J. Appl. Cryst.の発刊に際して

Radiation damage inflicted during diffraction data collection in macromolecular crystallography has re-emerged in the last decade as a major experimental and computational challenge, as even for crystals held at 100 K it can result in severe data-quality degradation and the appearance in solved structures of artefacts which affect biological interpretations. Here, the observable symptoms and basic physical processes involved in radiation damage are described and the concept of absorbed dose as the basic metric against which to monitor the experimentally observed changes is outlined. Investigations into radiation damage in macromolecular crystallography are ongoing and the number of studies is rapidly increasing. The current literature on the subject is compiled as a resource for the interested researcher.

/pdf/radiation-damage-in-macromolecular-crystallography-what-is-3rnta29rro.pdf

Radiation damage in macromolecular crystallography: what is it and why should we care?

https://journals.iucr.org/m/issues/2015/02/00/it5004/it5004.pdf

Serial femtosecond crystallography: the first five years

The determination of protein crystal structures is hampered by the need for macroscopic crystals. X-ray free-electron lasers (FELs) provide extremely intense pulses of femtosecond duration, which allow data collection from nanometre- to micrometre-sized crystals in a 'diffraction-before-destruction' approach. So far, all protein structure determinations carried out using FELs have been based on previous knowledge of related, known structures. Here we show that X-ray FEL data can be used for de novo protein structure determination, that is, without previous knowledge about the structure. Using the emerging technique of serial femtosecond crystallography, we performed single-wavelength anomalous scattering measurements on microcrystals of the well-established model system lysozyme, in complex with a lanthanide compound. Using Monte-Carlo integration, we obtained high-quality diffraction intensities from which experimental phases could be determined, resulting in an experimental electron density map good enough for automated building of the protein structure. This demonstrates the feasibility of determining novel protein structures using FELs. We anticipate that serial femtosecond crystallography will become an important tool for the structure determination of proteins that are difficult to crystallize, such as membrane proteins.

De novo protein crystal structure determination from X-ray free-electron laser data.

Many advances in the understanding of radiation damage to protein crystals, particularly at cryogenic temperatures, have been made in recent years, but with this comes an expanding literature, and, to the new breed of protein crystallographer who is not really interested in X-ray physics or radiation chemistry but just wants to solve a biologically relevant structure, the technical nature and breadth of this literature can be daunting. The purpose of this paper is to serve as a rough guide to radiation damage issues, and to provide references to the more exacting and detailed work. No attempt has been made to report precise numbers (a factor of two is considered satisfactory), and, since there are aspects of radiation damage that are demonstrably unpredictable, the `worst case scenario' as well as the `average crystal' are discussed in terms of the practicalities of data collection.

/pdf/a-beginner-s-guide-to-radiation-damage-49nm0g5h18.pdf

A beginner’s guide to radiation damage

A detailed analysis of radiation-damage-induced structural and intensity changes is presented on the model protein thaumatin. Changes in reflection intensities induced by irradiation display a parabolic character. The most pronounced structural changes observed were disulfide-bond breakage and associated main-chain and side-chain movements as well as decarboxylation of aspartate and glutamate residues. The structural changes induced on the sulfur atoms were successfully used to obtain high-quality phase estimates via an RIP procedure. Results obtained with ACORN suggest that the contribution originating from the partial structure may play an important role in phasing even at less than atomic resolution.

https://mcl1.ncifcrf.gov/dauter_pubs/230.pdf

Structural effects of radiation damage and its potential for phasing.

Substructure-solution and phasing procedures using a combination of anomalous scattering and radiation-damage-induced isomorphous differences have been investigated. The tyrosine residues in thaumatin were iodinated with N-­iodosuccinimide in the crystalline form as well as prior to crystallization. Several data sets were collected from both forms and used for substructure solution and phasing using various protocols, employing anomalous, isomorphous or both these signals. It was shown that combination of the anomalous and isomorphous signals in the form of the RIPAS (radiation-damage-induced phasing with anomalous scattering) strategy is beneficial for both locating the substructure and subsequent phasing.

Petrus H. Zwart

Papers

Structural effects of radiation damage and its potential for phasing.

Radiation-damage-induced phasing with anomalous scattering: substructure solution and phasing.