RESUMO
All evidence to date indicates that at T = 100â K all protein crystals exhibit comparable sensitivity to X-ray damage when quantified using global metrics such as change in scaling B factor or integrated intensity versus dose. This is consistent with observations in cryo-electron microscopy, and results because nearly all diffusive motions of protein and solvent, including motions induced by radiation damage, are frozen out. But how do the sensitivities of different proteins compare at room temperature, where radiation-induced radicals are free to diffuse and protein and lattice structures are free to relax in response to local damage? It might be expected that a large complex with extensive conformational degrees of freedom would be more radiation sensitive than a small, compact globular protein. As a test case, the radiation sensitivity of 70S ribosome crystals has been examined. At T = 100 and 300â K, the half doses are 64â MGy (at 3â Å resolution) and 150â kGy (at 5â Å resolution), respectively. The maximum tolerable dose in a crystallography experiment depends upon the initial or desired resolution. When differences in initial data-set resolution are accounted for, the former half dose is roughly consistent with that for model proteins, and the 100/300â K half-dose ratio is roughly a factor of ten larger. 70S ribosome crystals exhibit substantially increased resolution at 100â K relative to 300â K owing to cooling-induced ordering and not to reduced radiation sensitivity and slower radiation damage.
Assuntos
Ribossomos/efeitos da radiação , Thermus thermophilus/efeitos da radiação , Cristalização , Cristalografia por Raios X , Tolerância a Radiação , Temperatura , Raios XRESUMO
Damage from ice and potential toxicity of ice-inhibiting cryoprotective agents (CPAs) are key issues in assisted reproduction of humans, domestic and research animals, and endangered species using cryopreserved oocytes and embryos. The nature of ice formed in bovine oocytes (similar in size to oocytes of humans and most other mammals) after rapid cooling and during rapid warming were examined using synchrotron-based time-resolved x-ray diffraction. Using cooling rates, warming rates and CPA concentrations of current practice, oocytes show no ice after cooling but always develop large ice fractions - consistent with crystallization of most free water - during warming, so most ice-related damage must occur during warming. The detailed behavior of ice at warming depended on the nature of ice formed during cooling. Increasing cooling rates allows oocytes soaked as in current practice to remain essentially ice free during both cooling and warming. Much larger convective warming rates are demonstrated and will allow routine ice-free cryopreservation with smaller CPA concentrations. These results clarify the roles of cooling, warming, and CPA concentration in generating ice in oocytes and establish the structure and grain size of ice formed. Ice formation can be eliminated as a factor affecting post-thaw oocyte viability and development in many species, improving outcomes and allowing other deleterious effects of the cryopreservation cycle to be independently studied.
RESUMO
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.
Assuntos
Temperatura Baixa , Espalhamento a Baixo Ângulo , Difração de Raios X , Aldose-Cetose Isomerases/química , Animais , Soluções Tampão , Galinhas , Crioprotetores/farmacologia , Relação Dose-Resposta à Radiação , Polietilenoglicóis/química , Soluções , Vitrificação/efeitos dos fármacosRESUMO
A series of studies that provide a consistent and illuminating picture of global radiation damage to protein crystals, especially at temperatures above â¼200â K, are described. The radiation sensitivity shows a transition near 200â K, above which it appears to be limited by solvent-coupled diffusive processes. Consistent with this interpretation, a component of global damage proceeds on timescales of several minutes at 180â K, decreasing to seconds near room temperature. As a result, data collection times of order 1â s allow up to half of global damage to be outrun at 260â K. Much larger damage reductions near room temperature should be feasible using larger dose rates delivered using microfocused beams, enabling a significant expansion of structural studies of proteins under more nearly native conditions.
