Eukaryotic fatty acid synthases (FASs) are large multifunctional enzymes that catalyze all the reaction steps in the essential biosynthesis of fatty acids. Recent X-ray crystallographic studies on the mammalian and fungal enzymes have established a structural basis for understanding the architectural organization, molecular function and evolution of these megasynthases. Fungal FAS is an α6
heterododecameric complex with a molecular weight of 2.6 MDa that forms a 27 nm barrel-shaped particle in which fatty acid production is compartmentalized in two large reaction chambers (Jenni et al.
; Leibundgut et al.
; Lomakin et al.
; Johansson et al.
), whereas mammalian FAS is an X-shaped 540 kDa α2
homodimer with two semicircular reaction chambers on both sides of the molecule (Maier et al.
Although the crystallization of yeast FAS was reported almost 40 years ago (Oesterhelt et al.
), it was not until recently that structural information was extracted from crystals of this large assembly. This was because solution of the structures of crystals with large asymmetric units presents significant challenges. Such difficulties can now be overcome by the advancement of methods in macromolecular crystallography and the ability to collect data from weakly diffracting crystals using X-ray beams from synchrotron-radiation sources (Mueller et al.
). An important step towards the atomic structure of fungal FAS was the solution of the crystallographic phase problem, which yielded interpretable electron-density maps at 5 Å resolution from Thermomyces lanuginosus
FAS (Jenni et al.
). This phase information subsequently allowed further X-ray crystallographic and electron-microscopic (EM) studies of Saccharomyces cerevisiae
FAS (Fig. 1).
Figure 1 Structural studies of fungal fatty acid synthase (FAS). The figure summarizes the flow of phase information between the different X-ray crystallographic and electron-microscopic (EM) systems for structural determination of fungal FAS at progressively (more ...)
To reliably identify the folds of individual enzymatic FAS domains in crystallographic electron-density maps, it was necessary to reach a resolution of about 5 Å, at which α-helices are clearly resolved and β-sheets are visible as flat surfaces. T. lanuginosus FAS crystals that diffracted to this resolution were only obtained after dehydration, which occurred during stabilization. The improvement of the diffraction properties upon dehydration unfortunately also resulted in a space-group transition from orthorhombic to monoclinic, leading to imperfect pseudo-merohedral twinning, which complicated structure determination. Here, we describe how we identified the twinning in the monoclinic crystal form and how we processed the diffraction images acquired from twinned crystals, which allowed us to calculate accurate intensity statistics for a quantitative description of the twinning. We also show how molecular-replacement solutions were obtained for the two crystal forms at very low resolution, which established the packing of the FAS molecules and rationalized the observed twinning.