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Improving crystal quality
Revision as of 21:33, 12 February 2009
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21:33, 12 February 2009
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==Improving diffraction quality of membrane protein crystals==
Perma-Link to original CCP4bb thread: [https://www.jiscmail.ac.uk/cgi-bin/webadmin?A2=CCP4BB;AJMLIg;20090205170801%2B1300]
===Target protein concentration===
# Concentrate protein using a higher molecular weight cutoff (e.g. 50-100 kDa). If the target protein is large enough, or oligomerises into a larger structure, a higher cutoff should allow detergent micelles to pass through. This will minimise concurrent concentration of the detergent.
# Dialyse protein overnight (routinely or after centrifugal concentration) to reduce and define the detergent concentration. This can get expensive, as relatively large buffer volumes require more detergent.
# Target can be concentrated by elution (in a small buffer volume) from a small volume of an appropriate affinity resin. This avoids detergent concentration altogether.
===Improving crystal contacts===
# 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). Detergents might also be added, below their CMC, as an additive to the crystallisation drop.
#* Use shorter detergents (e.g. Cymal-3 to -6) or mixed detergent micelles
#* Sparse matrix screens should be reconstructed for each different detergent
#* See Lemieux ''et al''. (2003) "Importance of detergent and phospholipid in the crystallization of the human erythrocyte anion-exchanger membrane domain." Protein Science 137: 322-332.
# Identify any native membrane lipids associated with the target 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)
# Post-translational modifications, such as glycosylation, are usually removed prior to crystallisation. As an alternative to removing glycans completely (e.g. as with PNGase F digestion), the target protein could be digested with Endolgycosidase H, which leaves one GlcNac residue at each glycosylation site. This aims to improve crystal contacts.
#* See Chang, V.T. ''et al''. (2007) "Glycoprotein structural genomics: solving the glycosylation problem." Structure 15(3):267-73
# [[Modifying_the_protein_to_crystallize_better | 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
#* Adding salt (or PEG) to reservoir solution may promote crystal growth in the aqueous phase, rather than the ‘oil/gel’ phase.
#* In addition to alcohols and amphiphiles, other additives should be screened for stablilisation of weak crystal forms. These might include glycerol or 0.1% agarose (low gelling-temperature).
#* Test crystallisation conditions at low temperatures (e.g. 4°C)
===Working with poor quality crystals===
#* Detergent concentration should be maintained in reservoir-based cryoprotectants. Alternatively test oils (paraffin or paraton-N) as cryoprotectants.
#* Crystal dehydration may improve quality, through reduction in solvent content and improved contacts.
#* Attempt to collect a low resolution dataset and try molecular replacement with a close homolog.
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