Assuntos
Proteínas/efeitos da radiação , Cristalização , Cristalografia por Raios X , Radiação Eletromagnética , Proteínas de Plantas/efeitos da radiação , Proteínas/química , Temperatura , Fatores de TempoRESUMO
When pure water is cooled at ~10(6) K / s, it forms an amorphous solid (glass) instead of the more familiar crystalline phase. The presence of solutes can reduce this required (or "critical") cooling rate by orders of magnitude. Here, we present critical cooling rates for a variety of solutes as a function of concentration and a theoretical framework for understanding these rates. For all solutes tested, the critical cooling rate is an exponential function of concentration. The exponential's characteristic concentration for each solute correlates with the solute's Stokes radius. A modification of critical droplet theory relates the characteristic concentration to the solute radius and the critical nucleation radius of ice in pure water. This simple theory of ice nucleation and glass formability in aqueous solutions has consequences for general glass-forming systems, and in cryobiology, cloud physics, and climate modeling.
RESUMO
For roughly two decades, cryocrystallography has been the overwhelmingly dominant method for determining high-resolution biomolecular structures. Competition from single-particle cryo-electron microscopy and micro-electron diffraction, increased interest in functionally relevant information that may be missing or corrupted in structures determined at cryogenic temperature, and interest in time-resolved studies of the biomolecular response to chemical and optical stimuli have driven renewed interest in data collection at room temperature and, more generally, at temperatures from the protein-solvent glass transition near 200â K to â¼350â K. Fischer has recently reviewed practical methods for room-temperature data collection and analysis [Fischer (2021), Q. Rev. Biophys. 54, e1]. Here, the key advantages and physical principles of, and methods for, crystallographic data collection at noncryogenic temperatures and some factors relevant to interpreting the resulting data are discussed. For room-temperature data collection to realize its potential within the structural biology toolkit, streamlined and standardized methods for delivering crystals prepared in the home laboratory to the synchrotron and for automated handling and data collection, similar to those for cryocrystallography, should be implemented.
Assuntos
Proteínas , Síncrotrons , Cristalografia por Raios X , Temperatura , Microscopia Crioeletrônica , Proteínas/químicaRESUMO
Biliverdin Reductase B (BLVRB) is an NADPH-dependent reductase that catalyzes the reduction of multiple substrates and is therefore considered a critical cellular redox regulator. In this study, we sought to address whether both structural and dynamics changes occur between different intermediates of the catalytic cycle and whether these were relegated to just the active site or the entirety of the enzyme. Through X-ray crystallography, we determined the apo BLVRB structure for the first time, revealing subtle global changes compared to the holo structure and identifying the loss of a critical hydrogen bond that "clamps" the R78-loop over the coenzyme. Amide and Cα chemical shift perturbations were used to identify environmental and secondary structural changes between intermediates, with more distant global changes observed upon coenzyme binding compared to substrate interactions. NMR relaxation rate measurements provided insights into the dynamic behavior of BLVRB during the catalytic cycle. Specifically, the inherently dynamic R78-loop that becomes ordered upon coenzyme binding persists through the catalytic cycle while similar regions experience dynamic exchange. However, the dynamic exchange processes were found to differ through the catalytic cycle with several groups of residues exhibiting similar dynamic responses. Finally, both local and distal structural and dynamic changes occur within BLVRB that are dependent solely on the oxidative state of the coenzyme. Thus, through a comprehensive analysis here, this study revealed structural and dynamic alterations in BLVRB through its catalytic cycle that are not simply relegated to the active site, but instead, are allosterically coupled throughout the enzyme.
