Re: [ccp4bb] Truncated protein structure

[junfeng liu]

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 <[EMAIL PROTECTED]>:

   
 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 <[EMAIL PROTECTED]>
*To:* CCP4BB@JISCMAIL.AC.UK
*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