Ice formation within protein crystals is a significant obstacle to cryocrystallographic study of protein structure and has long prevented studies of how a protein's structural ensemble evolves with temperature in the most biophysically interesting range from 260 K to the protein-solvent glass transition near 200 K, where non-harmonic motions cease.Using protein crystals with solvent cavities as large as 68 A, larger than 98% of deposited structures in the Protein Data Bank, we study ice formation in response to quenches to temperatures between 260 K and 180 K as a function of glycerol concentration using time-resolved X-ray diffraction.Maximum temperatures at which ice formation is observed are consistent with expected melting point suppression due to nanoconfinement.Internal crystalline solvent forms neither pure cubic or hexagonal ice, but a complex mixture where atomic planes of cubic and hexagonal ice stack in a disordered manner.The fraction of cubic planes in this ice increases with the addition of glycerol.Comparison of diffraction intensities from internal ice and the protein lattice allows the maximum ice volume fraction to be determined, and crystallography allows the solvent volume fraction within the unit cell to be determined.Combining these results, we determine the maximum crystallizable solvent fraction within three different protein crystal systems, and these fractions indicate that roughly a monolayer of water adjacent to the protein surface cannot be crystallized.Of greatest practical importance in crystallography, we show that with temperature steps and fast data collection before ice nucleation, ice free crystallographic data sets can be obtained with high probability from crystals with supercooled cryoprotectant-free liquid solvent at temperatures as low as 200 K.Moreover, this supercooled solvent facilitates post-cooling crystal relaxations from kinetically-favored states toward a new low-temperature equilibrium, and this can lead to substantial increases in crystal order.

Small angle X-ray scattering (SAXS) is an increasingly popular technique for obtaining low resolution structural information from macromolecules and complexes in solution. Biomolecular SAXS signals can rapidly degrade due to radiation damage, so that flow or oscillating cells and large total sample volumes may be required. For particularly sensitive or hard to produce samples, such as of light sensitive proteins, metalloenzymes, and large complexes, and studies where multiple buffer conditions are probed sample consumption may be prohibitive. We describe cryo-cooling of samples to 100 K to prevent X-ray induced radiation damage. We identify SAXS-friendly cryoprotectant conditions that suppress ice formation upon cooling, and compare cryoSAXS profiles obtained in window-free variable-path-length cells with room temperature measurements for a variety of standard molecules. We obtain data sufficient for envelope reconstructions using scattering volumes as small as 20 nL, and find good agreement between cryoSAXS data and known atomic structures. We also discuss work on developing low-volume fixed path-length sample holders for cryoSAXS. Cryo-cooled samples can withstand doses that are 2-3 orders of magnitude higher than typically used for SAXS at room temperature, comparable to those used in cryo-crystallography. While practical challenges remain, cryoSAXS opens the possibility of studies exploiting high brightness X-ray sources and mail-in high-throughput SAXS. This work is funded by the NSF (DBI-1152348).

Small angle x-ray scattering (SAXS) is a versatile and widely used technique for obtaining low-resolution structures of macromolecules and complexes. SAXS experiments measure molecules in solution, without the need for labeling or crystallization. However, radiation damage currently limits the application of SAXS to molecules that can be produced in microgram quantities; for typical proteins, 10-20 μL of solution at 1 mg/mL is required to accumulate adequate signal before irreversible x-ray damage is observed. Here, we show that cryocooled proteins and nucleic acids can withstand doses at least two orders of magnitude larger than room temperature samples. We demonstrate accurate T = 100 K particle envelope reconstructions from sample volumes as small as 15 nL, a factor of 1000 smaller than in current practice. Cryo-SAXS will thus enable structure determination of difficult-to-express proteins and biologically important, highly radiation-sensitive proteins including light-activated switches and metalloenzymes.

