Re: [ccp4bb] ligand refinemnet

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CCP4bb <-- 1999 <-- November 1999 <-- 30 November 1999

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Subject: ligand refinemnet
From: peter hudson peter {- dot -} hudson {- dot -} peter {- at -} GMAIL {- dot -} COM
Date: 2009-04-02

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Subject: Re: ligand refinemnet
From: Tim Gruene tg {- at -} SHELX {- dot -} UNI-AC {- dot -} GWDG {- dot -} DE
Date: 2009-04-02

Subject: Re: ligand refinemnet
From: Kontopidis George gkontopidis {- at -} VET {- dot -} UTH {- dot -} GR
Date: 2009-04-02

Peter from you description seems that big blob is not your ligand. In 3.0
resolution molecules with similar size, including ligands, tend to look
similar in shape as well. I suggest you refine further your structure and
check again later this N-terminal blob of density. It may tern out to be a
bias from your molecular replacement model. If it is still there I will
suggest to rum a MS and compare the MW with the one from your protein
structure. That will give you an indication that probable you have something
attached to N-terminal of your protein.

George

_____

From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of peter
hudson
Sent: Thursday, April 02, 2009 9:55 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] ligand refinemnet

Hello all

I have refined and built model a dataset of 3.0A resolution dataset. This
model is assocaied with a ligand. After final refinement and model building
i found a big blob of Fo-Fc density of around 4sigma level at the
N-terminal of the fianl model. My protein doesnot carry any tag at the
N-terminal. But, the template i have used for the molecular replacement
carrying a tag at the N-termianl, which can occupy only 1/4th of this
positive density after superpostion. My crystallisation conditions component
cannot occupy such higher level of positive density. Since my protein binds
to a ligand, i looked carefully to the positive density and it seems very
similar to the ligand density but its obscure. Refinement after fitting the
ligand leads to very high B-factor of the ligan, which vary for various
atoms from 50-90 and simultaneously positive density goes off. I also tried
to change the occupancy of the ligand but as i reduce the occupancy the B
factor comes down at the noraml expected average B factor value but again
Fo-Fc density appears in the map over ligand. If i leave to refine the
occupancy to the programme automatically, this lead to the zero occupancy of
the few atoms in the ligand and avearge B factor remains normal.
suggestions would be appreciated.

Thanks in advance
Peter

CCP4bb navigation

CCP4bb <-- 1999 <-- November 1999 <-- 30 November 1999

Previous message:
Subject: ligand refinemnet
From: peter hudson peter {- dot -} hudson {- dot -} peter {- at -} GMAIL {- dot -} COM
Date: 2009-04-02

Next message:
Subject: Re: ligand refinemnet
From: Tim Gruene tg {- at -} SHELX {- dot -} UNI-AC {- dot -} GWDG {- dot -} DE
Date: 2009-04-02