Jan Kmetko

Microfabricated mounts for high-throughput macromolecular cryocrystallography

A new approach is described for mounting microcrystals of biological macromolecules for cryocrystallography. The sample mounts are prepared by patterning thin polyimide films by standard microfabrication techniques. The patterned structures contain a small hole for the crystal connected to a larger hole via a drainage channel, allowing removal of excess liquid and easier manipulation in viscous solutions. These polyimide structures are wrapped around small metal rods. The resulting curvature increases their rigidity and allows a convenient scoop-like action in retrieving crystals. The polyimide contributes minimally to X-ray background and absorption, and can be treated to obtain desired hydrophobicity or hydrophilicity. The new mounts are fully compatible with existing automated sample-handling hardware for cryocrystallography. Their potential advantages include completely reproducible sample hole sizes to below 10 µm; accurate and reproducible sample positioning and good sample-to-mount contrast, simplifying alignment; more convenient manipulation of small crystals; easier removal of excess liquid and reduced background scatter; reduced thermal mass and more rapid flash-cooling; and easy design customization and mass production. They are especially well suited to data collection from the smaller crystals produced in high-throughput crystallization trials, and are suitable for automated crystal retrieval. They should be more generally useful for X-ray data collection from small organic and inorganic crystals of all types.

Quantifying X-ray radiation damage in protein crystals at cryogenic temperatures

The dependence of radiation damage to protein crystals at cryogenic temperatures upon the X-ray absorption cross-section of the crystal has been examined. Lysozyme crystals containing varying heavy-atom concentrations were irradiated and diffraction patterns were recorded as a function of the total number of incident photons. An experimental protocol and a coefficient of sensitivity to absorbed dose, proportional to the change in relative isotropic B factor, are defined that together yield a sensitive and robust measure of damage. Radiation damage per incident photon increases linearly with the absorption coefficient of the crystal, but damage per absorbed photon is the same for all heavy-atom concentrations. Similar damage per absorbed photon is observed for crystals of three proteins with different molecular sizes and solvent contents.

A new sample mounting technique for room-temperature macromolecular crystallography

A new method for mounting protein crystals and other environmentally sensitive samples for room-temperature diffraction measurements is described. A crystal is retrieved using a microfabricated sample mount as recently reported, and the mount is inserted into a modified goniometer-compatible base. A transparent thin-wall polyester tube sealed at one end and filled with stabilizing liquid is then drawn over the crystal and sealed to the goniometer base. Compared with mounting using glass capillaries, this method can provide lower-background X-ray scattering, especially at higher resolutions; dramatically improved ease of crystal mounting with minimal chance of damage; accurate and reproducible crystal positioning relative to the goniometer base; improved crystal visibility and ease of alignment, especially for very small crystals; and compatibility with high-throughput approaches. Crystals can be rapidly screened and eliminated earlier in the data collection pipeline, and the cause of poor low-temperature diffraction can be diagnosed.

Can radiation damage to protein crystals be reduced using small-molecule compounds?

Recent studies have defined a data-collection protocol and a metric that provide a robust measure of global radiation damage to protein crystals. Using this protocol and metric, 19 small-molecule compounds (introduced either by cocrystallization or soaking) were evaluated for their ability to protect lysozyme crystals from radiation damage. The compounds were selected based upon their ability to interact with radiolytic products (e.g. hydrated electrons, hydrogen, hydroxyl and perhydroxyl radicals) and/or their efficacy in protecting biological molecules from radiation damage in dilute aqueous solutions. At room temperature, 12 compounds had no effect and six had a sensitizing effect on global damage. Only one compound, sodium nitrate, appeared to extend crystal lifetimes, but not in all proteins and only by a factor of two or less. No compound provided protection at T=100 K. Scavengers are ineffective in protecting protein crystals from global damage because a large fraction of primary X-ray-induced excitations are generated in and/or directly attack the protein and because the ratio of scavenger molecules to protein molecules is too small to provide appreciable competitive protection. The same reactivity that makes some scavengers effective radioprotectors in protein solutions may explain their sensitizing effect in the protein-dense environment of a crystal. A more productive focus for future efforts may be to identify and eliminate sensitizing compounds from crystallization solutions.

Order in molecular liquids near solid-liquid interfaces

Abstract Much less is known about structure at solid–liquid interfaces than at solid or liquid surfaces, largely because it is more difficult for experimental probes to penetrate into an interface. In recent years, the availability of synchrotron radiation (which is intense and collimated, and whose energy and thus penetration depth can be varied) has made scattering experiments at such interfaces possible. This article reviews some recent studies of ordering at the liquid side of a solid–liquid interface.

Using X-rays to characterize the process of self-assembly in real time

Abstract We have performed in situ X-ray reflectivity studies of the growth of octadecyltrichlorosilane (OTS) monolayers on oxidized Si(111) from solutions in heptane. We find that for all concentrations, the film grows through the formation of islands of vertical molecules. The coverage follows a simple Langmuir form as a function of time, except for very low concentration solutions at early times, where a time offset is required to fit the curve. We have also examined films removed from solution, and we find that rinsing removes molecules and causes the remaining molecules to tilt. Thus, samples studied using the ‘interrupted growth’ technique are not representative of the actual growth process.

Quantifying X-ray radiation damage in protein crystals

We have examined how radiation damage to protein crystals at cryogenic temperatures depends upon the crystals X-ray absorption cross-section [1]. Lysozyme crystals containing varying heavy atom concentrations were irradiated, and diffraction patterns recorded as a function of the total number of incident photons. We define an experimental protocol that monitors degradation of the relative, isotropic B-factor and a coefficient of sensitivity to absorbed dose, and show that together they yield a sensitive and robust measure of damage. Radiation damage per incident photon increases linearly with the crystals absorption coefficient, but damage per absorbed photon is the same for all heavy atom concentrations. Using this protocol and damage metric, we have examined crystals of five proteins with a wide range of molecular weights, solvent contents and room-temperature radiation sensitivities. At cryogenic temperatures, all five show similar damage per absorbed photon. X-ray radiation sensitivity of protein crystals — properly defined — may thus be roughly independent of the crystal composition at cryogenic temperatures, consistent with results in electron diffraction. This will simplify automated optimization of data collection protocols to minimize X-ray damage. We have also examined the effects of a wide variety of free radical scavengers on radiation sensitivity of simple model proteins at room and cryogenic temperatures. Only one — nitrate ion — shows protective effects at room temperature, and none show protective effects at cryogenic temperatures.

Interfacial Diffusion in a MOCVD Grown Barium Titanate Film

A combination of two nondestructive techniques, Grazing Incidence X-ray Reflectivity and High Resolution X-ray Diffraction, is used to study (at around 10{angstrom} resolution) the composition profile across a 500{angstrom} thick film of BaTiO{sub 3} grown epitaxially on (100) MgO by MOCVD. Results form both studies indicate diffusion of Mg to about 250{angstrom} into the film at film-substrate interface, consistent with the diffuse ferroelectric phase transition observed in this film. The lattice parameter a shows a progressive decrease as the authors move into the film from the interface, and an anomalously low value in the Mg-free portion of the film.