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
>>
>>
>
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