Helping to solve the mystery of RSV

Earlier this year, the labs of Xavier Saelens at Ghent University and VIB in Belgium and Jason S. McLellan at Dartmouth College made an important breakthrough by identifying two single-domain antibodies that neutralize the human respiratory syncytial virus (RSV). This work was published in Nature Communications in February, 2017(1). RSV is a leading cause of respiratory tract infections in infants and young children. Roughly 9% of children infected with RSV annually develop more serious complications, such as bronchiolitis, necessitating hospitalization. 
 
This work describes the identification of two single-domain antibodies (VHH) that bind to the RSV protein F in its prefusion state. RSV F, a trimeric class I fusion protein, is essential for infection.  Both VHHs, F-VHH-4 and F-VHH-L66, are potent in neutralizing RSV and bind to the RSV prefusion F protein with picomolar affinity. To define the epitopes on the RSV F protein recognized by each VHH, the co-crystal structures of each of these antibodies bound to the RSV F protein were determined. These structures show that F-VHH-4 and F-VHH-L66 bind in a nearly identical manner to a cavity formed by the boundary of two F protomers. This work is an important step forward in the development of effective therapeutics against RSV.
 
Crystals of the F-VHH-L66 in complex with the RSV F protein (PDB: 5TOK) were obtained using the free-interface diffusion method in the Microlytic Crystal Former. Initial hits of this complex were identified in vapor diffusion, but after extensive optimization, diffraction was limited to approximately 4.5 Å. The crystallization conditions identified in vapor diffusion were setup in the Microlytic Crystal Former, harvested directly from the capillary, and diffraction improved to 3.8 Å.
 
The Microlytic Crystal Former
 
As an alternative to vapor diffusion and other crystallization methods, the Microlytic Crystal Former utilizes the method of free-interface diffusion to increase crystallization returns in an easy to use and efficient manner (2). Comprising 96 individual microchannels the Microlytic Crystal Former is a fully SBS-compliant crystallization plate, meaning that it is compatible with the most common crystallization and imaging robots, as well as manual set-up and inspection. By using the Microlytic Crystal Former you can screen a much greater area of crystallization space on one plate with fewer conditions and less protein consumption. 

The end result? An increased probability of crystallization success by at least two-fold over vapor diffusion. 

References:

1. Rossey, I., et. al (2017) Nature Communications 13(8), 14158.
2. Stojanoff, V., et. al (2011) Acta Crystallogr Sect F Struct Biol Cryst Commun. 67(Pt 8), 971-975.