For higher completeness - the NMR structure that showed FH19-20 is a
monomer in solution is in AP Herbert et al, JBC 2006. I agree that
it's a very impressive example of how deceiving crystal assembly can
be. Although I think with regard to the original question it is much
more common to observe such apparent higher order oligomers in
crystals when compared to solution than the opposite scenario for
which Sebastian asked.

Bärbel


Zitat von Adrian Goldman <[log in to unmask]>:

> 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 <[log in to unmask]> 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: [log in to unmask]
>> ****************************************************************
>>
>>
>>
>>
>> On Apr 8, 2015, at 10:52 AM, "Fischmann, Thierry"
>> <[log in to unmask]> 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:[log in to unmask]] On Behalf
>>> Of R. M. Garavito
>>> Sent: Wednesday, April 08, 2015 10:04 AM
>>> To: [log in to unmask]
>>> 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: [log in to unmask]
>>> ****************************************************************
>>>
>>>
>>>
>>>
>>> On Apr 8, 2015, at 9:28 AM, Roger Rowlett <[log in to unmask]> 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: [log in to unmask]
>>>
>>> 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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>>
>
>



--
Bärbel Blaum, Ph.D.
Interfakultäres Institut für Biochemie (IFIB)
Hoppe-Seyler-Strasse 4
D-72076 Tübingen
Germany
+49 70 71 29 75 359
http://www.ifib.uni-tuebingen.de/research/blaum.html