A comparison of gas stream cooling and plunge cooling of macromolecular crystals.

Cryocooling for macromolecular crystallography is usually performed via plunging the crystal into a liquid cryogen or placing the crystal in a cold gas stream. These two approaches are compared here for the case of nitro-gen cooling. The results show that gas stream cooling, which typically cools the crystal more slowly, yields lower mosaicity and, in some cases, a stronger anomalous signal relative to rapid plunge cooling.

Structural characterization of a prolyl aminodipeptidase (PepX) from Lactobacillus helveticus.

Prolyl aminodipeptidase (PepX) is an enzyme that hydrolyzes peptide bonds from the N-terminus of substrates when the penultimate amino-acid residue is a proline. Prolyl peptidases are of particular interest owing to their ability to hydrolyze food allergens that contain a high percentage of proline residues. PepX from Lactobacillus helveticus was cloned and expressed in Escherichia coli as an N-terminally His-tagged recombinant construct and was crystallized by hanging-drop vapor diffusion in a phosphate buffer using PEG 3350 as a precipitant.

The impact of cryosolution thermal contraction on proteins and protein crystals: volumes, conformation and order.

Cryocooling of macromolecular crystals is commonly employed to limit radiation damage during X-ray diffraction data collection. However, cooling itself affects macromolecular conformation and often damages crystals via poorly understood processes. Here, the effects of cryosolution thermal contraction on macromolecular conformation and crystal order in crystals ranging from 32 to 67% solvent content are systematically investigated.

Elucidating factors important for monovalent cation selectivity in enzymes: E. coli β-galactosidase as a model.

Many enzymes require a specific monovalent cation (M(+)), that is either Na(+) or K(+), for optimal activity. While high selectivity M(+) sites in transport proteins have been extensively studied, enzyme M(+) binding sites generally have lower selectivity and are less characterized. Here we study the M(+) binding site of the model enzyme E.

In crystallo optical spectroscopy (icOS) as a complementary tool on the macromolecular crystallography beamlines of the ESRF.

The analysis of structural data obtained by X-ray crystallography benefits from information obtained from complementary techniques, especially as applied to the crystals themselves. As a consequence, optical spectroscopies in structural biology have become instrumental in assessing the relevance and context of many crystallographic results. Since the year 2000, it has been possible to record such data adjacent to, or directly on, the Structural Biology Group beamlines of the ESRF.

: a computation tool to aid in the visualization and characterization of solvent channels in macromolecular crystals.

A computation tool is described that facilitates visualization and characterization of solvent channels or pores within macromolecular crystals. A scalar field mapping the shortest distance to protein surfaces is calculated on a grid covering the unit cell and is written as a map file. The map provides a multiscale representation of the solvent channels, which when viewed in standard macromolecular crystallographic software packages gives an intuitive sense of the solvent channel architecture.

Efficient cryoprotection of macromolecular crystals using vapor diffusion of volatile alcohols.

Macromolecular X-ray crystallography, usually done at cryogenic temperature to limit radiation damage, often requires liquid cryoprotective soaking that can be labor intensive and damaging to crystals. Here we describe a method for cryoprotection that uses vapor diffusion of volatile cryoprotective agents into loop-mounted crystals. The crystal is mounted into a vial containing a small volume of an alcohol-based cryosolution.

Improved reproducibility of unit-cell parameters in macromolecular cryocrystallography by limiting dehydration during crystal mounting.

In macromolecular cryocrystallography unit-cell parameters can have low reproducibility, limiting the effectiveness of combining data sets from multiple crystals and inhibiting the development of defined repeatable cooling protocols. Here, potential sources of unit-cell variation are investigated and crystal dehydration during loop-mounting is found to be an important factor. The amount of water lost by the unit cell depends on the crystal size, the loop size, the ambient relative humidity and the transfer distance to the cooling medium.

Tetra-ethyl-ammonium 7,12-di-cyano-1-carba-closo-dodeca-borate.

In the title compound, C8H20N(+)·C3H10B11N2 (-), the carborane anion cage displays nearly-perfect Cs symmetry, with the two CN groups lying on a noncrystallographic mirror plane that bis-ects the cage. In the crystal, the anions form extended chains along the a-axis direction, with C-H⋯N hydrogen bonds linking consecutive anions. The C N bond lengths (and B-C N angles) in the nitrile moities are 1.

Copper-promoted cyanation of a boron cluster: synthesis, X-ray structure, and reactivity of 12-CN-closo-CHB11H10-.

Microwave-assisted cross-coupling reactions of boron-iodinated derivatives of 1-carba-closo-dodecaborate(1-) (1) with CuCN is shown to cyanate boron vertices of this anion. Clusters with one or two CN groups can be prepared: syntheses of 12-CN-CHB11H10(-) (3) and 7,12-(CN)2-CHB11H9(-) (6) gave yields of 80% and 81%, respectively. The [Et4N](+) salts of 3 and 6 were characterized by NMR, IR, and mass spectroscopies, and the crystal structure of [Et4N]3 was determined by single-crystal X-ray diffraction.

LacZ β-galactosidase: structure and function of an enzyme of historical and molecular biological importance.

This review provides an overview of the structure, function, and catalytic mechanism of lacZ β-galactosidase. The protein played a central role in Jacob and Monod's development of the operon model for the regulation of gene expression. Determination of the crystal structure made it possible to understand why deletion of certain residues toward the amino-terminus not only caused the full enzyme tetramer to dissociate into dimers but also abolished activity.

