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Hi ,Haitao , If I were you ,the first thing I will do is to check xtal using MS and N-terminal sequencing to know what it is . These need to be done anyway. At the same time ,try to use the ways describe by other people to handle the data and search for solution using truncated model without flexible region and see what will be obtained . Maybe you will be lucky and build your model soon. Good luck! leo Eleanor Dodson wrote: > If the structure factors are available for the original protein you > can use ALMN to check if there is an agreement between > the two data sets. > > (Very old technology but a useful trick) > > Other things to check - Does the crystallographic 2fold in P43212 > generate a tight dimer? > > is there a non-crystallographic 2fold or a non-cryst translation in > the P2 21 21 crystal? > > (If so maybe you should search with the P43212 dimer..) > > I would expect the MR to give a solution even if your molecule is > truncated. You will need to turn off the packing checks but there > still should be a strong signal. > > Eleanor > > > Lijun Liu wrote: >> The convention for "P22121"s is P21212, which is used by both CNS and >> CCP4 >> and many else, if not all. The unit cell needs to be reindexed >> (a-b-c --> >> b-c-a in your case). Then please try again. Lijun >> >> >> >>> Thank you for your suggestions. >>> 1. The unit cell of my crystal is 47.41 99.67 114.97 90 90 90 space >>> group >>> P22121, different from PDB structure 64 64 113 90 90 90 P43212, >>> which has >>> the same growth condition. >>> 2. Mathhews_coef indicate my crystal should be dimer if ~30kD. I >>> used all >>> monomer, dimer and tetramer PDB templates and their truncated >>> models, but >>> all high R-factor. >>> 3. My protein has only one domain(ligand binding domain), and there are >>> both >>> structures reported with or without ligand. >>> >>> I think even though my protein was degraded, the structure can be >>> determined >>> due to its remained same sequence. >>> So now do I have to turn to mass spec? >>> >>> 2008/7/8 Zhijie Li <[log in to unmask]>: >>> >>> >>>> Hi Haitao, >>>> >>>> I need to ask you a few questions first: >>>> >>>> 1. Did you mean you could not solve your structure by molecular >>>> replacement? Did you compare your crystal's unit cell with the PDB >>>> file? >>>> Are >>>> they significantly different? If the assymetric unit has more than one >>>> monomer, have you tried doing a molecular replacement search with one >>>> monomer only? >>>> >>>> 2. Can you give us the PDB number so that we can take a look at the >>>> protein? The reason for that is, I suspect that your protein was not >>>> degraded from either end, but only got clipped some where on the >>>> surface >>>> - >>>> so the structure is basically unperturbed. >>>> >>>> If the PDB structure turns out to be single-domain, then you should do >>>> your >>>> molecular replacement search with the whole protein. If it is a >>>> two-domain >>>> structure, and one of them is ~20kD, then try use that 20kD domain >>>> to do >>>> the >>>> search again. >>>> >>>> R-fac~=0.5 is probably saying that your current solution is totally >>>> wrong. >>>> As I remember, R-factor for a totally radom acentric (for example, >>>> protein) >>>> structure is 0.59. >>>> >>>> Also, Even if you follow the published crystallization conditions, >>>> your >>>> protein may still crystallize in a totally different way. But >>>> unless the >>>> protein itself has changed its shape (which normally does not happen), >>>> you >>>> should be able to do a molecular replacement. >>>> >>>> If molecular replacement does not work at all, then maybe it is time >>>> to send your sample to mass spec to see what it really is. But I >>>> highly >>>> suspect what you need to do now is nothing but to optimize your >>>> molecular >>>> replacement parameters. >>>> >>>> Zhijie Li >>>> Graduate student, Univeristy of Toronto >>>> >>>> >>>> >>>> ----- Original Message ----- >>>> >>>> *From:* Haitao ZHANG <[log in to unmask]> >>>> *To:* [log in to unmask] >>>> *Sent:* Monday, July 07, 2008 10:27 PM >>>> *Subject:* [ccp4bb] Truncated protein structure >>>> >>>> Dear all, >>>> I repeated a protein crystallization which is reported in PDB with >>>> the >>>> almost same condition, and got a 2.6A data. But the problem is that I >>>> can >>>> not determine the right phases with neither CCP4 nor CNS. >>>> Then I found the protein in my crystal had been degraded from ~30kD to >>>> ~20kD by SDS-PAGE, but did not know from which terminus it was >>>> truncated. >>>> So I used truncated PDB templates which N-, C- or both terminal >>>> were cut >>>> to >>>> fit the length of my shorter protein, and tried many different >>>> templates. >>>> But always high R-factor (~0.5). >>>> With COOT I found the backbone did not fill the electron density map >>>> perfectly. >>>> The only difference of my crystallization condition is 277K(mine) >>>> *vs*295K(reported) in temprature. >>>> >>>> Any suggestions will be appreciated. >>>> >>>> >>>> Haitao ZHANG, Ph.D Student, >>>> Shanghai Institute of Materia Medica, >>>> Chinese Academy of Sciences >>>> >>>> >>>> >> >> >> >