Fig 1.
Cryo-electron microscopy of AOX.
(a) Micrograph of AOX collected on a JEOL 3200 FSC electron microscope using a K2 direct electron detector. The defocus was determined as 1100 nm. The scale bar represents 20 nm. (b) 2D class averages calculated using Relion show the octameric particle in different orientations.
Fig 2.
Map quality and validation of map and model.
(a) Orientation distribution of the particles. One asymmetric unit of the D4 symmetric map is shown. The height of the cylinders is proportional to the number of particles in the orientation indicated. In green the unsharpened final map is shown. Top views along the 4-fold axis (top) and side views (bottom) are overrepresented in the dataset. (b) Local resolution analysis by ResMap [45] shows that the complex is rigid without flexible parts. (c) Gold-standard FSC of the final map after masking as determined by the post-processing procedure in RELION [44] indicates a resolution of 3.37 Å. A B factor of −147 Å2 was determined and applied to sharpen the map. (d) FSC curves between the model and the cryo-EM map. The FSC curve between the final refined model and the reconstruction from all particles (blue) indicates a resolution of 3.5 Å. FSC between the model refined in the reconstruction from half the particles and the reconstruction from that same half (FSCwork, red), and between this model and the reconstruction from the other half of the particles (FSCfree, green) both indicate the same resolution (3.7 Å), showing that no overfitting of the model to the map has taken place.
Fig 3.
The map was sharpened by a negative B-factor of -147 Å2. Each of the eight monomers is shown in a different colour. (a) Top view along the fourfold axis. (b) View in the most common orientation. (c) Side view along a twofold axis. (d) Side view along another twofold axis. (e) Same view as (c), with the front two monomers removed to show the interior of the octamer.
Fig 4.
(a) α-helix H10 with model fitted. (b) The FAD cofactor and its binding pocket. Domain colors as in Fig 5a.
Fig 5.
(a) Structural domains. The domain assignment for the GMC family [2]) is as follows: FAD-binding domain (red); FAD covering lid (pink); extended FAD-binding domain (yellow); flavin attachment loop (green) and intermediate region (light green); substrate-binding domain (cyan). Helix H12, which forms crystal contacts, is shown in orange. The longest AOX-specific insert 481–548 is shown in dark blue, other AOX-specific sequences are colored purple. The FAD cofactor is blue. The location of the five β-sheets A-E and some α-helices (nomenclature from S1 Fig) and sequence mentioned in the text is indicated. The view of the monomer is along the fourfold axis from inside the complex. (b) Conservation of residues. Residues conserved in the GMC family (see S1 Fig) are shown in red and residues conserved in AOX are green; unconserved residues are white. Structural elements unique for AOX are yellow. Note that the FAD binding domain (right; red in (a)) has high conservation in the GMC family, while the substrate binding domain centered around β-sheet C (left; cyan in (a)) is conserved between AOX species.
Fig 6.
The octamer is shown in (a) with every subunit in a different color and in (b) in the same orientation with domain colors as in Fig 5a. One monomer (the green one in (a)) is shown in (c) with the location of residues discussed in the text indicated. The interface of two tetrameric rings is almost exclusively made of the AOX-specific insert 481–548 (dark blue); the only exception is the dimer contact between the loops 408–409. (d) A view rotated 90° relative to (b). Only one tetrameric ring is shown for clarity. The C-terminal AOX-unique tail (purple) extends from the long helix H25 (red) of the FAD binding domain. The four copies form a ring around the fourfold axis and the last four amino acids are buried inside the complex.
Fig 7.
Domain colors as in Fig 5a. His567 and Asn616 play a role in catalysis. The side chains of the other indicated residues restrict substrate access to the active site and are probably involved in the specificity of AOX for small alcohols. His515 is part of a different subunit (grey).
Fig 8.
A slice of the density near the fourfold surface.
A salt bridge between Glu656 and Arg214 of different subunits (gold and light blue) may contribute to intersubunit contacts.
Fig 9.
Peroxisomal AOX crystal packing.
(a) Schematic representation of the peroxisomal AOX crystal packing (reprinted from [14] under a CC BY license, with permission from the American Society for Microbiology, original copyright 1992). AOX octamers are shown as square blocks with a cross indicating the fourfold axis. One unit cell of space group I432 with cell dimensions 228x228x228 Å is shown as a cube and contains 6 octamers (shaded grey). Each octamer has four neighbours, which are 90° rotated. This packing creates rows of octamers in three perpendicular directions, while the fourfold faces of six octamers line a hole (located in the center and at the eight vertices of the cube). (b) Class averages of a subset of picked particles containing a second molecule in the box. Classes showing a side view (1, 2, 4, 5, 9, 10, 11, 12) have a convex neighbour (top view) and classes showing a top view (3, 7, 8) a concave one (side view), suggesting a preferential 90° rotation between neighbours. (c) Part of a micrograph showing several AOX molecules arranged as in the crystals. Note the alternating arrangement of top view and side views. (d,e) Model of the crystal packing. The EM map was filtered to 10 Å for clarity. Neighbouring molecules are 90° rotated relative to each other and shifted 114 Å (half a unit cell). Molecules in the same orientation are shown in the same color. The yellow molecules are viewed along the fourfold axis. (d) shows one layer and (e) two layers. A unit cell is indicated in (e). (f, g) The crystal packing is mediated by helix H12. Two models are shown with each subunit in a different shade for clarity. In (f), the molecules are in the same orientation as in (d) and (e) and in (g) 45° rotated.