RESUMO
Damage from ice and potential toxicity of ice-inhibiting cryoprotective agents (CPAs) are key issues in assisted reproduction using cryopreserved oocytes and embryos. We use synchrotron-based time-resolved x-ray diffraction and tools from protein cryocrystallography to characterize ice formation within bovine oocytes after cooling at rates between â¼1000 °C/min and â¼600,000°C /min and during warming at rates between 20,000 and 150,000 °C /min. Maximum crystalline ice diffraction intensity, maximum ice volume, and maximum ice grain size are always observed during warming. All decrease with increasing CPA concentration, consistent with the decreasing free water fraction. With the cooling rates, warming rates and CPA concentrations of current practice, oocytes may show no ice after cooling but always develop substantial ice fractions on warming, and modestly reducing CPA concentrations causes substantial ice to form during cooling. With much larger cooling and warming rates achieved using cryocrystallography tools, oocytes soaked as in current practice remain essentially ice free during both cooling and warming, and when soaked in half-strength CPA solution oocytes remain ice free after cooling and develop small grain ice during warming. These results clarify the roles of cooling, warming, and CPA concentration in generating ice in oocytes, establish the character of ice formed, and suggest that substantial further improvements in warming rates are feasible. Ice formation can be eliminated as a factor affecting post-thaw oocyte viability and development, allowing other deleterious effects of the cryopreservation cycle to be studied, and osmotic stress and CPA toxicity reduced. Significance Statement: Cryopreservation of oocytes and embryos is critical in assisted reproduction of humans and domestic animals and in preservation of endangered species. Success rates are limited by damage from crystalline ice, toxicity of cryoprotective agents (CPAs), and damage from osmotic stress. Time-resolved x-ray diffraction of bovine oocytes shows that ice forms much more readily during warming than during cooling, that maximum ice fractions always occur during warming, and that the tools and large CPA concentrations of current protocols can at best only prevent ice formation during cooling. Using tools from cryocrystallography that give dramatically larger cooling and warming rates, ice formation can be completely eliminated and required CPA concentrations substantially reduced, expanding the scope for species-specific optimization of post-thaw reproductive outcomes.
RESUMO
The spatial distribution of radiation damage (assayed by increases in atomic B factors) to thaumatin and urease crystals at temperatures ranging from 25 to 300 K is reported. The nature of the damage changes dramatically at approximately 180 K. Above this temperature the role of solvent diffusion is apparent in thaumatin crystals, as solvent-exposed turns and loops are especially sensitive. In urease, a flap covering the active site is the most sensitive part of the molecule and nearby loops show enhanced sensitivity. Below 180 K sensitivity is correlated with poor local packing, especially in thaumatin. At all temperatures, the component of the damage that is spatially uniform within the unit cell accounts for more than half of the total increase in the atomic B factors and correlates with changes in mosaicity. This component may arise from lattice-level, rather than local, disorder. The effects of primary structure on radiation sensitivity are small compared with those of tertiary structure, local packing, solvent accessibility and crystal contacts.
Assuntos
Cristalografia por Raios X/métodos , Urease/análise , Enterobacter aerogenes/enzimologia , Modelos Moleculares , Estrutura Quaternária de Proteína , Estrutura Terciária de Proteína , Temperatura , Urease/químicaRESUMO
Global radiation damage to 19 thaumatin crystals has been measured using dose rates from 3 to 680 kGyâ s⻹. 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 â¼1â s. However, at T = 260â K approximately half of the global damage manifested at dose rates of â¼10â kGyâ s⻹ can be outrun by collecting data at 680â kGyâ s⻹. 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.
Assuntos
Proteínas de Plantas/química , Plantas/química , Cristalização , Cristalografia por Raios X , Doses de Radiação , Raios XRESUMO
Interpretation of X-ray fluorescence images of archeological artifacts is complicated by the presence of surface relief and roughness. Using two symmetrically arranged fluorescence detectors in a back-reflection geometry, the proper X-ray fluorescence yield can be distinguished from intensity variations caused by surface topography. This technique has been applied to the study of Roman inscriptions on marble.
RESUMO
Ice formation on warming is of comparable or greater importance to ice formation on cooling in determining survival of cryopreserved samples. Critical warming rates required for ice-free warming of vitrified aqueous solutions of glycerol, dimethyl sulfoxide, ethylene glycol, polyethylene glycol 200 and sucrose have been measured for warming rates of order 10-104 K/s. Critical warming rates are typically one to three orders of magnitude larger than critical cooling rates. Warming rates vary strongly with cooling rates, perhaps due to the presence of small ice fractions in nominally vitrified samples. Critical warming and cooling rate data spanning orders of magnitude in rates provide rigorous tests of ice nucleation and growth models and their assumed input parameters. Current models with current best estimates for input parameters provide a reasonable account of critical warming rates for glycerol solutions at high concentrations/low rates, but overestimate both critical warming and cooling rates by orders of magnitude at lower concentrations and larger rates. In vitrification protocols, minimizing concentrations of potentially damaging cryoprotectants while minimizing ice formation will require ultrafast warming rates, as well as fast cooling rates to minimize the required warming rates.
