Extended Data Fig. 2: Identification, data collection, and processing of Cv-HAS bound to two nanobodies and UDP.

(a) Increased melting temperature of Cv-HAS in the presence of Nb872. Protein melting was measured based on enzymatic activity detected by quantifying the release of UDP in real time. (b) HA biosynthesis in the presence of the indicated nanobodies and based on quantification of ³H-labelled HA by scintillation counting. Data is normalized relative to product yields in the absence of nanobodies. Error bars represent deviations from the means with n = 3 independent experiments. (c) Representative autoradiography of ¹⁴C-labelled HA produced in the presence of the indicated nanobodies. The experiment has been repeated at least 4 times with essentially identical results. NC: Negative control in the absence of UDP-GlcNAc substrate (for panel b) or UDP-GlcA (for panel c). PC: Positive control in the absence of nanobody. Lyase: Hyaluronan lyase treatment prior to SDS-PAGE. (d) This workflow produced the UDP-bound Cv-HAS structure.