3D cryo-electron microscopy and molecular model of two arthropod hemocyanins
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Abstract
This work presents 3D-reconstructions of two arthropod hemocyanin molecules produced by single particle cryo-EM. The 3D-reconstruction of the 8x6mer hemocyanin of the horseshoe crab Limulus polyphemus was built upon a newly acquired dataset under oxygenated conditions and improved the previous results. The achieved resolution of 8,7 Å (0,5 criterion) allowed for further refinement of the existing molecular model by flexible fitting into the 3D density map. Also newly available additional authentic sequences of Limulus polyphemus hemocyanin as well as a more suitable source for homology models of subunits with unknown sequence remaining were used to update the molecular model and verify previously identified interface sites and in turn molecular mechanisms of cooperativity by chemo-mechanical signal transduction.
Compared to a conventional rigid body fitting, the secondary structure aware flexible fitting algorithm especially improved the overall fit in domain #1 of each subunit, which contains mainly well resolved α-helices. Domain #2 also contains α-helices as well as the active site. Changes in this region were generally less frequent with the active site left intact. Domain #3 is mainly comprised of β-sheets which lead to few discernible improvements as the observed level of detail contained within the reconstruction was insufficient to provide clear target masses for the fitting algorithm.
Secondly both coarse and fine structural details of the 4x6mer hemocyanin of the ghost shrimp Callianassa truncata were revealed by an unbiased reference-free reconstruction method yielding a resolution of 9,8 Å (0,5 criterion). In combination with available sequence data, a molecular model of the 4x6mer was constructed. This revealed a total of twelve contact points facilitated by four distinct types of interface. Most contacts are densely packed in the center of the 4x6mer.
The interfaces are the sites of interaction between the hexamers, both in terms of stabilization of the quaternary structure of the molecule as well as possible ways for chemo-mechanical signal transduction. Overall there are three subunits involved in contacts with subunit b of type CtrHc2 participating in every contact but one. This indicates its importance to the assembly of the 2x6mers and 4x6mer. Also, in combination with published low-resolution 3D-reconstrcutions of crustacean 2x6mers, insight into the evolutionary development of crustacean hemocyanin was gained.