Figure 8.

Single particle cryo-electron microscopy of E123Q UMPS.A and B, a representative motion corrected micrograph from a sample of the OPRT active site mutant of UMPS (E123Q) shows a good distribution of particles (A, defocus −2.4, circle diameter 100 Å). The CTF fit of the micrograph in (A) is shown in B. C, the preliminary 2D classification from ∼20,000 particles shows an obvious S-shaped structure (the five classes shown represent about 3700 particles—additional 2D classes from a larger dataset are shown in Fig. S7). A model of HsUMPS was manually built into the 2D class (D) using the known crystal structures of HsOMPDC (green, 2JGY) and HsOPRT (blue, 2WNS). The position of the N terminus of the OPRT domains and the C terminus of the OMPDC domains are shown on the figure as N and C, respectively. D and E, the orientation of the domains shown in (D) allows for dimerization of OPRT (E, additional OPRT protomer given in gray). Scale bar in (C) is 50 Å, and the box size is 256 pixels at 0.89 Å/pixel. HsUMPS, human UMP synthase; OMPDC, orotidine 5′-monophosphate decarboxylase; OPRT, orotate phosphoribosyltransferase; UMP, uridine 5′-monophosphate.