TRD 2. Phasing and refinement - Penczek (Lead) Summary Similarly as X-ray crystallography, microcrystal electron diffraction (MicroED) delivers amplitudes of the imaged object Fourier transform and the phase information is lost. Several procedures have been developed in X-ray crystallography for solving the phase problem for de novo structure determination. These include Patterson difference, molecular replacement, ab initio statistical methods, heavy metal (multi- and single isomorphous replacement), damage-based phasing, and anomalous dispersion. We already successfully applied MicroED to the determination of new protein structures using two approaches: (1) direct ab initio and molecular replacement using idealized models and (2) molecular replacement using homologues. However, ab initio methods are feasible only in cases when the obtained resolution is better than ~1.2Å while molecular replacement is only possible if the new protein is homologous with another already known structure. Other X-ray methods are either not applicable to MicroED (for example anomalous dispersion) or were not yet adapted and implemented for MicroED. Here we will expand the pallet of available tools by developing dedicated MicroED phasing methods for routine de novo structure determination in cases in which the diffraction spots do not reach spatial frequencies required by ab initio methods and molecular replacement is not possible due to lack of homologous structural information. To facilitate phasing, we will develop comprehensive MicroED data scaling and integration methodologies taking advantage of maximum likelihood approaches to deliver accurate intensity values. For de novo phasing we will adapt two methods that were historically most successful in X-ray crystallography: (1) heavy metal isomorphous replacement strategies and (2) phasing by specific radiation damage. The aims are: 1. Development of a comprehensive MicroED data scaling and integration methodology; 2. De novo phasing using isomorphous replacement; 3. De novo phasing using radiation damage. The successful completion of the Aims listed will bring MicroED to an equal footing with X-ray crystallography. In the long-run, the proposed developments will lead to establishment of MicroED as a method capable of phasing and solving structures of entirely new and biologically important systems that currently are not tractable by X-ray crystallography.