.9185 email: j-kell...@northwestern.edu ***
