Improving crystal quality

Improving diffraction quality of membrane protein crystals

Perma-Link to original CCP4bb thread: [1]

Target protein concentration

1. 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.
2. 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.
3. 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

1. 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.
2. 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)
3. 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
4. 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

Crystallisation micro-environment

1. • 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

1. • 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.