cryoEM Administrative Supplement for Equipment

ABSTRACT Funds are requested for the purchase of a revolutionary new camera, the Apollo, from Direct Electron Ltd. that would enable the collection of large amounts of image data, currently obtained in the form of ?movies?.

The ability to collect large amounts of data would broaden the types of questions addressable in the PI's R35 grant currently focused on obtaining atomic resolution images of thick filaments from striated muscle.

Generally, thick filaments from all striated muscles have four structurally distinct regions: (1) a 160 nm long bare zone in the center where myosin tails are packed antiparallel to each other, (2) an A-band follows where myosin molecules are arranged in a helical or quasi helical structure with the myosin tails packed in a parallel arrangement, (3) a tapered end where the connecting filament, e.g. titin, connects the thick filament to the Z-disk, and (4) features along the thick filament A-band from invertebrates in particular, but also from vertebrates, that fail to follow the myosin molecule arrangement.

The helical arrangement of myosin in the A-band of invertebrate thick filaments facilitates an atomic structure of the myosin tail and head arrangement by using all of the myosin axial repeats from the end of the bare zone to the beginning of the tapered ends.

However, the bare zone, the transition from bare zone to helical arrangement, the tapered ends as well as additional features that do not follow the myosin helical symmetry perform important roles in the assembly of thick filaments.

Features, such as the paramyosin of invertebrate thick filaments fail to follow the myosin helical symmetry in the A-band and are poorly represented in cryoEM reconstructions.

Paramyosin in concert with other non-myosin proteins may function to determine the thick filament length in invertebrates.

Titin, myosin binding protein C and other non-myosin proteins of vertebrate thick filaments also do not follow a regular pattern along the entire A-band.

Acquisition of a much faster camera capable of recording a 60-frame movie in a single second, will provide the tool needed to obtain high resolution 3-D images of these features that are not well represented if at all in structures solved by enforcing helical symmetry.