Fig. 3
From: Enzyme intermediates captured “on the fly” by mix-and-inject serial crystallography
Ceftriaxone density in the active site in 10 × 10 × 3 μm3 shard and 5 × 2 × 2 μm3 needle crystal forms at various times after mixing with 200–300 mmol/L CEF. The main species is displayed in blue, the minor species in gray. First two columns: shard crystal form, mFo-DFc SA-omit density (green) contoured at 2.5 σ. Third column: needle crystal form. SA omit maps were calculated using extrapolated structure factors. Time delays are arranged from top (30 ms) to bottom (2 s). Black arrows show the electron density of the covalently bound acyl adduct (see also Additional file 1: Figure S2 for details). a, b, c The ES complex at 30 ms. The full-length CEF model (blue) is displayed. The ES complex can be observed in needles or shards (both subunits). Blue arrows: features of the leaving group sulfur, red arrows: dioxo-triaxine ring feature. d, e, f Early phases of the formation of a covalently bound CEF adduct at 100 ms. The full-length CEF model (blue) is displayed together with the minor E-CFO* species (gray), where the β-lactam ring is open and attached to Ser-70 in subunit-B (shard crystal form, panel d) and the needle crystal form (panel f). In the shard crystal form subunit D (panel e) the acyl adduct is not yet observed. g, h, i Covalently bound adduct (E-CFO* in blue) formation at 500 ms with a small contamination of full-length CEF (gray). The red arrow points to electron density that may favor the interpretation by an OH group. j, k, l Mixture of the non-covalently bound, full-length CEF (blue arrow shows the leaving group sulfur feature) and covalently bound E-CFO* in the shard crystal form (both subunits) at 2 s. The electron density in the needle crystal form favors only the full-length CEF species