[RASMB] sedfit vs SOMO vs structure

Fri Jul 8 21:02:46 PDT 2011

Mark,

I have a lot of experience that the crystal structure of a protein is dimer but it is monomer in
solution.
The Kd of monomer-dimer equilibrium sometimes come close to hundreds of micro-molar or mM , in such
case it's quite difficult to show dimer presence correctly even in AUC-SE because we have to
increase concentration of protein, where non-ideal effect become dominant.
In case of AUC-SV, concentration dependence of s-value in C(s) is also very small.

I think that your protein is monomer in solution, because s-value from SOMO generally quite agree
with that derived from AUC-SV.
I agree with John about double minimum is surprising and should be solved by checking sedfit procedure.

Susumu

(2011/07/09 0:22), John Philo wrote:
> 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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