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The baseline version is composed solely of one rigid module with an inverted cupola for observations.
The goal of the LO-refinement is to resolve a higher resolution structure of each rigid module.
The lumped parameter, rigid module, flexible connector (RMFC) model and a more detailed finite element shell model were used.
Starting from the preliminary alignment parameters, we focus on a single rigid module, optimize its orientation and position and thereafter compute a new reconstruction with the refined parameters.
The main idea of the LO-refinement procedure is to focus on each rigid module and optimize its orientation and position individually.
For the orientation of a rigid module, there are five parameters to be optimized, three orientation angles φ, θ, ψ and two in-plane translations x, y.
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Besides the ribosome molecules with two assumed rigid modules, this LO-refinement procedure could in principle be applicable to the complexes with multiple modules.
And the multiple conformations of the complex can be regarded as slightly varied relative positions and orientations among different rigid modules.
The LO-refinement is based on the assumption that the imaged macromolecular complex comprises a number of rigid modules with slightly varying relative orientations and positions between different modules.
In a test case, we used the ribosome (80S or 70S) that has two rigid modules (60S/50S or 40S/30S) with the fluctuating relative orientations and positions to prove the concept of this method.
For a proof of principle of our LO-refinement procedure, we generated two types of datasets using the structures of ribosomes with two relative rigid modules (large and small subunits).
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