Global radiation damage to 19 thaumatin crystals has been measured using dose rates from 3 to 680 kGy s⁻¹. At room temperature damage per unit dose appears to be roughly independent of dose rate, suggesting that the timescales for important damage processes are less than ∼1 s. However, at T = 260 K approximately half of the global damage manifested at dose rates of ∼10 kGy s⁻¹ can be outrun by collecting data at 680 kGy s⁻¹. Appreciable sample-to-sample variability in global radiation sensitivity at fixed dose rate is observed. This variability cannot be accounted for by errors in dose calculation, crystal slippage or the size of the data sets in the assay.

X-ray transparent crystallization plates based upon a novel drop-pinning technology provide a flexible, simple and inexpensive approach to protein crystallization and screening. The plates consist of open cells sealed top and bottom by thin optically, UV and X-ray transparent films. The plates do not need wells or depressions to contain liquids. Instead, protein drops and reservoir solution are held in place by rings with micrometre dimensions that are patterned onto the bottom film. These rings strongly pin the liquid contact lines, thereby improving drop shape and position uniformity, and thus crystallization reproducibility, and simplifying automated image analysis of drop contents. The same rings effectively pin solutions containing salts, proteins, cryoprotectants, oils, alcohols and detergents. Strong pinning by rings allows the plates to be rotated without liquid mixing to 90° for X-ray data collection or to be inverted for hanging-drop crystallization. The plates have the standard SBS format and are compatible with standard liquid-handling robots.

Capillary tubes have many advantages over multi-well plates for macromol-ecular crystal growth and handling, including the possibility of in situ structure determination. To obtain complete high-resolution X-ray data sets, cryopreservation protocols must be developed to prevent crystalline ice formation and preserve macromolecular crystal order. The minimum glycerol concentrations required to vitrify aqueous solutions during plunging into liquid nitrogen and liquid propane have been determined for capillary diameters from 3.3 mm to 150 microm. For the smallest diameter, the required glycerol concentrations are 30%(w/v) in nitrogen and 20%(w/v) in propane, corresponding to cooling rates of approximately 800 and approximately 7000 K s(-1), respectively. These concentrations are much larger than are required in current best practice using crystals in loops or on microfabricated mounts. In additon, the relation between the minimum cooling rate for vitrification and glycerol concentration has been estimated; this relation is of fundamental importance in developing rational cryopreservation protocols.

Sessions 4This Bijvoet difference, however, is still small and highly accurate data collection is essential.One of the experimental difficulties using longer wavelength is the increased absorption.Therefore we have developed a crystal mounting technique to eliminate absorption by the frozen cryo-buffer around protein crystal (Kitago et al., 2005), and the practical applicability of this mounting method was examined using several novel proteins at CrKα radiation of 2.29 Å (Watanabe, 2006).In order to utilize this mounting method at the synchrotron beamlines, we made this mounting tool compatible to the standard Hampton CrystalCap.With a special magnet base for this cap, it becomes possible to mount and remount frozen crystals as the standard CrystalCap.We have tested its applicability by sending several frozen crystals to the beamline BL13B1 at NSRRC, Taiwan.This mounting method is also very useful at synchrotron beamlines to mount tiny crystals that are difficult to center because of the lens-shaped frozen buffer in the cryoloop.In this development, we also use a loop made of a polyimide film microfabricated by photolithography.

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.

A modified capillary-growth method is described that has substantial advantages for standard and high-throughput protein crystal growth. Protein-containing drops are injected into vapor-permeable flexible X-ray-transparent polyester tubing. The protein concentration in the drop increases over time by water transport through the tubing wall at a rate controlled by the wall thickness and ambient relative humidity. Unlike in conventional vapor-diffusion growth, the evaporation rate from the drop is constant over a longer time period, providing more suitable conditions for nucleation, and can be controlled by varying the tubing thickness and surrounding humidity. In situ X-ray diffraction can be performed at room temperature or, by flash-cooling, at low temperatures. Compared with glass capillaries or thick-wall plastic tubing, sealing and handling the tubing and extracting crystals are much easier.