Crystallization of an AMPA receptor binding domain without agonist: importance of carbohydrate content and flash-cooling conditions.
An AMPA-specific ionotropic glutamate receptor binding domain was overexpressed using the baculovirus system and purified by immunoaffinity and metal-affinity chromatography. Purified protein was enzymatically deglycosylated. Both glycosylated and deglycosylated proteins crystallized under the same conditions and in the same space group (P2). In both cases, it was observed that the use of MPD as a cryoprotectant induced a significant reduction in the unit-cell volume compared with glycerol or sucrose. For crystals of deglycosylated protein, cryoprotection with MPD also yielded a dramatic improvement in resolution.
Féthière
J
J
Ion Channel Structure Research Group, Max Planck Institute for Medical Research, Jahnstrasse 29, 69120 Heidelberg, Germany.
Andersson
A
A
Keinänen
K
K
Madden
D R
DR
D000818:Animals; D002241:Carbohydrates / Q000032:analysis / Q000737:chemistry*; D003460:Crystallization; D006031:Glycosylation; D007313:Insecta; D011487:Protein Conformation; D018091:Receptors, AMPA / Q000819:agonists / Q000737:chemistry* / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry; D014961:X-Ray Diffraction
D002241:Carbohydrates; D018091:Receptors, AMPA; D011994:Recombinant Proteins; C115953:glutamate receptor type B