Here are
a couple points on the process of this discovery- for those of you who are
interested in methodology and lab protocols!
- The zebrafish α1 GlyR (NP_571477) was isolated for
structural elucidation as it shares 92% sequence similarity with the human α1
GlyR and modified to resemble the human phenotype.
- GlyREM was then subcloned into a pGEM vector for transcription using Ambion, to be used for two-electrode voltage clamp electrophysiology (TEVC). TEVC is used to measure the ion currents through cell membranes.
- Crystalline structures of GlyREM were
obtained using the hanging-drop vapour diffusion method with the data being collected using a
Titan Krios cryo-electron microscope. Super resolution images were then
determined using the Gatan K2 Summit direct electron detector.
And a little background about cryo-electron microscopy (cryo-EM)...
Cryo-EM is transmission electron microscopy at cryogenic temperatures. Contrary to X-ray crystallography, this allows for structures to be determined in their native environment. Recent advances in cryo-EM have resulted in structural elucidation at very high resolutions. In June 2015, a map of a complex between E. coli β-galactosidase and inhibitor phenylethyl-β-D-thiogalactopyranoside was resolved to 2.2 Å, the highest resolution to date.
Read more about this here: http://www.nature.com/nmeth/journal/v12/n7/full/nmeth.3469.html.
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