Re: [ccp4bb] determining the domain for overexpression and crystallization

[Xianhui Wu]

Dear all,

Thank you very much for all of information about domain determination! I
think the limited proteolysis is a good choice for the domain determination
as we have no information about a protein.

  However, if we do have a domain information by the bioinformatics, how can
we truncate the domain? Are we need to keep three or five more amino acids
in two ends? Thanks.

Best regards,
Xianhui


On Tue, Mar 8, 2011 at 2:22 AM, Mark J van Raaij mjvanra...@cnb.csic.eswrote:

 for an experimental way to determine soluble domains see the following
 paper:
 ESPRIT: an automated, library-based method for mapping and soluble
 expression of protein domains from challenging targets.
 Yumerefendi H, Tarendeau F, Mas PJ, Hart DJ.
 J Struct Biol. 2010 Oct;172(1):66-74. Epub 2010 Mar 4. Review.
 PMID: 20206698 [PubMed - indexed for MEDLINE]



 Mark J van Raaij
 Laboratorio M-4
 Dpto de Estructura de Macromoleculas
 Centro Nacional de Biotecnologia - CSIC
 c/Darwin 3, Campus Cantoblanco
 E-28049 Madrid, Spain
 tel. (+34) 91 585 4616

 http://www.cnb.csic.es/content/research/macromolecular/mvraaij/index.php?l=1



 On 7 Mar 2011, at 19:18, gauri misra wrote:

  Hi,
  To start with it would be great if you look in to the secondary structure
 prediction of the sequence using any of the standard servers like PSIPRED,
 JPRED etc. Many more available at expasy site http://ca.expasy.org/tools/.
  Whatever construct you finally choose to make just remember the standard
 rule that we generally follow is to avoid deleting the alpha helices and
 beta sheets. You can design your initial primers so as to obtain the
 complete amplification of these secondary structures from any part within
 the protein.
  You can even use the various modules of the following online available
 server
  http://scratch.proteomics.ics.uci.edu/
   to have an idea of the intrinsically disordered regions in the protein,
 transmemebrane regions and disulfide bonds that would certainly help you in
 initiating in the right direction.
 
  Best wishes
  Gauri
 
  On Mon, Mar 7, 2011 at 4:10 AM, Xianhui Wu wuxian...@gmail.com wrote:
  Dear all,
 
Before we try to study the crystal structure of an unknown protein, we
 need to determine the sequence that can fold into a compact and stable 3D
 domain. What kinds of methods can we choose?
 
  --
  Best regards,
  XH Wu
 




-- 
Best regards,
Xianhui

[ccp4bb] determining the domain for overexpression and crystallization

[Xianhui Wu]

Dear all,

  Before we try to study the crystal structure of an unknown protein, we
need to determine the sequence that can fold into a compact and stable 3D
domain. What kinds of methods can we choose?

-- 
Best regards,
XH Wu

[ccp4bb] molecular replacement

[Xianhui Wu]

Dear All,

   I am trying to resolve a protein structure with the use of Molecular
Replacement. However, some part of protein are overlap in the interface of
homodimer. Would someone please give me suggestions? Thank you!

-- 
Best regards,
WuXH

Re: [ccp4bb] Unexplained density

[Xianhui Wu]

I think it could be zinc ion.

On Fri, Apr 30, 2010 at 5:38 AM, Daniel Bonsor bon...@bbri.org wrote:

 Hello again

 I currently have some unexplained density in my structure. As you can
 hopefully see from the images (see file), the density is dumbbell shaped.
 Whatever it is, it is coordinated by Asp and Glu residues. To me it looks
 like each lobe is a ring structure.


 The crystallization condition was:
 6.5% PEG 8K, 10mM ZnSO4, 100mM sodium cacodylate pH 6.5, 100mM Am2SO4, 1%
 glycerol, with 20% glycerol as cryo.
 Protein was originally in 50mM Tris, 50mM NaCl pH 7.5.


 I originally placed a single Zn at the center of each lobe. Though after
 refmac, the Zn was displaced to one side. Two zincs in each dumbbell may
 have worked, but I am dubious about two zinc atoms being 4A apart and there
 is still some unexplained density. Are there any possible cyclization
 reactions of Tris, cacodylate or glycerol may have undergone to explain the
 density? Or is it simply a highly ordered water network? Or is there some
 other explanation?

 Thanks in advance

 Dan




-- 
Best regards,
Xianhui