Correction: Taberman, H. Radiation Damage in Macromolecular Crystallography—An Experimentalist’s View. Crystals 2018, 8, 157

The author wishes to make the following corrections to this paper [1]:[...

Radiation Damage in Macromolecular Crystallography—An Experimentalist’s View

Radiation damage still remains a major limitation and challenge in macromolecular X-ray crystallography. Some of the high-intensity radiation used for diffraction data collection experiments is absorbed by the crystals, generating free radicals. These give rise to radiation damage even at cryotemperatures (~100 K), which can lead to incorrect biological conclusions being drawn from the resulting structure, or even prevent structure solution entirely. Investigation of mitigation strategies and the effects caused by radiation damage has been extensive over the past fifteen years. Here, recent understanding of the physical and chemical phenomena of radiation damage is described, along with the global effects inflicted on the collected data and the specific effects observed in the solved structure. Furthermore, this review aims to summarise the progress made in radiation damage studies in macromolecular crystallography from the experimentalist’s point of view and to give an introduction to the current literature

Correction: Taberman, H. Radiation Damage in Macromolecular Crystallography—An Experimentalist’s View. Crystals 2018, 8, 157

The author wishes to make the following corrections to this paper [1]:[...

Radiation Damage in Macromolecular Crystallography—An Experimentalist’s View

Radiation damage still remains a major limitation and challenge in macromolecular X-ray crystallography. Some of the high-intensity radiation used for diffraction data collection experiments is absorbed by the crystals, generating free radicals. These give rise to radiation damage even at cryotemperatures (~100 K), which can lead to incorrect biological conclusions being drawn from the resulting structure, or even prevent structure solution entirely. Investigation of mitigation strategies and the effects caused by radiation damage has been extensive over the past fifteen years. Here, recent understanding of the physical and chemical phenomena of radiation damage is described, along with the global effects inflicted on the collected data and the specific effects observed in the solved structure. Furthermore, this review aims to summarise the progress made in radiation damage studies in macromolecular crystallography from the experimentalist’s point of view and to give an introduction to the current literature

Biomolecular Click Reactions Using a Minimal pH‐Activated Catcher/Tag Pair for Producing Native‐Sized Spider‐Silk Proteins**

A type of protein/peptide pair known as Catcher/Tag pair spontaneously forms an intermolecular isopeptide bond which can be applied for biomolecular click reactions. Covalent protein conjugation using Catcher/Tag pairs has turned out to be a valuable tool in biotechnology and biomedicines, but it is essential to increase the current toolbox of orthogonal Catcher/Tag pairs to expand the range of applications further, for example, for controlled multiple-fragment ligation. We report here the engineering of novel Catcher/Tag pairs for protein ligation, aided by a crystal structure of a minimal CnaB domain from Lactobacillus plantarum. We show that a newly engineered pair, called SilkCatcher/Tag enables efficient pH-inducible protein ligation in addition to being compatible with the widely used SpyCatcher/Tag pair. Finally, we demonstrate the use of the SilkCatcher/Tag pair in the production of native-sized highly repetitive spider-silk-like proteins with >90 % purity, which is not possible by traditional recombinant production methods

Purification, crystallization and preliminary X-ray diffraction analysis of a novel keto-deoxy-D-galactarate (KDG) dehydratase from Agrobacterium tumefaciens

d-Galacturonic acid is the main component of pectin. It could be used to produce affordable renewable fuels, chemicals and materials through biotechnical conversion. Keto-deoxy-d-galactarate (KDG) dehydratase is an enzyme in the oxidative pathway of d-galacturonic acid in Agrobacterium tumefaciens (At). It converts 3-deoxy-2-keto-l-threo-hexarate to α-ketoglutaric semialdehyde. At KDG dehydratase was crystallized by the hanging-drop vapour-diffusion method. The crystals belonged to the monoclinic space group C2, with unit-cell parameters a = 169.1, b = 117.8, c = 74.3 Å, β = 112.4° and an asymmetric unit of four monomers. X-ray diffraction data were collected to 1.9 Å resolution using synchrotron radiation. The three-dimensional structure of At KDG dehydratase will provide valuable information on the function of the enzyme and will allow it to be engineered for biorefinery-based applications

Biomolecular Click Reactions Using a Minimal pH-Activated Catcher/Tag Pair for Producing Native-Sized Spider-Silk Proteins

