# Technology development for biological imaging with x-ray free electron lasers

> **NIH NIH R01** · LAWRENCE LIVERMORE NATIONAL SECURITY, LLC · 2020 · $541,329

## Abstract

Determining the structure and imaging the dynamical behavior of large protein complexes as well
as other biological nanoparticles at room temperature with near atomic resolution has the
potential to greatly impact structural biology and our knowledge of biomolecular function and
interactions. A major bottleneck in structural biology is that many of the proteins performing
critical cellular functions are membrane proteins that have proven intractable to structure
determination by traditional x-ray crystallography, in which x-ray radiation damage is mitigated by
spreading the radiation dose over many molecules in a crystal. Consequently, most membrane
protein structures remain unknown to date. Similarly, determining high-resolution x-ray structures
of single, non-periodic biological nanoparticles by x-ray diffraction imaging has been hampered by
radiation damage. While cryo-electron microscopy (cryo-EM) has been successful in obtaining
high-resolution structural information from large biomolecules and nanoparticles, it requires
freezing of the sample as a way to mitigate electron-induced radiation damage and the cryogenic
temperatures make it impossible to visualize fast conformational changes.
X-ray free electron lasers (XFELs), which produce ultra-short and ultra-bright x-ray pulses, allow
to break this nexus between resolution and absorbed dose by utilizing the “diffraction-before-
destruction” principle and promise imaging at unprecedented spatio-temporal resolution. Since
the commissioning of the Linac Coherent Light Source (LCLS) at SLAC National Accelerator
Laboratory only five years ago, both protein structure determination at room temperature to near-
atomic resolution by serial-femtosecond nanocrystallography (SFX) and modest resolution
structural studies by single nanoparticle diffraction imaging (SPI) have been demonstrated.
However, several challenges and limitations remain that need to be overcome to allow more fully
utilizing these new light sources for structural biology.
The overall objective of this proposed work is to address several of the current technological and
methodological challenges in coherent x-ray diffraction imaging of biological samples with XFELs,
in particular, in the areas of sample preparation and introduction for membrane proteins and
biological nanoparticles. Our work aims to drastically reduce sample consumption and to open up
this imaging method to a much broader range of research groups and to samples including a
diversity of membrane proteins and other biological nano-objects that are not abundant and/or
difficult to crystallize. The proposed work will lay the groundwork for future time-resolved
structural studies at XFELs that require efficient use of available sample.
If successful, this work would greatly aid our experimental capabilities to study and understand
function of protein complexes and biological nanoparticles in a wide range of fields including
human health and biosecurity.

## Key facts

- **NIH application ID:** 9869758
- **Project number:** 5R01GM117342-05
- **Recipient organization:** LAWRENCE LIVERMORE NATIONAL SECURITY, LLC
- **Principal Investigator:** MATTHIAS FRANK
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $541,329
- **Award type:** 5
- **Project period:** 2016-05-01 → 2021-05-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/9869758

## Citation

> US National Institutes of Health, RePORTER application 9869758, Technology development for biological imaging with x-ray free electron lasers (5R01GM117342-05). Retrieved via AI Analytics 2026-05-22 from https://api.ai-analytics.org/grant/nih/9869758. Licensed CC0.

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