The loss of trimer contacts and the Gln226→Leu mutation contribute to destabilize the 220 loop in the H5.3-H5hd_rdt_Vn structure. In gray, with the 220 loop highlighted in yellow, is the trimeric VN/1203 rdt H5 ectodomain (PDB ID code 4BH2). Intersubunit H-bonds are shown as dotted lines between the 220 loop (yellow) and a neighboring protomer (blue-gray). These interactions are lost in monomeric head domain constructs. Additionally, the loss of the hydrogen bond between Gln226 and Asp103 likely further destabilizes the 220 loop. In the figure, the H5.3-H5hd_rdt_Vn structure (H5hd_rdt_Vn in light green, H5.3 heavy chain in teal, H5.3 light chain in purple) is aligned to both wt_H5hd (gold) and a protomer of the VN/1203 rdt trimeric H5 ectodomain (PDB ID code 4BH2) protomer (gray). Additional H5 ectodomain protomers are shown as blue-gray (front, left) and light gray (back, left). The last structured residues in the 220 loop from the H5.3-H5hd_rdt_Vn structure are circled.