Assuntos
Crioprotetores/química , Gelo/análise , Água/química , Criopreservação , Dimetil Sulfóxido/química , Etilenoglicol/química , Congelamento , Glicerol/química , Calefação , Polietilenoglicóis/química , VitrificaçãoRESUMO
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.
Assuntos
Cristalografia por Raios X/instrumentação , Proteínas/análise , Automação Laboratorial/instrumentação , Automação Laboratorial/métodos , Cristalização , Cristalografia por Raios X/métodosRESUMO
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=100â 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.
Assuntos
Cristalografia por Raios X/métodos , Proteínas/química , Proteínas/efeitos da radiação , Cristalização , Radical Hidroxila , Muramidase/química , Nitratos/química , Conformação Proteica , Soluções/química , TemperaturaRESUMO
Can radiation damage to protein crystals be `outrun' by collecting a structural data set before damage is manifested? Recent experiments using ultra-intense pulses from a free-electron laser show that the answer is yes. Here, evidence is presented that significant reductions in global damage at temperatures above 200â K may be possible using conventional X-ray sources and current or soon-to-be available detectors. Specifically, `dark progression' (an increase in damage with time after the X-rays have been turned off) was observed at temperatures between 180 and 240â K and on timescales from 200 to 1200â s. This allowed estimation of the temperature-dependent timescale for damage. The rate of dark progression is consistent with an Arrhenius law with an activation energy of 14â kJâ mol(-1). This is comparable to the activation energy for the solvent-coupled diffusive damage processes responsible for the rapid increase in radiation sensitivity as crystals are warmed above the glass transition near 200â K. Analysis suggests that at T = 300â K data-collection times of the order of 1â s (and longer at lower temperatures) may allow significant reductions in global radiation damage, facilitating structure solution on crystals with liquid solvent. No dark progression was observed below T = 180â K, indicating that no important damage process is slowed through this timescale window in this temperature range.
Assuntos
Cristalografia por Raios X/métodos , Proteínas/química , TemperaturaRESUMO
Diffraction data acquired from cryocooled protein crystals often include diffraction from ice. Analysis of ice diffraction from crystals of three proteins shows that the ice formed within solvent cavities during rapid cooling is comprised of a stacking-disordered mixture of hexagonal and cubic planes, with the cubic plane fraction increasing with increasing cryoprotectant concentration and increasing cooling rate. Building on the work of Thorn and coworkers [Thorn et al. (2017), Acta Cryst. D73, 729-727], a revised metric is defined for detecting ice from deposited protein structure-factor data, and this metric is validated using full-frame diffraction data from the Integrated Resource for Reproducibility in Macromolecular Crystallography. Using this revised metric and improved algorithms, an analysis of structure-factor data from a random sample of 89â 827 PDB entries collected at cryogenic temperatures indicates that roughly 16% show evidence of ice contamination, and that this fraction increases with increasing solvent content and maximum solvent-cavity size. By examining the ice diffraction-peak positions at which structure-factor perturbations are observed, it is found that roughly 25% of crystals exhibit ice with primarily hexagonal character, indicating that inadequate cooling rates and/or cryoprotectant concentrations were used, while the remaining 75% show ice with a stacking-disordered or cubic character.
