[RASMB] sedfit vs SOMO vs structure

John Philo jphilo at mailway.com
Fri Jul 8 08:22:35 PDT 2011


Mark,

I agree with both Arthur and Ewa that either SEC-MALS or a sedimentation
equilibrium experiment would settle this quickly, and I too have seen
proteins that are dimeric in crystals but monomeric in solution. 

But regarding the existing velocity data, what you haven't said is what
happens as a function of concentration. For any new molecule it is really
essential for proper interpretation of velocity data to do a dilution
series. It may well be that you have a weak rapid monomer-dimer association
and your 2.60 S c(s) "species" represents a dynamic mixture with a small
amount of dimer.

A further point is that c(s) is really not the best way to determine a MW
from velocity data. If the c(s) says you have only a few species, then you
should fit the data by an explicit model with that number of species (in
SEDFIT the 'non-interacting discrete species' model) to directly get the
mass and sedimentation coefficient of your main species. This will also
allow you to determine the confidence limits for those quantities, which you
cannot do with c(s).

It also seems rather surprising to me that you find a double minimum for
f/f0 in the c(s) fits. If you are fitting the meniscus and/or cell base
positions, you should probably check whether there is possibly something odd
(non-physical) going on with those.

John

-----Original Message-----
From: rasmb-bounces at rasmb.bbri.org [mailto:rasmb-bounces at rasmb.bbri.org] On
Behalf Of Arthur Rowe
Sent: Friday, July 08, 2011 7:38 AM
To: Ewa Folta-Stogniew
Cc: rasmb at server1.bbri.org
Subject: Re: [RASMB] sedfit vs SOMO vs structure

Hi Mark

The energetics of monomer-monomer interaction in the crystalline state
differ greatly from those applicable in solution. The fact that you see as
dimer by XRC is not hard evidence for the presence of a dimer in solution. I
recall working in the past on CAT (chloramphenicol
acetyltransferase) which we found to be trimeric in solution under a range
of solvent conditions: yet by XRC in the presence of Mn two of these
trimeric units were clearly associated to form a hexameric structure within
the unit cell. So I don't think that you have any real evidence from XRC to
support the presence of dimer in solution.

It's fascinating that you can get two minimal positions by fitting with
SEDFIT, but I cannot believe that the fact that initialising the f/f0 value
close to 2.3 give you a credible dimer value via c(M). But all this shows I
think is the danger of letting curve-fitting numerology overwhelm common
sense. If your dimer has so large an f/f0, then your monomer must be either
hugely swollen, immensely assymmetrical, or both. I guess that inspection of
the monomer structure within the XRC structure shows nothing of the sort?

SEC column MW's are not credible, for well-known reasons. And I don;t agree
with Ewa that having DLS on-line would totally resolve the issue, since
SEC-DLS remains a non-absolute method. SEC-MALLS of course would be
excellent - if you have the kit. But you do have an AUC, and doing a simple
overnight SE run and analysing by even the simplest software approach would
immediately tell you, on firm theoretical grounds, whether you had a 30 kDa
or a 60 kDa entity.

Regards to you, and to all

Arthur

************************************************************************
*******
Arthur J Rowe
Professor of Biomolecular Technology / Director NCMH Business Centre School
of Biosciences University of Nottingham Sutton Bonington Leics LE12 5RD

TEL:  0115 9516156

************************************************************************
*******

On Jul 8, 2011, at 13:54, Ewa Folta-Stogniew wrote:

> At 06:08 AM 7/8/2011, Mark Agacan wrote:
>
>> Hello RASMB,
>> Sedfit c(s) analysis of a protein I'm working with shows a 30 kDa 
>> peak with S = 2.60 and f/f0 of 1.2, that corresponds to monomer.
>> SOMO in Ultrascan using the pdb file for this protein predicts a 30 
>> kDa monomer at 2.42, with f/f0 of 1.19.  It's good agreement with the 
>> sedimentation velocity results.
>> However, the crystal structure, SEC, etc. indicate this protein 
>> should be a dimer.
>> Starting with the default value of 1.2 for the frictional ratio and 
>> floating in sedfit returns a value of 1.2, with MW that corresponds 
>> to monomer.  RMSD = 0.009756 Doubling this value to 2.4 and floating 
>> gives a value of 2.3, and assigns a mass that corresponds to dimer to 
>> the main peak in the c(s) analysis.  RMSD = 0.009324 Thus there 
>> appear to be two energy minima for the frictional ratio, depending 
>> which initial value is used.  The RMSD value is lower when the higher 
>> value of f/f0 is used.
>> Would you, on the basis of RMSD values, assign monomer or dimer to 
>> this peak?
>
> I would run SEC/MALLS with DLS on line- to get direct measurement of 
> MW and Rh. which will allow you to determine f/fo value. .
>
> See attached.
>
> Ewa
>
>
>> Thanks,
>> Mark
>>
>> ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
>> Dr Mark Agacan, Scientific Officer for the Division of Biological 
>> Chemistry and Drug Discovery, Wellcome Trust Biocentre, College of 
>> Life Sciences, University of Dundee, Dundee, DD1 5EH
>> Tel: +44 1382 386095    Fax: +44 1382 345764    Mobile: 07525 451 117
>> ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
>>  ************************************************************
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>>
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