Re: [ccp4bb] detergent crystals

- Protein crystallography

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

Previous message:
Subject: Beamline Scientist PROXIMA 2
From: SHEPARD William william {- dot -} shepard {- at -} SYNCHROTRON-SOLEIL {- dot -} FR
Date: 2009-08-04

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Subject: Re: detergent crystals
From: Pascal Egea pascal {- at -} MSG {- dot -} UCSF {- dot -} EDU
Date: 2009-08-04

Subject: Re: detergent crystals
From: "Van Den Berg, Bert" Lambertus {- dot -} VandenBerg {- at -} UMASSMED {- dot -} EDU
Date: 2009-08-04

Hi Jose,

how do you know that those crystals were detergent and not protein? My impression is that it is really hard to crystallize DDM, and even harder for DM (solubilities > 20% in water). The easiest (?) way to check this may be to take some crystals, wash them well and run them out on a PAGE gel. If you don't see anything and you've taken enough crystals, then you're probably dealing with pure detergent crystals. As for your second point, you're right. For most low-cmc detergents the total detergent concentration will be substantially higher than reported, since a substantial amount is always bound to your protein. For 1 mM DDM, you would have only ~ 20 uM micelles, assuming an aggregation # of 50 (its higher). I don't think people measure the total detergent concentration in the end; for maltosides one could in principle do a Fehling's based assay to get the concentration.

Cheers, Bert

Bert van den Berg
University of Massachusetts Medical School
Program in Molecular Medicine
Biotech II, 373 Plantation Street, Suite 115
Worcester MA 01605
Phone: 508 856 1201 (office); 508 856 1211 (lab)
e-mail: bert.vandenberg@umassmed.edu
http://www.umassmed.edu/pmm/faculty/vandenberg.cfm

-----Original Message-----
From: CCP4 bulletin board on behalf of Jose Antonio Cuesta Seijo
Sent: Tue 8/4/2009 12:24 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] detergent crystals

Hi Parveen.

DDM crystals can look a bit like that. Like a pencil that has been
sharpened from both ends until there is very little pencil left, but often
the ends are flatter than that. You can get tons of these (also with DM) in
conditions with medium weight PEGs. Of course many proteins will also
crystallize as hexagonal rods with PEGs :-)
I'll take this chance to shre a thought with the community. When we say
"contains x% of detergent y", are we sure of what there is in the protein
stock? Most membrane protein stocks for crystallization will undergo a
concentration step in a filter, and the membrane proteins themselves can be
excellent detergent concentrators. For example, 0.05% DDM is about 1 mM
DDM, hardly enough to make a belt around proteins that are 100 uM or
higher.
Do people measure how much detergent there really is in the final
crystallization stock? It doesn't seem to get published, instead the
concentration in the last purification step is often mentioned.

Jose.

"Parveen Goyal" wrote:
> Hi All,
>
> I got some hexagonal crystals in one of my crystal condition. The protein
is
> a membrane protein and contains 0.05% DDM. Has anybody seen DDM crysals
and
> if yes, how do they look like?
>
> thanks in advance
>
> Parveen Goyal
>

--
***************************
Jose Antonio Cuesta-Seijo

Biophysical
Chemistry Group
Department of Chemistry
University of Copenhagen
Tlf:
+45-35320261
Universitetsparken 5
DK-2100 Copenhagen,
Denmark
***************************

CCP4bb navigation

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

Previous message:
Subject: Beamline Scientist PROXIMA 2
From: SHEPARD William william {- dot -} shepard {- at -} SYNCHROTRON-SOLEIL {- dot -} FR
Date: 2009-08-04

Next message:
Subject: Re: detergent crystals
From: Pascal Egea pascal {- at -} MSG {- dot -} UCSF {- dot -} EDU
Date: 2009-08-04