Crystallization of an AMPA receptor binding domain without agonist: importance of carbohydrate content and flash-cooling conditions

• Published in: Acta crystallographica. Section D, Biological crystallography. Vol. 56; no. 12; pp. 1625 - 1629
• Main Authors: Féthière, James, Andersson, Arnold, Keinänen, Kari, Madden, Dean R.
• Format: Journal Article
• Language: English
• Published: 5 Abbey Square, Chester, Cheshire CH1 2HU, England Munksgaard International Publishers 01.12.2000
• Subjects: AMPA receptor domain; Animals; Carbohydrates - analysis; Carbohydrates - chemistry; Crystallization; Glycosylation; Insecta; Protein Conformation; Receptors, AMPA - agonists; Receptors, AMPA - chemistry; Receptors, AMPA - genetics; Recombinant Proteins - chemistry; X-Ray Diffraction
• ISSN: 1399-0047; 0907-4449; 1399-0047
• DOI: 10.1107/S0907444900014104

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.