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14 pages, 9 figures, 2 tables., Counterdiffusion crystallization in capillary is a very simple, cost-effective, and practical procedure for obtaining protein crystals suitable for X-ray data analysis. Its principles have been derived using well-known concepts coupling the ideas of precipitation and diffusion mass transport in a restricted geometry. The counterdiffusion process has been used to simultaneously screen for optimal conditions for protein crystal growth, incorporate strong anomalous scattering atoms, and mix in cryogenic solutions in a single capillary tube. The crystals obtained in the capillary have been used in situ for X-ray analysis. The implementation of this technique linked to the advancement of current crystallography software leads to a powerful structure determination method consolidating crystal growth, X-ray data collection, and ab initio phase determination into one without crystal manipulation. We review the historical progress of counterdiffusion crystallization, its application to X-ray crystallography, and ongoing tool development for high-throughput protein structure determination., This work was supported by NASA, Alabama Structural Biology Consortium NSF-EPSCoR, NIH NIGMS Structural Genomic Project, Alabama Space Grant Consortium, and the Secretaria de Educación y Ciencia of Spain. We thank Zhi-Jie Liu and Bi-Cheng Wang for the usage and guidance of the ISAS crystallography package. We also express our gratitude to Craig Ogata for his invaluable assistance at beam line X4A at the National Synchrotron Light Source, Brookhaven National Laboratory, and Zbigniew Dauter and Edward Meehan for many insightful discussions. We greatly appreciate the technical support of Joyce Looger and Diana Toh. The fabrication of the crystallization cassette would not have been possible without the engineering expertise of Mark Wells and Greg Jenkins. Finally we thank the laboratories that have shared their results and experiences with counterdiffusion capillary crystallization.

Pressures in the 100 MPa range are known to have an enormous number of effects on the action of proteins, but straightforward means for determining the structural basis of these effects have been lacking. Here, crystallography has been used to probe effects of pressure on sperm whale myoglobin structure. A comparison of pressure effects with those seen at low pH suggests that structural changes under pressure are interpretable as a shift in the populations of conformational substates. Furthermore, a novel high-pressure protein crystal-cooling method has been used to show low-temperature metastability, providing an alternative to room temperature, beryllium pressure cell-based techniques. The change in protein structure due to pressure is not purely compressive and involves conformational changes important to protein activity. Correlation with low-pH structures suggests observed structural changes are associated with global conformational substates. Methods developed here open up a direct avenue for exploration of the effects of pressure on proteins.

Background: [NiFeSe] hydrogenases are metalloenzymes that catalyze the reaction H 2 ↔2H + +2e - .They are generally heterodimeric, contain three iron–sulfur clusters in their small subunit and a nickel–iron-containing active site in their large subunit that includes a selenocysteine (SeCys) ligand. Results: We report here the X-ray structure at 2.15 A resolution of the periplasmic [NiFeSe] hydrogenase from Desulfomicrobium baculatum in its reduced, active form. A comparison of active sites of the oxidized, as-prepared, Desulfovibrio gigas and the reduced D. baculatum hydrogenases shows that in the reduced enzyme the nickel-iron distance is 0.4 A shorter than in the oxidized enzyme. In addition, the putative oxo ligand, detected in the as-prepared D.gigas enzyme, is absent from the D.baculatum hydrogenase. We also observe higher-than-average temperature factors for both the active site nickel–selenocysteine ligand and the neighboring Glu18 residue, suggesting that both these moieties are involved in proton transfer between the active site and the molecular surface. Other differences between [NiFeSe] and [NiFe] hydrogenases are the presence of a third [4Fe4S] cluster replacing the [3Fe4S] cluster found in the D.gigas enzyme, and a putative iron center that substitutes the magnesium ion that has already been described at the C terminus of the large subunit of two [NiFe] hydrogenases. Conclusions: The heterolytic cleavage of molecular hydrogen seems to be mediated by the nickel center and the selenocysteine residue. Beside modifying the catalytic properties of the enzyme, the selenium ligand might protect the nickel atom from oxidation. We conclude that the putative oxo ligand is a signature of inactive ‘unready' [NiFe] hydrogenases.