Protein Cryocrystallography Research Articles

The rise of neutron cryo-crystallography.

The use of boiled-off liquid nitrogen to maintain protein crystals at 100 K during X-ray data collection has become almost universal. Applying this to neutron protein crystallography offers the opportunity to significantly broaden the scope of biochemical problems that can be addressed, although care must be taken in assuming that direct extrapolation to room temperature is always valid. Here, the history to date of neutron protein cryo-crystallography and the particular problems and solutions associated with the mounting and cryocooling of the larger crystals needed for neutron crystallography are reviewed. Finally, the outlook for further cryogenic neutron studies using existing and future neutron instrumentation is discussed.

Thermal contraction of aqueous glycerol and ethylene glycol solutions for optimized protein-crystal cryoprotection.

The thermal contraction of aqueous cryoprotectant solutions on cooling to cryogenic temperatures is of practical importance in protein cryocrystallography and in biological cryopreservation. In the former case, differential contraction on cooling of protein molecules and their lattice relative to that of the internal and surrounding solvent may lead to crystal damage and the degradation of crystal diffraction properties. Here, the amorphous phase densities of aqueous solutions of glycerol and ethylene glycol at T = 77 K have been determined. Densities with accuracies of <0.5% to concentrations as low as 30%(w/v) were determined by rapidly cooling drops with volumes as small as 70 pl, assessing their optical clarity and measuring their buoyancy in liquid nitrogen-argon solutions. The use of these densities in contraction matching of internal solvent to the available solvent spaces is complicated by several factors, most notably the exclusion of cryoprotectants from protein hydration shells and the expected deviation of the contraction behavior of hydration water from bulk water. The present methods and results will assist in developing rational approaches to cryoprotection and an understanding of solvent behavior in protein crystals.

Crystal sample pins and a storage cassette system compatible with the protein crystallography beamlines at both the Photon Factory and SPring-8

Protein crystallography beamlines at the two major Japanese synchrotron facilities (SPring-8 and Photon Factory) employ different crystal-exchange robots. The robots SPACE and PAM, installed at SPring-8 and the Photon Factory, respectively, accelerate crystal exchange and assist in the efficient use of beamtime. SPACE also supports customizable automatic data collection, in which an X-ray beam is irradiated at a manually determined position during the crystal evaluation process. However, this data collection requires special crystal-mounting screw pins made of polyacetal, which are not compatible with PAM. In order to give users the flexibility to collect diffraction data sets at either of the two synchrotrons, we have developed a compatible cassette system. Pins stored in the cassette can be mounted or demounted using either SPACE or PAM, and are compatible with the customizable automatic data collection provided by SPACE. Users can handle the pins and cassettes easily, employing common tools and techniques for efficient protein cryocrystallography experiments.

A fibre-based crystal mounting technique for protein cryocrystallography

The CryoFibre, a crystal mounting tool, has been developed for protein cryocrystallography. The technique attaches single crystals to the tips of polyester fibres, allowing removal of excess liquid around each crystal. Single-wavelength anomalous dispersion phasing using a Cu Kα X-ray source (Cu SAD) was applied to crystals from five proteins without any derivatization, demonstrating a clear improvement in the success rate of Cu SAD compared with the conventional loop technique. In addition, a xylanase crystal on the surface of a synthetic zeolite as a hetero-epitaxic nucleant was directly mounted on the CryoFibre without separation treatment of the crystal from the zeolite. The crystal had a lower mosaicity than that observed using the conventional technique, indicating that the fibre technique is suitable for high-quality data collection from zeolite-mediated crystals.

The Carbohydrate-Binding Site in Galectin-3 Is Preorganized To Recognize a Sugarlike Framework of Oxygens: Ultra-High-Resolution Structures and Water Dynamics

The recognition of carbohydrates by proteins is a fundamental aspect of communication within and between living cells. Understanding the molecular basis of carbohydrate–protein interactions is a prerequisite for the rational design of synthetic ligands. Here we report the high- to ultra-high-resolution crystal structures of the carbohydrate recognition domain of galectin-3 (Gal3C) in the ligand-free state (1.08 Å at 100 K, 1.25 Å at 298 K) and in complex with lactose (0.86 Å) or glycerol (0.9 Å). These structures reveal striking similarities in the positions of water and carbohydrate oxygen atoms in all three states, indicating that the binding site of Gal3C is preorganized to coordinate oxygen atoms in an arrangement that is nearly optimal for the recognition of β-galactosides. Deuterium nuclear magnetic resonance (NMR) relaxation dispersion experiments and molecular dynamics simulations demonstrate that all water molecules in the lactose-binding site exchange with bulk water on a time scale of nanoseconds or shorter. Nevertheless, molecular dynamics simulations identify transient water binding at sites that agree well with those observed by crystallography, indicating that the energy landscape of the binding site is maintained in solution. All heavy atoms of glycerol are positioned like the corresponding atoms of lactose in the Gal3C complexes. However, binding of glycerol to Gal3C is insignificant in solution at room temperature, as monitored by NMR spectroscopy or isothermal titration calorimetry under conditions where lactose binding is readily detected. These observations make a case for protein cryo-crystallography as a valuable screening method in fragment-based drug discovery and further suggest that identification of water sites might inform inhibitor design.

