In standard X-ray diffraction experiments, errors caused by absorption are small and easily accounted for. However, they become a more serious limitation in experiments conducted at long wavelengths. Diamond’s I23 beamline is a unique synchrotron instrument, operating in a wavelength range between 1.1 and 5.9 Å to give access to the absorption edges of several light elements of biological significance.
Together with the Scientific Computing group at the University of Oxford, I23 beamline scientists have developed a new technique for advanced absorption correction, using X-ray tomography integrated into an innovative new software program (AnACor). Their work, recently published in the Journal of Applied Crystallography, shows that AnACor provides superior results to standard spherical harmonics corrections.
The team at Oxford have since released a significant upgrade in AnACor2.0, improving the computational speed up to 180 times. The whole innovative solution can facilitate analytical absorption corrections for any data set for which a voxel-annotated file is available and the relationship between the coordinate system of the 3D model and the diffraction experiment is clearly defined.
The new technique was a joint effort from the team at I23 as well as contibutors from and the Department of Life Sciences at Imperial College London and Harwell Campus's Rosalind Franklin Institute and Rutherford Appleton Laboratory, highlighting the success of cross-campus collaboration.
X-ray diffraction (XRD) is a commonly used technique for structural characterisation of materials. A beam of X-rays will be scattered by a sample crystal, for example, depending on the kind and arrangement of atoms in the crystal structure. Recording the diffraction pattern created by the crystal at different orientations allows us to determine the crystal structure. Some X-rays will be absorbed by the sample, but for most experiments, the error caused by absorption is small and easily accounted for using standard computational methods. However, if the sample contains heavy elements, or the X-ray wavelength is very long, the absorption effect increases and can affect the data quality.
Fig. 1: Tomography projection images for background [(a) and (d)], sample [(b) and (e)] and flat-field-corrected images [(c) and (f)] of OmpK36 (top) and Cld samples (bottom).
Principal Beamline Scientist Armin Wagner said:
I23 is Diamond’s long-wavelength macromolecular crystallography beamline. It's a unique instrument that can be tuned to very long wavelengths, to give access to the absorption edges of light elements of biological significance, such as calcium, potassium, chlorine, sulfur and phosphorus. So, the absorption effect does become an issue.
This is a long-standing problem, solved to a certain extent by using computational methods to account for absorption. There have been various attempts to develop improved techniques using X-ray tomography. Creating a 3D model of the sample allows us to trace the path of the X-rays through the sample, and more accurately account for absorption. However, adding tomography onto an XRD beamline is not a simple matter; the experiments are different, and the endstations can become very crowded. Past attempts have not resulted in high quality data.
This is a long-standing problem, solved to a certain extent by using computational methods to account for absorption. There have been various attempts to develop improved techniques using X-ray tomography. Creating a 3D model of the sample allows us to trace the path of the X-rays through the sample, and more accurately account for absorption. However, adding tomography onto an XRD beamline is not a simple matter; the experiments are different, and the endstations can become very crowded. Past attempts have not resulted in high quality data.
The I23 team solved those issues, producing accurate tomographic reconstructions and segmentations of the crystals and their surrounding materials. The next challenge to tackle was using the resulting 3D models and processing the data to provide accurate corrections for the absorption effect.
DPhil student Yishun Lu and Prof. Wesley Armour of the Oxford e-Research Centre use a ray-tracing method to perform an analytical absorption correction strategy based on the 3D model of the sample derived from X-ray tomography. The ray-tracing method accurately determines individual path lengths through the different sample materials: the crystal, sample mount, mother liquor, and the oils or glues used to mount the crystals. In addition, the ray-tracing method is combined with advanced image processing techniques and statistical methods to effectively remove noise and enhance the accuracy of absorption coefficient calculations. Everything is integrated in the software AnACor, enabling analytical absorption corrections for any dataset with a voxel-annotated file and a clearly defined relationship between the 3D model's coordinate system and the diffraction experiment.
AnACor was tested with two very long wavelength experiments, on a crystal of the membrane protein OmpK36 GD, and a crystal of the heme-binding enzyme chlorite dismutase (Cld). The results showed that the new software provides superior results to standard spherical harmonics corrections. AnACor is also able to correct data in multiple crystal orientations and for cases where the beam is smaller than the sample. The ray-tracing method is slow, so Oxford researchers developed AnACor 2.0, an updated version that employs advanced computational strategies and GPU acceleration. This significantly improves computational speed, making it up to 180 times faster than AnACor 1.0 while still performing accurate analytical absorption corrections. This innovative solution will facilitate analytical absorption corrections on I23 and beyond Diamond.
![Figure 3: Volume renderings of segmentations of OmpK36 [(a)–(c)] and Cld [(d)–(f)]. Transparent blue: mother liquor; gold: loop; pink: crystal; green: protein/detergent aggregate](/dam/jcr:2dd4a89c-af91-4e9a-9fea-e7e4a3fc9a10/I23-20250214-figure2.jpg)
To find out more about the I23 beamline or discuss potential applications, please contact Principal Beamline Scientist Armin Wagner: [email protected].
Lu Y et al. Ray-tracing analytical absorption correction for X-ray crystallography based on tomographic reconstructions. Journal of Applied Crystallography 57.3 (2024). DOI:10.1107/S1600576724002243
Lu Y et al. AnACor2.0: A GPU-accelerated open-source software package for analytical absorption corrections in X-ray crystallography. Journal of Applied Crystallography 57.6 (2024). DOI:10.1107/S1600576724009506
Image credits: 10.1107/S1600576724002243 under CC BY 4.0 license
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