A comparison of an X-ray diffraction and electron diffraction experiment from a single protein microcrystal lamella

Authors: Adam Crawshaw (Diamond Light Source) , C. David Owen (Diamond Light Source; Research Complex at Harwell) , Melissa R. Whyte (University of St Andrews) , Anna J. Warren (Diamond Light Source) , Pedro Nunes (Diamond Light Source) , Jose Trincao (Diamond Light Source) , C. Alistair Siebert (Diamond Light Source) , Gwyndaf Evans (Diamond Light Source; Rosalind Franklin Institute)
Co-authored by industrial partner: No

Type: Conference Paper
Conference: 12th International Workshop on Radiation Damage to Biological Samples
Peer Reviewed: No

State: Published (Approved)
Published: June 2025

Abstract: X-ray diffraction (XRD) of microcrystals is signal-to-noise limited by due to the inherently weak diffraction. Therefore, it is key that the beamline instrumentation and the sample itself introduce minimal noise. The VMXm beamline, at Diamond Light Source, has been optimised for maximising the S:N in experiments with a variable focus high-energy (>20 KeV) X-ray beam, with in-vacuum endstation and the use of low background cryoTEM grids for crystal mounting [1], [2]. This has allowed VMXm to collect high-resolution rotation data from single crystals measuring ~1.2 μm which were only previously tractable using an X-ray Free Electron Laser [3]. This has pushed the amenable sample envelope at synchrotrons to new dimensions and perhaps near to the practical limit of XRD. Indeed, simulations have predicted the limit to be ~0.5 μm thick in the case of lysozyme, assuming photoelectron escape [4]. Electron diffraction (ED) is frequently used to measure diffraction data from submicron crystals. Many samples which are too thin for XRD are often too thick for ED using the currently available electron beam energies (<300 keV) and hence require thinning by focussed ion beam milling (FIB). In addition to determining structures from nanocrystals, ED provides Coulomb potential data which are complementary to that obtained with XRD. As such ED data may be necessary to answer particular scientific questions. In this work we present data from cubic human insulin crystals that have been thinned by FIB milling from ~10 μm to various submicron thicknesses. 200 kV ED data were then collected from these lamellae before XRD data were measured from the same lamellae using VMXm. It was possible to obtain a complete XRD dataset to 2.45 Å using a 1.68 μm3 illuminated volume and a 2.04 Å ED dataset from the same 0.25 μm lamella. We have demonstrated that the data quality is comparable between ED and VMXm from the same crystal, while giving an opportunity to directly compare X-ray and electron derived maps. This includes the comparison of the radiation damage each experiment imparts on the sample [5] as well as the information content [6]. This work indicates that the usable sample envelope for synchrotron X-rays extends to much thinner samples than had been previously thought. It is also the first demonstration of ED and XRD measured from the same crystal volume enabling direct comparison of X-ray and electron derived data. Ultimately, the work will inform the design and use of high energy (MeV) ED instruments such as HeXI and how those can be complemented by XRD derived information from beamlines such as VMXm.

Subject Areas: Biology and Bio-materials


Instruments: VMXm-Versatile Macromolecular Crystallography microfocus

Added On: 18/06/2025 12:15

Discipline Tags:

Structural biology Life Sciences & Biotech

Technical Tags: