CCP4 wiki - User contributions [en]

Improving crystal quality

2009-02-12T22:44:33Z

D Colbert: /* Improving diffraction quality of membrane protein crystals */

==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

===Crystallisation micro-environment===

#* 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.

Improving crystal quality

2009-02-12T21:33:36Z

D Colbert:

==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

===Crystallisation micro-environment===

#* 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.

Crystals

2009-02-12T19:50:22Z

D Colbert: /* Crystal Growth */

== Crystal Characterization ==
* [[Birefringence]]

== Crystal Imperfections ==
* [[Diffuse Scattering]]
* [[Incommensurate Lattices]]

== Crystal Growth ==

* [[Modifying the protein to crystallize better]]
* [[Crystallization screens and methods]]
* [[I have crystals, but are they salt?]]
* [[Conditions prone to salt crystallization]]
* [[Biophysical methods]] for evaluation of crystallization tendency
* [[Improving crystal quality]]
* [[Robots for crystallization setup]]
* [[Visualization of crystal growth]]
* [[Crystal mounting]]
* [[Robots for crystal mounting]]
* [[Robots for crystallization monitoring]]
* [[Crystal growth: Tips and Tricks]]

== Crystal handling ==
* [[Sticky crystals]]

== Data collection ==
* [[X-ray generators and detectors]] (experiences)
* [[Synchrotrons]]
* [[Cryo]]
* [[Strategies]]
* [[Data quality]]
* [[Radiation damage]]
* [[Data collection: Tips and Tricks]]

== References ==

* crystallization of soluble proteins

* crystallization of membrane proteins
# Wiener MC (2004) A pedestrian guide to membrane protein crystallization. Methods 34(3), 364-372.
# Loll PJ (2003) Membrane protein structural biology: the high throughput challenge. J. Struct. Biol. 142(1), 144-153.
# Methods and Results in Crystallization of Membrane Proteins Iwata S (Ed.), San Diego, CA: International University Line. (2003)
# Macromolecular Crystallography Protocols, Volume 1: Preparation and Crystallization of Macromolecules. Doublie S (Ed.), Totowa, NJ: Humana Press. (2007)
# Structural Biology of Membrane Proteins. Grisshammer R, & Buchanan SK (Eds.), Cambridge, UK: Royal Society of Chemistry. (2006)