Phosphoenolpyruvate carboxykinase (PEPCK), an essential enzyme that converts oxaloacetate to phosphoenolpyruvate and gates the gluconeogenesis pathway has recently been found to be upregulated in certain cancers.(1,2)Utilizing MMQX (Millisecond Mix-and-Quench Crystallography) we collected time-resolved crystallography data at timepoints of 40ms, 120ms, and 200ms.These datasets were able to capture PEPCK motions associated with substrate binding and catalysis as well as binding positions of the phosphoenolpyruvate and carbon dioxide products.(3)In addition to time-resolved crystallography, we also performed multi-temperature crystallography of PEPCK to better understand the energy landscape in steady-state conditions.These experiments captured the opening of the omega active site gating loop in PEPCK.Taken together, these experiments greatly improve our understanding of PEPCK's structural fluctuations.

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 nitrogen immediately before cryo-EM sample preparation, and cryocooled samples must be transferred to liquid nitrogen 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 ∼250 000 K s−1, at the low end of earlier estimates, and that the dominant factor that has limited cooling rates of small samples in liquid nitrogen 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 nitrogen and obtain apoferritin datasets and refined structures with 2.65 Å resolution. The use of liquid nitrogen as the primary coolant may allow manual and automated workflows to be simplified and may reduce sample stresses that contribute to beam-induced motion.

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 40 ms. By lowering the entry barrier to time-resolved crystallography, MMQX should enable a broad expansion in structural studies of protein dynamics.

We report the identification of the ter gene cluster responsible for the formation of the p-terphenyl derivatives terfestatins B and C and echoside B from the Appalachian Streptomyces strain RM-5-8. We characterize the function of TerB/C, catalysts that work together as a dual enzyme system in the biosynthesis of natural terphenyls. TerB acts as a reductase and TerC as a dehydratase to enable the conversion of polyporic acid to a terphenyl triol intermediate. X-ray crystallography of the apo and substrate-bound forms for both enzymes provides additional mechanistic insights. Validation of the TerC structural model via mutagenesis highlights a critical role of arginine 143 and aspartate 173 in catalysis. Cumulatively, this work highlights a set of enzymes acting in harmony to control and direct reactive intermediates and advances fundamental understanding of the previously unresolved early steps in terphenyl biosynthesis.

Watching biomolecules in motion on biologically relevant time scales has been a long-standing goal of structural biology.Current methodologies allowing for time-resolved crystallographic data collection are mostly through serial methods using microcrystalswhich are technically challenging experiments with elaborate synchrotron beamline setups, consumption of large amounts of sample, and requiring contributions from many researchers.Here, we propose an alternative methodological setup in order to collect timeresolved data in the millisecond time regime (>5ms), suitable for measuring relatively large structural changes that may be ratelimiting in particular cases.Our approach has been to leverage rapid freeze-quenching by robotically plunging our crystals of choice through a substrate film prior to hyperquenching in liquid nitrogen.This method affords many quality of life improvements over current time-resolved methods, such as the potential for a single crystal use per time-point, divorcing the reaction initiation from data collection, and the ability to use the standard mail-in remote data collection available at all synchrotron sources.In order to show proof-of-concept, we used a well characterized metabolic enzyme phosphoenolpyruvate carboxykinase which converts oxaloacetic acid to phosphoenolpyruvate.Our initial experiments uncovered a previously hypothesized state believed to occur directly after phosphoryl transfer and prior to product release.We hope that this method, with its simplicity and ease of access, can allow many structural biology labs to begin time-resolved exploration of suitable systems to uncover further molecular details of enzymes of interest.

Small-angle X-ray scattering (SAXS) is a key tool for probing the structure and function of proteins, nucleic acids, and macromolecular complexes.Storage and transport of biomolecular solutions at or near room temperature where they may be unstable, large sample volume requirements per measurement, and low synchrotron data collection duty cycles are critical bottlenecks in expanding the application of BioSAXS, especially in high-throughput screening applications.CryoSAXS -SAXS performed on samples cryocooled to T ≈ 100 K, has the potential to address key issues by allowing preparation of samples in the home lab immediately after biomolecule purification, reducing radiation damage and sample consumption per measurement, and allowing the use of sample holders compatible with standard macromolecular cryocrystallography infrastructure for sample storage, shipping, and automated highthroughput data collection.Demonstrations of CryoSAXS have shown the potential of this technique1,2, but the lack of a robust experimental platform has prevented it from becoming a routine method.We are continuing the development of sample cell arrays and methods for high throughput CryoSAXS.Using cell arrays with sample volumes per cell < 0.2 microliters, we can now obtain high quality data using T=100 K gas stream cooling, robust buffer subtraction using biomolecule and buffer scattering profiles obtained from different cells in the same array, and exposures per sample that can be increased to several minutes to maximize signal to noise.Precision cell array assembly, careful management of upstream parasitic X-ray scatter and shadowing of that scatter by the sample cell array, and performing experimental checks for cooling related artifacts are among the keys to obtaining high quality biomolecular profiles.

The bilin-containing photoreceptor TePixJ, a member of the cyanobacteriochrome (CBCR) family of phytochromes, switches between blue-light-absorbing and green-light-absorbing states in order to drive phototaxis in Thermosynechococcus elongatus. Its photoswitching process involves the formation of a thioether linkage between the C10 carbon of phycoviolobilin and the sidechain of Cys494 during the change in state from green-absorbing to blue-absorbing forms. Complex changes in the binding pocket propagate the signal to other domains for downstream signaling. Here, we report time-resolved circular dichroism experiments in addition to pump-probe absorption measurements for interpretation of the biophysical mechanism of the green-to-blue photoconversion process of this receptor.

