Structure of Human Complement C8, a Precursor to Membrane Attack

Complement component C8 plays a pivotal role in the formation of the membrane attack complex (MAC), an important antibacterial immune effector. C8 initiates membrane penetration and coordinates MAC pore formation. High-resolution structures of C8 subunits have provided some insight into the function of the C8 heterotrimer; however, there is no structural information describing how the intersubunit organization facilitates MAC assembly. We have determined the structure of C8 by electron microscopy and fitted the C8α-MACPF (membrane attack complex/perforin)-C8γ co-crystal structure and a homology model for C8β-MACPF into the density. Here, we demonstrate that both the C8γ protrusion and the C8α-MACPF region that inserts into the membrane upon activation are accessible.


Supplementary figure legends
Fig S1. Purification of C8. Human plasma was clarified of less soluble proteins using a PEG precipitation. C8 was then isolated from the remaining solution using monoclonal anti-C8 immuno-affinity columns as described previously 1   Initial models for refinement. A Gaussian blob with the approximate dimensions measured from the reference-free averages of C8 (a; grey) was generated using EMAN 2 .
Other starting models (a; cyan and yellow) were calculated experimentally using the Random Conical Tilt (RCT) method 3 . Eighteen tilt pairs (0 and 50°) were taken at a magnification of 59,000 on a Tecnai F30 microscope, as described in Fig. 1. The 50° image was recorded first, followed by the 0° image. 1409 windowed tilt pairs were selected interactively in WEB 4 , CTF-corrected using the phase-flip option in BSOFT 5 , and subjected to 5 rounds of reference-free alignment 6 before converging on a stable global average. Using hierarchical classification techniques in SPIDER 4 , 0° images were separated into 100 groups generating 2D class averages. 3D reconstructions were computed from the 50° images corresponding to class averages using the back-projection method in SPIDER 4 . (b) Multiple seed models initiate independent refinements of C8.
To minimize distortions of the RCT structures, RCT starting models were used as references for Euler angular assignment of reference-free generated 2D class averages in EMAN 2 . Resulting 3D reconstructions exhibited density features similar to those found in the final reconstruction starting from the Gaussian blob (grey). These improved RCT structures were then used as templates for the refinement of single particles (see Fig. S3).
Scale bar, 45 Å. The C8 reconstruction refined from the Gaussian blob (grey) is compared with a reconstruction refined using one of the models from the random conical tilt method (yellow, as colored in Fig. S2). Scale bar, 45 Å.   Table S1. For each placement, C8β-MACPF is rendered as an isosurface filtered to 25 Å (green). Crystal structures of C8α-MACPF and C8γ are shown as blue and yellow ribbons, respectively.
The C8 reconstruction is highlighted as a grey mesh. N and C termini of C8α-MACPF are cyan spheres, N and C termini of C8β-MACPF are brown spheres. Scale bar, 45Å. The hairpin extension of C8α-MACPF that is covalently linked to C8γ is shown as residues 176-208. Tables   Table S1. Results of fitting and refining atomic models of C8 subunits into a 3D reconstruction of C8 at 24 Å resolution

Supplementary
38 84 * The model was generated in a stepwise fashion beginning with manual placement of the C8α-MACPF-C8γ crystal structure, γ-α(MACPF). Its position was refined as a single rigid-body using PHENIX 7 . Two possible placements of a homology model of C8β MACPF, β(MACPF) a and β(MACPF) b , were manually fit in the remaining density and subsequently rigid-body refined. These two placements are related by a 180° rotation interchanging the arms of the "L". ¶ R factor is the crystallographic R factor minimized during the PHENIX 7 refinement. § CC is the real space correlation coefficient between the EM reconstruction and the map calculated from the model. The value was generated using the BSOFT 5 program suite.