Real-space analysis of radiation-induced specific changes with independent component analysis

Abstract

A method of analysis is presented that allows for the separation of specific radiation-induced changes into distinct components in real space. The method relies on independent component analysis (ICA) and can be effectively applied to electron density maps and other types of maps, provided that they can be represented as sets of numbers on a grid. Here, for glucose isomerase crystals, ICA was used in a proof-of-concept analysis to separate temperature-dependent and temperature-independent components of specific radiation-induced changes for data sets acquired from multiple crystals across multiple temperatures. ICA identified two components, with the temperature-independent component being responsible for the majority of specific radiation-induced changes at temperatures below 130 K. The patterns of specific temperature-independent radiation-induced changes suggest a contribution from the tunnelling of electron holes as a possible explanation. In the second case, where a group of 22 data sets was collected on a single thaumatin crystal, ICA was used in another type of analysis to separate specific radiation-induced effects happening on different exposure-level scales. Here, ICA identified two components of specific radiation-induced changes that likely result from radiation-induced chemical reactions progressing with different rates at different locations in the structure. In addition, ICA unexpectedly identified the radiation-damage state corresponding to reduced disulfide bridges rather than the zero-dose extrapolated state as the highest contrast structure. The application of ICA to the analysis of specific radiation-induced changes in real space and the data pre-processing for ICA that relies on singular value decomposition, which was used previously in data space to validate a two-component physical model of X-ray radiation-induced changes, are discussed in detail. This work lays a foundation for a better understanding of protein-specific radiation chemistries and provides a framework for analysing effects of specific radiation damage in crystallographic and cryo-EM experiments.

High-resolution cryo-EM reconstructions in the presence of substantial aberrations

“…Glucose isomerase (GI), also called xylose isomerase, from Streptomyces rubiginosus was purchased from Hampton Research (23). Protein slurry was dialyzed three times against excess of dH 2 O and concentrated to ∼40 mg/ml with Amicon filter.…”

High-resolution cryo-EM reconstructions in the presence of substantial aberrations

“…Glucose isomerase (GI), also called xylose isomerase, from Streptomyces rubiginosus was purchased from Hampton Research. Protein slurry was dialyzed three times against an excess of distilled H 2 O and concentrated to $40 mg ml À1 with an Amicon filter (Borek et al, 2018).…”

Electronic Excitations and Radiation Damage in Macromolecular Crystallography

“…These phenomena are then followed by various chemical changes involving, for instance, the elongation and eventual breaking of disulphide bonds, the decarboxylation of glutamate and aspartate residues and the breaking of covalent bonds between the protein backbone and the metal centres. Gerstel et al [1] provide an exhaustive methodological discussion concerning different susceptibilities empirically observed for all the different types of specific damage described in the literature, while Borek and collaborators recently provided a promising method to discern temperature-dependent changes from other types of change [5]. On the other hand, combined measurements based on single-crystal X-ray diffraction, small angle scattering and radiolysis experiments have shown that hydrogen gas formed in the sample during irradiation, rather than bond cleavage, is mainly responsible for the loss of high-resolution information [6].…”

“…In this work, we will outline how specific damage events in crystallographic models can be interpreted in terms of local structural deformations caused by the population and depopulation of virtual and occupied electronic states in the close vicinity of the Fermi energy of the perfect crystal. Previous reports in the literature have addressed and quantified the local changes, but unfortunately, due to the lack of a unifying model, a quantification of this effect has proved to be very subjective and system dependent (although procedures for radiation damage corrections have been refined empirically to a point where a systematic and automated assessment is possible [1,5,15]). It is our view that single crystal macromolecular crystallography oscillation datasets are unsuitable for assessing the structural changes caused by specific damage, since they rely on information obtained too long (seconds) after the initial interaction of the X-ray beam with the sample.…”

“…In our model, the electronic and structural changes caused by the interaction of X-rays with a macromolecular crystal structure are therefore akin to typical non-thermal melting phenomena in semiconductors and insulators [21] and Refs. [1][2][3][4][5][6][7][8][9] therein. In this communication, we describe a phenomenological model of how different classes of specific damage induced by X-rays can be rationalized in terms of the basic assumptions of this model, and how the appearance of specific damage features in X-ray measurements and sample degradation relate to the timescales of the electronic and nuclear processes described above.…”