Hyperquenching for protein cryocrystallography.

When samples having volumes characteristic of protein crystals are plunge cooled in liquid nitrogen or propane, most cooling occurs in the cold gas layer above the liquid. By removing this cold gas layer, cooling rates for small samples and modest plunge velocities are increased to 1.5 × 10(4) K s(-1), with increases of a factor of 100 over current best practice possible with 10 μm samples. Glycerol concentrations required to eliminate water crystallization in protein-free aqueous mixtures drop from ∼28% w/v to as low as 6% w/v. These results will allow many crystals to go from crystallization tray to liquid cryogen to X-ray beam without cryoprotectants. By reducing or eliminating the need for cryoprotectants in growth solutions, they may also simplify the search for crystallization conditions and for optimal screens. The results presented here resolve many puzzles, such as why plunge cooling in liquid nitrogen or propane has, until now, not yielded significantly better diffraction quality than gas-stream cooling.

How to Avoid Premature Decay of Your Macromolecular Crystal: A Quick Soak for Long Life

Radiation damage to biological samples is currently one of the major limiting factors in macromolecular X-ray crystallography, since it severely and irreversibly affects the quality of the data that can be obtained from a diffraction experiment. However, radiation damage can effectively be reduced by utilizing the electron and radical scavenging potential of certain small-molecule compounds. We propose an approach to protect macromolecular crystals prior to data collection by quick soaking with scavengers. This, in favorable cases, can more than double crystal lifetime in the X-ray beam. The approach has the potential to yield diffraction data of superior quality and hence to increase the amount of high-quality diffraction data and of structural information attainable from a single crystal.

Getting protein solvent structures down cold

Getting protein solvent structures down cold

Investigation of possible free-radical scavengers and metrics for radiation damage in protein cryocrystallography.

The introduction of highly intense wiggler and undulator beamlines has reintroduced the problem of X-ray radiation damage in protein crystals even at cryogenic temperatures. Several metrics for monitoring radiation damage are considered and unit-cell volume expansion is systematically investigated using crystals of three different types, but it is found to be too variable to be a useful metric. Radical scavengers of secondary radiation damage are investigated as possible mitigating agents. Styrene is found to be ineffective. A method of spectroscopically measuring the radiation damage with a microspectrophotometer was used and, in conjunction with crystallographic data, provided tentative but suggestive evidence for the efficacy of ascorbate as a free-radical scavenging agent in cryocooled hen egg-white lysozyme crystals.

Investigation of possible free radical scavengers in protein cryocrystallography

Investigation of possible free radical scavengers in protein cryocrystallography

Freeze-Trapping Isomorphous Xenon Derivatives of Protein Crystals

A simple and efficient method to prepare isomorphous derivatives of protein crystals with xenon as a heavy atom is described. The method consists of exposing a crystal to xenon gas of pressures above 5 atm (~ 0.5 MPa) for several minutes and subsequently shock-freezing the crystal to immobilize the xenon dissolved in the mother liquor and bound to the protein. Diffraction data can the be collected with the established techniques of protein cryocrystallography. Two types of high-pressure device are described to expose a protein crystal to the required xenon pressure, permitting rapid freeze-quenching after xenon exposure. One of these devices can be used for gas pressures up to and exceeding 5.0 MPa, with a gas consumption of a few millilitres of uncompressed gas. The technique has been tested with monoclinic crystals of sperm-whale metmyoglobin, which has four xenon binding sites. The results of these experiments are described and discussed. Potential applications of this technique include-besides the classical multiwavelength anomalous diffraction (MAD) or single isomorphous replacement with anomalous scattering (SIRAS) experiments-the derivation of low-angle phase information by modifying the electron density of the solvent regions within the crystal.

A simple method for making reproducible fibre loops for protein cryocrystallography

A practical, step-by-step description of the construction of loops for protein cryocrystallography using pulled-out micro-injection capillaries is described. The advantages of this method are the high reproducibility of the loops, the ease of adjustment of the loop diameter and the formation of thin films owing to the narrow base of the loop. During assembly, the loop does not make contact with either glue or cement.