# TRD2: Phasing and refinement

> **NIH NIH P41** · UNIVERSITY OF CALIFORNIA LOS ANGELES · 2020 · $255,065

## Abstract

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.

## Key facts

- **NIH application ID:** 9935724
- **Project number:** 1P41GM136508-01
- **Recipient organization:** UNIVERSITY OF CALIFORNIA LOS ANGELES
- **Principal Investigator:** PAWEL A. PENCZEK
- **Activity code:** P41 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $255,065
- **Award type:** 1
- **Project period:** — → —

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/9935724

## Citation

> US National Institutes of Health, RePORTER application 9935724, TRD2: Phasing and refinement (1P41GM136508-01). Retrieved via AI Analytics 2026-05-24 from https://api.ai-analytics.org/grant/nih/9935724. Licensed CC0.

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