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Purification and crystallization of a non‐GluR2 AMPA‐receptor ligand‐binding domain: a case of cryo‐incompatibility addressed by room‐temperature data collection

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Glutamate is the major excitatory neurotransmitter in the brain. Among the cognate ionotropic glutamate receptors, the subfamily selective for AMPA (α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazole propionic acid) is responsible for most fast excitatory synaptic signaling and plays key roles in synaptic plasticity. AMPA receptors (AMPA‐Rs) have also been implicated in a number of neurological disorders. To investigate subunit‐specific differences in the ligand binding and activation of AMPA‐Rs, the GluR4 AMPA‐R ligand‐binding domain (LBD) was crystallized in complex with full and partial agonists. This is the first non‐GluR2 AMPA‐R LBD available for structural analysis. Standard cryoprotection protocols yielded high‐resolution diffraction from flash‐cooled crystals of the complex with the full agonist glutamate. However, for cocrystals with the partial agonist kainate, systematic screening and optimization of cryoprotection conditions yielded at best mosaic, weak diffraction at 100 K. In contrast, room‐temperature data collection from capillary‐mounted kainate cocrystals exhibited reproducible diffraction to better than 3 Å resolution. Together, these crystals lay the foundation for a structural comparison of LBD–agonist interactions in distinct AMPA‐R subunits.
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Keywords: AMPA‐receptor ion channel; GluR4 ligand‐binding domain; capillary mounting; ionotropic glutamate receptor; metal‐affinity chromatography; recombinant expression

Document Type: Research Article

Affiliations: Department of Biochemistry, Dartmouth Medical School, 7200 Vail Building, Hanover, NH 03755, USA

Publication date: September 1, 2008

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