The use of trimethylamine N-oxide as a primary precipitating agent and related methylamine osmolytes as cryoprotective agents for macromolecular crystallography.

Both crystallization and cryoprotection are often bottlenecks for high-resolution X-ray structure determination of macromolecules. Methylamine osmolytes are known stabilizers of protein structure. One such osmolyte, trimethylamine N-oxide (TMAO), has seen occasional use as an additive to improve macromolecular crystal quality and has recently been shown to be an effective cryoprotective agent for low-temperature data collection.

Similarities and differences in radiation damage at 100 K versus 160 K in a crystal of thermolysin.

The temperature-dependence of radiation damage in macromolecular X-ray crystallography is currently much debated. Most protein crystallographic studies are based on data collected at 100 K. Data collection at temperatures below 100 K has been proposed to reduce radiation damage and above 100 K to be useful for kinetic crystallography that is aimed at the generation and trapping of protein intermediate states.

Inducing phase changes in crystals of macromolecules: status and perspectives for controlled crystal dehydration.

The increase in the number of large multi-component complexes and membrane protein crystal structures determined over the last few years can be ascribed to a number of factors such as better protein expression and purification systems, the emergence of high-throughput crystallization techniques and the advent of 3rd generation synchrotron sources. However, many systems tend to produce crystals that can be extremely heterogeneous in their diffraction properties. This prevents, in many cases, the collection of diffraction data of sufficient quality to yield useful biological or phase information.

Gradual adaptive changes of a protein facing high salt concentrations.

Several experimental techniques were applied to unravel fine molecular details of protein adaptation to high salinity. We compared four homologous enzymes, which suggested a new halo-adaptive state in the process of molecular adaptation to high-salt conditions. Together with comparative functional studies, the structure of malate dehydrogenase from the eubacterium Salinibacter ruber shows that the enzyme shares characteristics of a halo-adapted archaea-bacterial enzyme and of non-halo-adapted enzymes from other eubacterial species.

Progress in rational methods of cryoprotection in macromolecular crystallography.

Cryogenic cooling of macromolecular crystals is commonly used for X-ray data collection both to reduce crystal damage from radiation and to gather functional information by cryogenically trapping intermediates. However, the cooling process can damage the crystals. Limiting cooling-induced crystal damage often requires cryoprotection strategies, which can involve substantial screening of solution conditions and cooling protocols.

Direct and indirect roles of His-418 in metal binding and in the activity of beta-galactosidase (E. coli).

The active site of ss-galactosidase (E. coli) contains a Mg(2+) ion ligated by Glu-416, His-418 and Glu-461 plus three water molecules. A Na(+) ion binds nearby.

Changes to crystals of Escherichia coli beta-galactosidase during room-temperature/low-temperature cycling and their relation to cryo-annealing.

Flash-cooling of macromolecular crystals often compromises diffraction quality by increasing the mosaicity. In some cases, cycling the crystal between low temperature (LT) and room temperature (RT) can reverse this increase in mosaicity. Previous studies of RT/LT cycling have focused on the quality of the crystal as it was repeatedly returned to the LT state.

Structural analysis of silanediols as transition-state-analogue inhibitors of the benchmark metalloprotease thermolysin.

Dialkylsilanediols have been found to be an effective functional group for the design of active-site-directed protease inhibitors, including aspartic (HIV protease) and metallo (ACE and thermolysin) proteases. The use of silanediols is predicated on its resemblance to the hydrated carbonyl transition-state structure of amide hydrolysis. This concept has been tested by replacing the presumed tetrahedral carbon of a thermolysin substrate with a silanediol group, resulting in an inhibitor with an inhibition constant K(i) = 40 nM.

Cryo-cooling in macromolecular crystallography: advantages, disadvantages and optimization.

The flash-cooling of crystals in macromolecular crystallography has become commonplace. The procedure makes it possible to collect data from much smaller specimens than was the case in the past Also, flash-cooled crystals are much less prone to radiation damage than their room-temperature counterparts, allowing data to be accumulated over extended periods of time. Notwithstanding the attractiveness of the technique, it does have potential disadvantages.

The role of solvent transport in cryo-annealing of macromolecular crystals.

Macromolecular crystals are usually cooled to approximately 100 K for X-ray diffraction experiments in order to diminish lattice damage arising from the ionizing radiation. Such cooling often produces lattice disorder, but this disorder can sometimes be substantially reduced by cycling the crystal between low and higher temperatures (called annealing). Here, two related aspects of cryocooling and annealing are investigated using crystals of beta-galactosidase and thermolysin.

Structural basis for the altered activity of Gly794 variants of Escherichia coli beta-galactosidase.

The open-closed conformational switch in the active site of Escherichia coli beta-galactosidase was studied by X-ray crystallography and enzyme kinetics. Replacement of Gly794 by alanine causes the apoenzyme to adopt the closed rather than the open conformation. Binding of the competitive inhibitor isopropyl thio-beta-D-galactoside (IPTG) requires the mutant enzyme to adopt its less favored open conformation, weakening affinity relative to wild type.

Crystallization of beta-galactosidase does not reduce the range of activity of individual molecules.

By use of a capillary electrophoresis-based procedure, it is possible to measure the activity of individual molecules of beta-galactosidase. Molecules from the crystallized enzyme as well as the original enzyme preparation used to grow the crystals both displayed a range of activity of 20-fold or greater. beta-Galactosidase molecules obtained from two different crystals had indistinguishable activity distributions of 31,600 +/- 1100 and 31,800 +/- 1100 reactions min(-1) (enzyme molecule)(-1).