Funding Information: We thank Eva Crosas for her help with the early phases of the structure refinement. Protein crystallization was performed at SPC facility at EMBL Hamburg and the CD spectroscopy and mass spectrometry of the crystallized proteins at the Center for Structural Systems Biology (CSSB, Deutsches Elektronen‐Synchrotron DESY). We acknowledge technical support by the SPC facility at EMBL Hamburg. The synchrotron data was collected at beamline operated by EMBL Hamburg at the PETRA III storage ring (DESY, Hamburg, Germany). This work was supported by the Academy of Finland through its Centres of Excellence Programme Life‐Inspired Hybrid Materials (LIBER, 2022–2029) under project no 346105 and Academy of Finland projects nos. 317395, 308772, and 333238. We are grateful for the support by the FinnCERES Materials Bioeconomy Ecosystem and use of the Bioeconomy Infrastructure at the Aalto University. Publisher Copyright: © 2023 Wiley-VCH GmbH.A type of protein/peptide pair known as Catcher/Tag pair spontaneously forms an intermolecular isopeptide bond which can be applied for biomolecular click reactions. Covalent protein conjugation using Catcher/Tag pairs has turned out to be a valuable tool in biotechnology and biomedicines, but it is essential to increase the current toolbox of orthogonal Catcher/Tag pairs to expand the range of applications further, for example, for controlled multiple-fragment ligation. We report here the engineering of novel Catcher/Tag pairs for protein ligation, aided by a crystal structure of a minimal CnaB domain from Lactobacillus plantarum. We show that a newly engineered pair, called SilkCatcher/Tag enables efficient pH-inducible protein ligation in addition to being compatible with the widely used SpyCatcher/Tag pair. Finally, we demonstrate the use of the SilkCatcher/Tag pair in the production of native-sized highly repetitive spider-silk-like proteins with >90 % purity, which is not possible by traditional recombinant production methods.Peer reviewe

Structure and function of Caulobacter crescentus aldose-aldose oxidoreductase

Aldose–aldose oxidoreductase (Cc AAOR) is a recently characterized enzyme from the bacterial strain Caulobacter crescentus CB15 belonging to the glucose-fructose oxidoreductase/inositol dehydrogenase/rhizopine catabolism protein (Gfo/Idh/MocA) family. Cc AAOR catalyses the oxidation and reduction of a panel of aldose monosaccharides using a tightly bound NADP(H) cofactor that is regenerated in the catalytic cycle. Furthermore, Cc AAOR can also oxidize 1,4-linked oligosaccharides. In the present study, we present novel crystal structures of the dimeric Cc AAOR in complex with the cofactor and glycerol, D-xylose, D-glucose, maltotriose and D-sorbitol determined to resolutions of 2.0, 1.8, 1.7, 1.9 and 1.8 Å (1 Å=0.1 nm), respectively. These complex structures allowed for a detailed analysis of the ligand-binding interactions. The structures showed that the C1 carbon of a substrate, which is either reduced or oxidized, is close to the reactive C4 carbon of the nicotinamide ring of NADP(H). In addition, the O1 hydroxy group of the substrate, which is either protonated or deprotonated, is unexpectedly close to both Lys104 and Tyr189, which may both act as a proton donor or acceptor. This led us to hypothesize that this intriguing feature could be beneficial for Cc AAOR to catalyse the reduction of a linear form of a monosaccharide substrate and the oxidation of a pyranose form of the same substrate in a reaction cycle, during which the bound cofactor is regenerated.</jats:p

Radiation damage in small molecule crystallography: fact not fiction

Traditionally small molecule crystallographers have not usually observed or recognised significant radiation damage to their samples during diffraction experiments. However, the increased flux densities provided by third generation synchrotrons have resulted in increasing numbers of observations of this phenomenon. The diversity of types of small molecule systems means it is not yet possible to propose a general mechanism for their radiation induced sample decay, however characterisation of the effects will permit attempts to understand and mitigate it. Here systematic experiments are reported on the effects that sample temperature and beam attenuation have on radiation damage progression, allowing qualitative and quantitative assessment of their impact on crystals of a small molecule test sample. To allow inter-comparison of different measurements, radiation damage metrics (diffraction intensity decline, resolution fall-off, scaling B-factor increase) are plotted against the absorbed dose. For ease of dose calculations, the software developed for protein crystallography, RADDOSE-3D, has been modified for use in small molecule crystallography. It is intended that these initial experiments will assist in establishing protocols for small molecule crystallographers to optimise the diffraction signal from their samples prior to the onset of the deleterious effects of radiation damage