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The article discusses a study on the successful structure determination of 293 K of fully perdeuterated type 3 antifreeze proteins (AFPs) by joint X-ray and neutron diffraction and provides a detailed description of the protein and its solvent structure. It cites the use of the synthetic gene of type 3 AFP that corresponds to the sequence of the 1HG7 PDB entry. It highlights the importance of neutron diffraction for a reliable identification of hydration features in macromolecules particularly those slightly disordered.

The article reports on the LADI-III, a diffractometer dedicated to neutron macromolecular crystallography that is operated by the Institut Laue-Langevin since March 2007. The miniature reader has an improved detector and readout system that enables it to scan the image plate, study crystal biological systems and reduce the time for data collection. An illustration of the study of antifreeze protein is presented.

Antifreeze proteins (AFPs) are found in different species from polar, alpine and subarctic regions, where they serve to inhibit ice-crystal growth by adsorption to ice surfaces. Recombinant North Atlantic ocean pout ( Macrozoarces americanus) AFP has been used as a model protein to develop protocols for amino-acid-specific hydrogen reverse-labelling of methyl groups in leucine and valine residues using Escherichia coli high-density cell cultures supplemented with the amino-acid precursor α-ketoisovalerate. Here, the successful methyl protonation (methyl reverse-labelling) of leucine and valine residues in AFP is reported. Methyl-protonated AFP was expressed in inclusion bodies, refolded in deuterated buffer and purified by cation-exchange chromatography. Crystals were grown in D2O buffer by the sitting-drop method. Preliminary neutron Laue diffraction at 293 K using LADI-III at ILL showed in a few 24 h exposures a very low background and clear small spots up to a resolution of 1.80 Å from a crystal of dimensions 1.60 × 0.38 × 0.38 mm corresponding to a volume of 0.23 mm3. [ABSTRACT FROM AUTHOR]

The highly homologous type III antifreeze protein (AFP) subfamily share the capability to inhibit ice growth at subzero temperatures. Extensive studies by X-ray crystallography have been conducted, mostly on AFPs from polar fishes. Although interactions between a defined flat ice-binding surface and a particular lattice plane of an ice crystal have now been identified, the fine structural features underlying the antifreeze mechanism still remain unclear owing to the intrinsic difficulty in identifying H atoms using X-ray diffraction data alone. Here, successful perdeuteration ( i.e. complete deuteration) for neutron crystallographic studies of the North Atlantic ocean pout ( Macrozoarces americanus) AFP in Escherichia coli high-density cell cultures is reported. The perdeuterated protein (AFP D) was expressed in inclusion bodies, refolded in deuterated buffer and purified by cation-exchange chromatography. Well shaped perdeuterated AFP D crystals have been grown in D2O by the sitting-drop method. Preliminary neutron Laue diffraction at 293 K using LADI-III at ILL showed that with a few exposures of 24 h a very low background and clear small spots up to a resolution of 1.85 Å were obtained using a `radically small' perdeuterated AFP D crystal of dimensions 0.70 × 0.55 × 0.35 mm, corresponding to a volume of 0.13 mm3. [ABSTRACT FROM AUTHOR]