Assuntos
Crioprotetores/química , Cristalização/métodos , Gelo , Substâncias Macromoleculares/química , Proteínas/química , Cristalografia por Raios XRESUMO
Time-resolved crystallography of biomolecules in action has advanced rapidly as methods for serial crystallography have improved, but the large number of crystals and the complex experimental infrastructure that are required remain serious obstacles to its widespread application. Here, millisecond mix-and-quench crystallography (MMQX) has been developed, which yields millisecond time-resolved data using far fewer crystals and routine remote synchrotron data collection. To demonstrate the capabilities of MMQX, the conversion of oxaloacetic acid to phosphoenolpyruvate by phosphoenolpyruvate carboxy-kinase (PEPCK) is observed with a time resolution of 40â ms. By lowering the entry barrier to time-resolved crystallography, MMQX should enable a broad expansion in structural studies of protein dynamics.
RESUMO
Based on work by Dubochet and others in the 1980s and 1990s, samples for single-particle cryo-electron microscopy (cryo-EM) have been vitrified using ethane, propane or ethane/propane mixtures. These liquid cryogens have a large difference between their melting and boiling temperatures and so can absorb substantial heat without formation of an insulating vapor layer adjacent to a cooling sample. However, ethane and propane are flammable, they must be liquified in liquid nitro-gen immediately before cryo-EM sample preparation, and cryocooled samples must be transferred to liquid nitro-gen for storage, complicating workflows and increasing the chance of sample damage during handling. Experiments over the last 15 years have shown that cooling rates required to vitrify pure water are only â¼250 000â Kâ s-1, at the low end of earlier estimates, and that the dominant factor that has limited cooling rates of small samples in liquid nitro-gen is sample precooling in cold gas present above the liquid cryogen surface, not the Leidenfrost effect. Using an automated cryocooling instrument developed for cryocrystallography that combines high plunge speeds with efficient removal of cold gas, we show that single-particle cryo-EM samples on commercial grids can be routinely vitrified using only boiling nitro-gen and obtain apoferritin datasets and refined structures with 2.65â Å resolution. The use of liquid nitro-gen as the primary coolant may allow manual and automated workflows to be simplified and may reduce sample stresses that contribute to beam-induced motion.
RESUMO
Serial synchrotron crystallography (SSX) is enabling the efficient use of small crystals for structure-function studies of biomolecules and for drug discovery. An integrated SSX system has been developed comprising ultralow background-scatter sample holders suitable for room and cryogenic temperature crystallographic data collection, a sample-loading station and a humid `gloveless' glovebox. The sample holders incorporate thin-film supports with a variety of designs optimized for different crystal-loading challenges. These holders facilitate the dispersion of crystals and the removal of excess liquid, can be cooled at extremely high rates, generate little background scatter, allow data collection over >90° of oscillation without obstruction or the risk of generating saturating Bragg peaks, are compatible with existing infrastructure for high-throughput cryocrystallography and are reusable. The sample-loading station allows sample preparation and loading onto the support film, the application of time-varying suction for optimal removal of excess liquid, crystal repositioning and cryoprotection, and the application of sealing films for room-temperature data collection, all in a controlled-humidity environment. The humid glovebox allows microscope observation of the sample-loading station and crystallization trays while maintaining near-saturating humidities that further minimize the risks of sample dehydration and damage, and maximize working times. This integrated system addresses common problems in obtaining properly dispersed, properly hydrated and isomorphous microcrystals for fixed-orientation and oscillation data collection. Its ease of use, flexibility and optimized performance make it attractive not just for SSX but also for single-crystal and few-crystal data collection. Fundamental concepts that are important in achieving desired crystal distributions on a sample holder via time-varying suction-induced liquid flows are also discussed.
Assuntos
Cristalografia por Raios X/instrumentação , Desenho de Equipamento , Proteínas/química , Manejo de Espécimes/métodos , Síncrotrons/instrumentaçãoRESUMO
In cryocrystallography, rapid sample cooling is generally deemed essential to prevent solvent crystallization and associated sample damage. We show that by carefully and completely removing all external solvent, many protein crystals can be successfully cooled to T = 100 K at only 0.1 K/s without additional penetrating cryoprotectants. Slow cooling provides an alternative when flash cooling fails, and enables diffraction studies of protein structure and function at all temperatures between T = 300 K and T = 100 K.