The serotonin 2A receptor (5-HT 2A R) is an important member of the G-protein coupled receptor (GPCR) family involved in an array of neuromodulatory functions. Although the high-resolution structures of truncated versions of GPCRs, captured in ligand-bound conformational states, are available, the structures lack several functional regions, which have crucial roles in receptor response. Here, in order to understand the structure and dynamics of the ligand-free form of the receptor, we have performed meticulous modeling of the 5-HT 2A R with the third intracellular loop (ICL3). Our analyses revealed that the ligand-free ground state structure of 5-HT 2A R has marked distinction with ligand-bound conformations of 5-HT 2 subfamily proteins and exhibits extensive backbone flexibility across the loop regions, suggesting the importance of purifying the receptor in its native form for further studies. Hence, we have standardized a strategy that efficiently increases the expression of 5-HT 2A R by infecting Sf9 cells with a very low multiplicity of infection of baculovirus in conjunction with production boost additive and subsequently, purify the full-length receptor. Furthermore, we have optimized the selective over-expression of glycosylated and nonglycosylated forms of the receptor merely by switching the postinfection growth time, a method that has not been reported earlier. K E Y W O R D S 5-HT 2A GPCR, baculoviral expression system, homology modeling, molecular dynamics simulation, protein over-expression and purification
A giant technological
leap in the field of cryo-electron microscopy
(cryo-EM) has assured the achievement of near-atomic resolution structures
of biological macromolecules. As a recognition of this accomplishment,
the Nobel Prize in Chemistry was awarded in 2017 to Jacques Dubochet,
Joachim Frank, and Richard Henderson, the pioneers in the field of
cryo-EM. Currently, the technique has become the method of choice
for structural analysis of heterogeneous and intrinsically dynamic
biological complexes. In the past few years, the most prolific branch
of cryo-EM, single particle analysis, has revolutionized the structural
biology field and structural investigation of membrane proteins, in
particular. To achieve high-resolution structures of macromolecules
in noncrystalline specimens, from sample and grid preparation to image
acquisition, image data processing, and analysis of 3D maps, methodological
advances in each of the steps play critical roles. In this Review,
we discuss two areas in single particle cryo-EM, the remarkable developments
in sample preparation strategies, particularly for membrane proteins,
and breakthroughs in methodologies for molecular model building on
the high-resolution 3D density maps that brought promises to resolve
high-resolution (<4 Å) structures of biological macromolecules.
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