Re: [ccp4bb] SUMMARY: Poor diffraction of eukaryotic membrane protein crystals

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

Previous message:
Subject: Issue on MOSFLM 7.0.4 with image collected on NOIR detector
From: Quentin Vicens quentin {- dot -} vicens {- at -} COLORADO {- dot -} EDU
Date: 2009-02-12

Next message:
Subject: Re: Issue on MOSFLM 7.0.4 with image collected on NOIR detector
From: harry powell harry {- at -} MRC-LMB {- dot -} CAM {- dot -} AC {- dot -} UK
Date: 2009-02-12

Subject: Re: SUMMARY: Poor diffraction of eukaryotic membrane protein crystals
From: Damon Colbert d {- dot -} colbert {- at -} AUCKLAND {- dot -} AC {- dot -} NZ
Date: 2009-02-12

On request, this summary (slightly amended) has been posted to the CPP4 wiki Crystal Growth page.

You can find it here: http://strucbio.biologie.uni-konstanz.de/ccp4wiki/index.php/Improving_crystal_quality

Regards,
Damon.

Damon Colbert schrieb:
>
> Thanks to everyone who responded with most helpful advice and
> suggestions. I have provided a summary of the suggestions (and
> clarifications to questions asked of me in return).
>
> Perma-Link to original question:
> _https://www.jiscmail.ac.uk/cgi-bin/webadmin?A2=CCP4BB;AJMLIg;20090205
> 170801%2B1300_
> __________________________________________________________________
>
>
> * Concentrate protein with a higher molecular weight cutoff (e.g.
> 50-100 kDa).
>
> * Protein is known to form a tetramer, and by approximation from gel
> filtration elution, exists as a 126 kDa species (~114 kDa tetramer
> and ~22 kDa OG micelle). It usually elutes as a single,
> well-resolved peak (unless, for example, I am using it to exchange
> detergent). DLS has shown monodispersity in samples, but I don't
> use it routinely.
>
>
>
> * Dialyse protein overnight (routinely or after centrifugal
> concentration) to reduce and define the detergent concentration.
>
> * This can get expensive, using relatively large volumes detergent
> to make the dialysis buffer. Nonetheless the most recent crystals
> were obtained from dialysed protein.
>
>
>
> * Trial extraction, purification, and crystallisation with different
> detergents (using desalting or Q-sepharose columns). Poor
> diffraction could be indicative of detergent-mediated crystal
> contacts (rather than protein-protein).
>
> * Use of shorter detergents (e.g. Cymal-3 to -6) or mixed detergent
> micelles
> * Reconstruct sparse matrix screens with each different detergent
> * See Lemieux /et al/. (2003), Protein Science.
>
>
>
> * Identify membrane lipids associated with protein (in-house by TLC
> or otherwise). Retaining some native lipid or adding it back in
> at crystallization may improve crystal quality. Conversely total
> delipidation may also help.
>
> * Need to correlate successful crystallisation with presence/absence
> of lipid
> * Could try using lipid-like detergents (FC or DHPC)
>
>
>
> * Deglycosylation is checked on SDS-PAGE, and confirmed by the loss
> of higher molecular weight smears (which are present before
> deglycosylation reaction).
>
> * Alternatively protein could be digested with Endolgycosidase H,
> which leaves one GlcNac residue on each glycosylation site. This
> could improve crystal contacts. See Chang, V.T. /et al/. (2007)
> "Glycoprotein structural genomics: solving the glycosylation
> problem." Structure 15(3):267-73
>
>
>
> * Chemical modification of surface residues may improve crystal
> contacts, for example lysine methylation.
>
> * See Walter /et al/. (2006) "Lysine methylation as a routine rescue
> strategy for protein crystallization." Structure 14(11):1617-22
> * Mutagenesis is another alternative, but we have not yet been
> successful producing a recombinant protein.
>
>
>
> * Adding salt (or PEG) to reservoir solution may promote crystal
> growth in the aqueous phase, rather than the 'oil/gel' phase.
>
> * Conditions producing the crystals grown in this 'gel' had PEG 1K
> or 2K as precipitant, and low [NaCl] present. (Is the suggestion
> 'to increase the concentration beyond that of the reservoir
> solution?').
>
>
>
> * Test crystallisation conditions at low temperature (e.g. 4°C)
>
>
>
> * Test oils (paraffin or paraton-N) as cryoprotectants.
> Alternatively maintain detergent concentration in cryoprotectant.
>
> * Paratone oil (softened with some mineral oil) was used with poorly
> diffracting native crystals, and showed no improvement in
> diffraction. It has not been attempted with more recent protein
> crystals grown in presence of ligand.
>
>
>
> * Attempt to collect a 10Ang dataset and try MR with a close homolog.
>
>
>
> Many thanks.
>
> Regards,
> Damon.

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

Previous message:
Subject: Issue on MOSFLM 7.0.4 with image collected on NOIR detector
From: Quentin Vicens quentin {- dot -} vicens {- at -} COLORADO {- dot -} EDU
Date: 2009-02-12

Next message:
Subject: Re: Issue on MOSFLM 7.0.4 with image collected on NOIR detector
From: harry powell harry {- at -} MRC-LMB {- dot -} CAM {- dot -} AC {- dot -} UK
Date: 2009-02-12