modelling a flexible peptide

From: intekhab alam
Date: 4 April 2012 02:37

Hi All

I have a 3.0A dataset (SG P1211) of a protein-protein complex having mol.wt 60 and 8 Kda respectively.

Molecular repalcement (60Kda protein as template) with Phaser gave a solution with 6 molecules in ASU.

A continuous density is also obersved near two different chains which i consider as the second protein.

I refined the density using a poly Alanine model but still i can't recognise the side chains confidently for modelling.

Considering the fact that the smaller protein partner is rich in lysine, arginine, Asp and Glutamate with only 3 tyr and 4 phe,

i tried to modell fragments one by one but the B-factor of the segments are quite high (in the range of 110)

what will be the best strategy to improve the map for modelling.

regards

--
INTEKHAB ALAM

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From: Zhijie Li

Hi Intekhab,

With 6 copies of the complex in ASU, NCS averaging might give you a better map. Uppsala software factory has everything you need to do that: http://xray.bmc.uu.se/usf/. Check the RAVE package. Particularly, have a look at the average.csh script listed in the RAVE package page in the "related freebies" section. That is a script made by Gerard Kleywegt and Tom Taylor for doing N cycles of map averaging. You can modify it for your own use. 

I owe my own thanks to the authors too.

Zhijie

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From: Bosch, Juergen

As already suggested you should use NCS averaging, but I would go the Parrot way http://www.ccp4.ac.uk/html/parrot.html or DM using a mask for your monomer of the hexamer and first ignoring the peptide. Do you have a ring or a dimer of trimers ?

If however your two peptides follow NCS as well you might define a separate NCS operator for two-fold averaging of the peptide.

The improved map can then be exposed to Buccaneer (http://www.ysbl.york.ac.uk/~cowtan/buccaneer/buccaneer.html)  for extension of your current model and possible automatic fitting of your two peptide chains.

Jürgen

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Jürgen Bosch