# Multicrystal data collection at the VMXm beamline at Diamond Light Source

## TL;DR

This paper describes a new beamline at Diamond Light Source that enables X-ray crystallography on very small protein crystals, improving data collection efficiency and quality.

## Contribution

The VMXm beamline introduces novel strategies for multicrystal data collection from micro- and nanocrystals using cryo-electron microscopy grids and optimized beam conditions.

## Key findings

- X-ray diffraction measurements were successfully performed on protein crystals as small as ∼1.2 μm.
- The beamline's strategies reduce background scatter and improve detection of weak signals from micro/nanocrystals.
- Higher X-ray energies extend crystal lifetime in the beam, enhancing data quality.

## Abstract

Determining the structure of a protein is essential for understanding its function. However, X-ray crystallography becomes increasingly difficult as the diffracting power of crystals decreases with a decrease in crystal size. This challenge is further exacerbated by the fact that more complex targets tend to crystallize on smaller scales, and efforts to produce larger crystals often fail. Over recent years, serial crystallography techniques at synchrotrons and X-ray free electron lasers (XFEL) have been developed to enable structure determination from smaller crystals and to carry out time-resolved experiments.1 Unfortunately, these methods require large sample quantities, which can be difficult, costly, and time-consuming to produce, particularly for novel systems where little prior information is known. For crystals smaller than 300 nm, micro-electron diffraction (microED) has emerged as a solution for structure determination.2 However, it can be challenging to confirm that a sample is of the correct size for these experiments, and often, samples are too large, necessitating focused ion beam milling to achieve the required sample thickness.3, 4, 5

The Versatile Macromolecular Crystallography Microfocus (VMXm)6 beamline was specifically designed to address these challenges by enabling rotation data collection from samples smaller than 20 μm, requiring only minimal sample volumes. This has been achieved through novel mounting of crystals on cryo-electron microscopy grids, blotting away excess liquid,7 conducting data collections in vacuum, and matching the beamsize to the crystal size. These strategies limit the background scatter, allowing weak signal from the micro/nanocrystals to be detected. An additional advantage comes from collecting diffraction data at higher X-ray energies (∼21 keV) to exploit photoelectron escape, extending the lifetime of the crystals in the beam.8, 9 To date, successful X-ray diffraction measurements have been performed on protein crystals as small as ∼1.2 μm and chemical crystallography samples down to 800 nm.

In this work we will present the beamline, and the novel strategies adopted to obtain multicrystal data from micro- and nanocrystals. In particular we will focus on comparisons between data collected on more traditional synchrotron beamlines, as well as XFELs to highlight the impact these strategies have on the production of high quality diffraction data.

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Source: https://tomesphere.com/paper/PMC12585467