Re: [ccp4bb] extra high B factor

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

Previous message:
Subject: Buried Surface Area - summary
From: Gloria Borgstahl gborgstahl {- at -} UNMC {- dot -} EDU
Date: 2007-04-30

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From: Paul Adams PDAdams {- at -} LBL {- dot -} GOV
Date: 2007-05-01

Subject: Re: extra high B factor
From: Jiamu Du jiamudu {- at -} GMAIL {- dot -} COM
Date: 2007-05-01

Thank you all.
I think i have known the reason for the low occupancy.
First, I co-crystallized the protein and peptide with a molar ratio 1:5. In
this situation, the peptide likely would fully occupy the protein binding
site. But the crystal is hard to freezen, so stepwise freezen method was
used. At this step, I did not add peptide in the cryo-protectant and this
step last for 24 hours. So, I think some peptdie in the crystal was lost at
this step.
Now, in the composite omit map, the protein-peptide binding site is clear
to identify. But 6 residues out of the 14 visible residues can only be seen
in the 2FoFc map but can not be seen in the composite omit map.

On 4/30/07, mesters wrote:
>
> Dear Jiamu Du,
>
> what were the exact concentrations (Molar values please) of protein and
> peptide in the co-crystallization experiment? This may help in
> estimating the (possible) occupancy of your peptide.
>
> Jeroen.
>
>
>
> JDwrote:
> > Does anyone know a program can perform the ocupancy refinement?
> > Or we always only refine B factor to reflect the occupancy?
> >
> > Thanks
> >
> >
> > On 4/30/07, *Eleanor Dodson*
> > > wrote:
> >
> > Well - it is extremely likely that the peptide is partially
> > occupied and
> > the occupancy may well be < 0.5..
> >
> > But at this resolution you are going to have great difficulty
> deciding
> > whether you should have
> > Occ=1.0
> >
> > Occ = 0.5
> >
> > Occ = 0.33
> >
> > As your Rfactors show it makes very little difference to any scoring
> > system..
> >
> > You can look at difference maps and try to see if one looks
> > flatter than
> > the other ..
> >
> > Even the overall Wilson plot B is not very well determined, so I
> > wouldnt
> > worry too much..
> >
> > Eleanor
> >
> > Jiamu Du wrote:
> > > Dear All:
> > > According to your suggestion, I have set the peptide's occupency
> to
> > > 0.5. Two strategies were employed.
> > > 1. Direct using Refmac restrained refinement for 10 cycles. The B
> > > factor only drops to around 100. R/Rf did not change, either.
> > > 2. Direct CNS B-fator refinemen. The B factor drops to a moderate
> > > level 60-80, and the R/Rf each increases about 2%.
> > > 3. First using CNS B-fator refinemen nad next Refmac restrained
> > > refinement. The B factor drops to 60-80, and the R/Rf did not
> > change.
> > >
> > > I think next step TLS refinement should be carried out.
> > >
> > >
> > > On 4/30/07, *Philippe DUMAS* < p.dumas@ibmc.u-strasbg.fr
> >
> > >
> > >> wrote:
> > >
> > > Jiamu
> > >
> > > According to the numbers you have mentioned I conclude that
> you
> > > peptide occupancy should be around 60-64 %
> > > I am interested to know what will be the value that you will
> > > obtain after refinement...
> > >
> > >
> > > Philippe Dumas
> > > IBMC-CNRS, UPR9002
> > > 15, rue René Descartes 67084 Strasbourg cedex
> > > tel: +33 (0)3 88 41 70 02
> > > p.dumas@ibmc.u-strasbg.fr
> >
> >>
> > >
> > >
> > > -----Message d'origine-----
> > > *De :* CCP4 bulletin board [mailto:
> > CCP4BB@JISCMAIL.AC.UK
> > >
> > >]*De la part de* Jiamu Du
> > > *Envoyé :* lundi 30 avril 2007 05:57
> > > *À :* CCP4BB@JISCMAIL.AC.UK
> >
> > >
> > > *Objet :* [ccp4bb] extra high B factor
> > >
> > > Dear All:
> > > I am refining a protein-peptide complex struture at 2.6
> > > angstrom resolution.
> > > The data was obtain from a co-crystal and the wilson B
> > factor
> > > of the data is about 70.
> > > The affinity between protein and peptide is about 10E-7 to
> > > 10E-8 molar.
> > > Protein fragment of the structure has a common B facor
> > about 50.
> > > But surprisingly, the average B factor of the peptide is
> as
> > > high as 130, although the peptide can be clearly traced
> > from
> > > the the electron density map. All residues of the
> > peptide have
> > > such a high B factor.
> > > My question is how can I reduce the abnormal high B
> > factor to
> > > a common level or if this high B factor acceptable.
> > > And another question is if this high B fator will
> influence
> > > the final refiment level.
> > >
> > > Thanks.
> > >
> > > --
> > > Jiamu Du
> > > State Key Laboratory of Molecular Biology
> > > Institute of Biochemistry and Cell Biology Shanghai
> > Institutes
> > > for Biological Sciences
> > > Chinese Academy of Sciences (CAS)
> > >
> > >
> > >
> > >
> > > --
> > > Jiamu Du
> > > State Key Laboratory of Molecular Biology
> > > Institute of Biochemistry and Cell Biology Shanghai Institutes for
> > > Biological Sciences
> > > Chinese Academy of Sciences (CAS)
> >
> >
> >
> >
> > --
> > Jiamu Du
> > State Key Laboratory of Molecular Biology
> > Institute of Biochemistry and Cell Biology Shanghai Institutes for
> > Biological Sciences
> > Chinese Academy of Sciences (CAS)
>
>
> --
> Jeroen Raymundus Mesters, Ph.D.
> Institut fuer Biochemie, Universitaet zu Luebeck
> Zentrum fuer Medizinische Struktur und Zellbiologie
> Ratzeburger Allee 160, D-23538 Luebeck
> Tel: +49-451-5004070, Fax: +49-451-5004068
> E-mail: mesters@biochem.uni-luebeck.de
> Http://www.biochem.uni-luebeck.de
> Http://www.iobcr.org
> Http://www.opticryst.org
> --
> If you can look into the seeds of time and say
> which grain will grow and which will not - speak then to me (Macbeth)
> --
>
>

--
Jiamu Du
State Key Laboratory of Molecular Biology
Institute of Biochemistry and Cell Biology Shanghai Institutes for
Biological Sciences
Chinese Academy of Sciences (CAS)

CCP4bb navigation
CCP4bb <-- 1999 <-- November 1999 <-- 30 November 1999
Previous message:
Subject: Buried Surface Area - summary
From: Gloria Borgstahl gborgstahl {- at -} UNMC {- dot -} EDU
Date: 2007-04-30 Next message:
Subject: Beam Time at ALS Sector 5.0
From: Paul Adams PDAdams {- at -} LBL {- dot -} GOV
Date: 2007-05-01