Extended Data Figure 1: Production of influenza A polymerase heterotrimer.

a, The heterotrimeric bat polymerase was recombinantly expressed in insect cells as a self-cleaving polyprotein. N-terminally it encodes the tobacco etch virus (TEV) protease that cleaves C-terminal to the amino-acid sequence ENLYFQ (in italics), and releases N-terminally His-tagged PA, PB1, C-terminally strep-tagged PB2 and cyan fluorescent protein (CFP) for facilitated monitoring of expression. Arrows indicate the N-to-C-terminal direction and the termini of each mature protein. The histidine and streptavidin tags are underlined. b, After ammonium sulphate precipitation, immobilized metal ion affinity chromatography, engineered streptavidin (strep-tactin) affinity and heparin chromatography, the final purification step consisted of size-exclusion chromatography. The elution profile (monitored by the absorbance at 280 nm) with a single and nearly symmetric peak suggests a homogeneous and monomeric polymerase complex. mAU, milli-absorption unit. c, Fractions of the final size-exclusion chromatography were subjected to 10% SDS–PAGE followed by Coomassie blue staining. Lane 1 contains the molecular mass markers and lanes 2–7 the eluate with PA (85.4 kilodaltons (kDa)), PB1 (87.8 kDa) and PB2 (91.0 kDa). d, Recombinant bat FluA polymerase was visualized by electron microscopy following negative staining with sodium silico-tungstate of a 0.02 mg ml⁻¹ protein sample. The image demonstrates that the sample is homogeneous and monodisperse with a V- or doughnut-like shape and central cavity.