a, Image processing workflow for pemetrexed-bound PCFT–nanobody. b, Local-resolution estimation of reconstructed map as determined within RELION. Detergent density omitted for clarity. Top, full map; bottom, central slab through map. c, Gold-standard FSC curves used for global resolution estimates within (i) cryoSPARC, (ii) RELION or (iii) 3DFSC. d, Close-up of side-chain density for all transmembrane helices. Volume contoured at threshold level of 0.3. e, Density for pemetrexed (PMX) and side chains surrounding PCFT. Volume contoured at threshold level of 0.4.