Natural products and natural product-derived compounds have been widely used for pharmaceuticals for many years, and the search for new natural products that may have interesting activity is ongoing. Abyssomicins are natural product molecules that have antibiotic activity via inhibition of the folate synthesis pathway in microbiota. These compounds also appear to undergo a required [4 + 2] cycloaddition in their biosynthetic pathway. Here we report the structure of an flavin adenine dinucleotide-dependent reductase, AbsH3, from the biosynthetic gene cluster of novel abyssomicins found in Streptomyces sp. LC-6-2.

Development of X-ray free electron laser sources (XFELs) and methods for serial crystallography have driven major advances in time-resolved (TR) protein crystallography.TR-crystallography is a wonderful tool for understanding protein dynamics and catalysis, allowing much greater understanding of structural motions and intermediate states in proteins.Early TR work was largely limited to proteins whose conformational changes could be triggered optically using an intrinsic chromophore, and whose motions were reversible in the crystal, so that a single crystal could be pumped and reset multiple times to generate the diffraction data.Serial crystallography using microcrystals has enabled non-reversible motions to be observed by utilizing large amounts of sample and getting one diffraction image per crystal.These experiments wouldn't have been possible without the extraordinary brightness of XFELs and the newest generation of synchrotron sources.More recently, serial crystallography has incorporated chemical triggering via diffusion on millisecond timescales, short enough to reveal biologically important intermediate states.However, the barrier to entry for current optically or chemically triggered TR crystallography techniques at XFELs and synchrotrons is high.Very large numbers of similar size and morphology crystals must be generated.Optical excitation and/or crystal mixing and delivery systems are complex and must be integrated into the beamline.Fine tuning for a given protein crystal system and efficient serial data collection using these methods requires multiple collaborators, knowledgeable beamline staff, and often large amounts of instrument time at the very few available beamlines suited to these experiments.This complexity puts TR crystallography beyond the reach of most investigators in the wider structural biology community.Alternative methods are needed to allow TR crystallography to be exploited fully by the field.Ideally, these methods should require far fewer crystals, allow data collection from standard MX beamlines without need for special sample delivery apparatus, and allow multiple routes to reaction initiation.We have been developing an alternative method to time-resolved serial crystallography that decouples the sample preparation and reaction evolution times from the data collection time, allows both optical and chemical triggering, and requires only remote data collection at standard synchrotron beamlines.Here we present preliminary data for our new TR-crystallography technique.

The structural conservation among methyltransferases (MTs) and MT functional redundancy is a major challenge to the cellular study of individual MTs. As a first step toward the development of an alternative biorthogonal platform for MTs and other AdoMet-utilizing enzymes, we describe the evaluation of 38 human methionine adenosyltransferase II-α (hMAT2A) mutants in combination with 14 non-native methionine analogues to identify suitable bioorthogonal mutant/analogue pairings. Enabled by the development and implementation of a hMAT2A high-throughput (HT) assay, this study revealed hMAT2A K289L to afford a 160-fold inversion of the hMAT2A selectivity index for a non-native methionine analogue over the native substrate l-Met. Structure elucidation of K289L revealed the mutant to be folded normally with minor observed repacking within the modified substrate pocket. This study highlights the first example of exchanging l-Met terminal carboxylate/amine recognition elements within the hMAT2A active-site to enable non-native bioorthgonal substrate utilization. Additionally, several hMAT2A mutants and l-Met substrate analogues produced AdoMet analogue products with increased stability. As many AdoMet-producing (e.g., hMAT2A) and AdoMet-utlizing (e.g., MTs) enzymes adopt similar active-site strategies for substrate recognition, the proof of concept first generation hMAT2A engineering highlighted herein is expected to translate to a range of AdoMet-utilizing target enzymes.

A major barrier to defining the structural intermediates that arise during the reversible photointerconversion of phytochromes between their biologically inactive and active states has been the lack of crystals that faithfully undergo this transition within the crystal lattice. Here, we describe a crystalline form of the cyclic GMP phosphodiesterases/adenylyl cyclase/FhlA (GAF) domain from the cyanobacteriochrome PixJ in Thermosynechococcus elongatus assembled with phycocyanobilin that permits reversible photoconversion between the blue light-absorbing Pb and green light-absorbing Pg states, as well as thermal reversion of Pg back to Pb. The X-ray crystallographic structure of Pb matches previous models, including autocatalytic conversion of phycocyanobilin to phycoviolobilin upon binding and its tandem thioether linkage to the GAF domain. Cryocrystallography at 150 K, which compared diffraction data from a single crystal as Pb or after irradiation with blue light, detected photoconversion product(s) based on Fobs - Fobs difference maps that were consistent with rotation of the bonds connecting pyrrole rings C and D. Further spectroscopic analyses showed that phycoviolobilin is susceptible to X-ray radiation damage, especially as Pg, during single-crystal X-ray diffraction analyses, which could complicate fine mapping of the various intermediate states. Fortunately, we found that PixJ crystals are amenable to serial femtosecond crystallography (SFX) analyses using X-ray free-electron lasers (XFELs). As proof of principle, we solved by room temperature SFX the GAF domain structure of Pb to 1.55-Å resolution, which was strongly congruent with synchrotron-based models. Analysis of these crystals by SFX should now enable structural characterization of the early events that drive phytochrome photoconversion.