Without wishing to get into the terminology, and going back to the OP’s question: our crystal structures of factor H 19-20 would be a citeable example. In our hands, the wt 19-20 preferentially crystallises as a well-ordered D2 tetramer (cf Jokiranta et al 2006, EMBO J) that is completely non-physiological as far as we know. In order to obtain complexes of FH19-20 with relevant ligands (C3D, OspE), we have used a double-mutant that eliminates some of the crystal packing contents (Kajander et al, PNAS; Bhattarchajee et al, JBC).
Adrian
On 8 Apr 2015, at 16:27, R. M. Garavito <[email protected]> wrote:
> Thierry,
>
> I need to point out there is no outside work as it is one system, but with
> multiple phases. Protein and nucleic acids are not true crystals in the
> classic sense, but highly hydrated ordered colloids (in the 1930's some
> called them "crystalloids" because bulk water is such a major and critical
> component, unlike small molecule crystals). It is colloidal physical
> chemistry at work. Thus, the water argument for a "force" does not hold,
> rather the system just comes to an energy minimum where two stable phases are
> formed (one being the crystal).
>
> My complaint is that we use terms that imply the wrong physical behavior,
> which then obscure the true issues. For example, every protein is packable
> from a purely physical standpoint; physical shape is not the issue, but the
> balancing of favorable and unfavorable interactions is. Crystallization is a
> balance between many global and local interactions.
>
> Michael
>
> ****************************************************************
> R. Michael Garavito, Ph.D.
> Professor of Biochemistry & Molecular Biology
> 603 Wilson Rd., Rm. 513
> Michigan State University
> East Lansing, MI 48824-1319
> Office: (517) 355-9724 Lab: (517) 353-9125
> FAX: (517) 353-9334 Email: [email protected]
> ****************************************************************
>
>
>
>
> On Apr 8, 2015, at 10:52 AM, "Fischmann, Thierry"
> <[email protected]> wrote:
>
>> Some counter-arguments to Michael :
>>
>> There is an “outside force doing the work”: macromolecule crystallization
>> except rare exceptions is driven by competition for water molecules between
>> the macromolecule and the precipitant. The exceptions are crystallization
>> against low salt buffer, in which case the process is driven by hydrophobic
>> “forces”.
>>
>> And “packable” may play a role. A molecule which is of such shape and
>> surface charge distribution that there is no way to pack it in a regular
>> lattice will never crystallize.
>>
>> Regarding the dimer vs. monomer debate, crystallization acts as a
>> purification step. It seems perfectly plausible that crystal growth would
>> “select” the monomeric state if dimers cannot be included in the growing
>> crystal lattice, regardless of whether one is more soluble than the other.
>> It all comes down to the initial crystal seed favored by the crystallization
>> conditions. On a separate note, protein which forms dimers in solution trend
>> to be more soluble in dimeric state than as monomers because dimerization
>> usually buries a significant hydrophobic patch of molecular surface. If
>> crystallization was only “selecting for the least soluble” oligomeric state
>> we would rarely crystallize proteins as dimers.
>>
>> Crystallization is such a confusing process J
>>
>> Thierry
>>
>> From: CCP4 bulletin board [mailto:[email protected]] On Behalf Of R. M.
>> Garavito
>> Sent: Wednesday, April 08, 2015 10:04 AM
>> To: [email protected]
>> Subject: Re: [ccp4bb] Crystallisation of a minority fraction monomers
>>
>> I just wanted to disagree with Roger's word choice, but not his argument
>> (this is a "flame"-free response). Forget about "packing" and "packable" as
>> there is no outside force doing the work. The molecules are just falling
>> into a local energy minimum where favorable intra- and intermolecular
>> interactions predominate. It is difference in the behavior of the ensemble
>> versus of a solubilized, dispersed species (be it monomer or dimer). It is
>> a phase behavior issue. Concerning Sebastian's case, while it is uncommon,
>> the idea that a monomer has a crystalline phase state while the dimer does
>> not is perfectly reasonable, and the crystals of the monomer grow due to
>> mass action. I am sure the number of verified examples of this are limited.
>> However, there are many cases where dimeric and tetrameric enzymes can be
>> shown to be fully saturated with one or another bound substrate in solution,
>> but show one or more empty active sites in the crystal. I know of several
>> cases where this occurs, showing that selection of the species with the best
>> set of favorable intra- and intermolecular interactions occurs.
>>
>> Regards,
>>
>> Michael
>>
>> ****************************************************************
>> R. Michael Garavito, Ph.D.
>> Professor of Biochemistry & Molecular Biology
>> 603 Wilson Rd., Rm. 513
>> Michigan State University
>> East Lansing, MI 48824-1319
>> Office: (517) 355-9724 Lab: (517) 353-9125
>> FAX: (517) 353-9334 Email: [email protected]
>> ****************************************************************
>>
>>
>>
>>
>> On Apr 8, 2015, at 9:28 AM, Roger Rowlett <[email protected]> wrote:
>>
>>
>> The problem with crystallization is that is selects for the least soluble,
>> most packable species. Sometimes that works against what you would like to
>> know. That could include oligomerization state as well as conformational
>> state. For example, some of the allosteric carbonic anhydrases stubbornly
>> crystallize only in the T-state, despite crystallization conditions that are
>> known to preferentially stabilize the R-state, and for which the predominant
>> R-state population can be confirmed by other methods.
>>
>> Cheers,
>>
>> _______________________________________
>> Roger S. Rowlett
>> Gordon & Dorothy Kline Professor
>> Department of Chemistry
>> Colgate University
>> 13 Oak Drive
>> Hamilton, NY 13346
>>
>> tel: (315)-228-7245
>> ofc: (315)-228-7395
>> fax: (315)-228-7935
>> email: [email protected]
>>
>> On 4/8/2015 9:07 AM, Sebastiaan Werten wrote:
>> Dear all,
>>
>> we are currently working on a protein that is known to exist in a
>> monomer-dimer equilibrium. At the high concentrations used for
>> crystallisation assays, the dimer is predominant and the monomer practically
>> undetectable.
>>
>> Nevertheless, one of the crystal forms that we have obtained contains the
>> monomeric species, not the dimer.
>>
>> I was wondering if anyone is aware of similar (published) cases, and if the
>> phenomenon as such has been discussed in detail anywhere?
>>
>> I did literature searches but so far couldn't find anything.
>>
>> Any pointers would be much appreciated!
>>
>> Best wishes,
>>
>> Sebastiaan Werten.
>>
>>
>>
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