Cryo-EM structure of the flight muscle thick filament from the bumble bee, Bombus ignitus, with novel non-myosin densities revealed.

Abstract

The cryo-EM structure of the flight muscle thick filament of Bombus ignitus (order Hymenoptera) has been resolved at 6 Å resolution. By the comparison with similar asynchronous flight muscle thick filament structures from Lethocerus (Diptera) and Lethocerus (Hemiptera), all three present highly consistent backbone structure with very similar tail structure and have closely similar helical parameters. The myosin heads in Bombus are disordered like those of Drosophila; both insects have higher wingbeat frequencies than Lethocerus where the myosin heads are ordered. The three filament structures differ mostly in their non-myosin proteins and make the interpretation of densities corresponding to flightin and myofilin uncertain. However, using Alphafold model prediction, multiple sequence alignment and mass spectrometry, a more complete profile of flightin and myofilin has been determined. The full length of that portion of flightin within the thick filament annulus of Bombus threads its way through two myosin curved layers, contacts three other myosin curved layers and has a close contact with the Skip 3 region. The previously-identified, highly-conserved WYR domain of Bombus flightin contacts three myosin tails. With assistance from Alphafold, an atomic model was built for Bombus flightin from residue 41 to residue 151 (C-terminus). The other non-myosin protein, myofilin is more diverse in sequence, length and shape among the three reconstructed filaments, although it also shows a highly conserved density and sequence at its N-terminus, which we have named the “LKG domain”. Both flightin and myofilin have close contact with the paramyosin core, these three non-myosin proteins are believed to work together to determine and stabilize the thick filament structure. Supported by NIH/NIGMS.