The emergence and spread of antibiotic resistance represent a growing threat to public health. Of particular concern is the appearance of β-lactamases, which are capable to hydrolyze and inactivate the most important class of antibiotics, the β-lactams. Effective β-lactamase inhibitors and mechanistic insights into their action are central in overcoming this type of resistance, and in this context boronate-based β-lactamase inhibitors were just recently approved to treat multidrug-resistant bacteria. Using boric acid as a simplified inhibitor model, time-resolved serial crystallography was employed to obtain mechanistic insights into binding to the active site serine of β-lactamase CTX-M-14, identifying a reaction time frame of 80-100 ms. In a next step, the subsequent 1,2-diol boric ester formation with glycerol in the active site was monitored proceeding in a time frame of 100-150 ms. Furthermore, the displacement of the crucial anion in the active site of the β-lactamase was verified as an essential part of the binding mechanism of substrates and inhibitors. In total, 22 datasets of β-lactamase intermediate complexes with high spatial resolution of 1.40-2.04 Å and high temporal resolution range of 50-10,000 ms were obtained, allowing a detailed analysis of the studied processes. Mechanistic details captured here contribute to the understanding of molecular processes and their time frames in enzymatic reactions. Moreover, we could demonstrate that time-resolved crystallography can serve as an additional tool for identifying and investigating enzymatic reactions. 

Time-resolved crystallography of boric acid binding to the active site serine of the β-lactamase CTX-M-14 and subsequent 1,2-diol esterification

Sample Preparation for Time-Resolved Serial Crystallography: Practical Considerations.

A systematic comparison of Kapton-based HARE chips for fixed-target serial crystallography

Understanding the enzyme reaction mechanism is important for identifying fundamental biochemical processes and for providing invaluable information for enzyme engineering for industrial applications. Serial femtosecond crystallography (SFX) enables determination structure of enzymes without radiation damage at room temperature. Time-resolved SFX with ligand mixing provides a molecular movie of enzyme reactions, including the transition states. This structural information provides insight into previously undiscovered molecular dynamics over various time periods. To observe enzyme reactions with SFX, it is essential to comprehend the device used for mixing the protein crystals and reaction solutions, as well as the mixing efficiency and available time delay provided by this equipment. In this review, we summarize the current mixing techniques for time-resolved SFX. We describe the configuration and experimental setup of the SFX sample environment unique to each experimental technique and discuss the technical possibilities and limitations of these techniques for performing time-resolved crystallography. 

Recent chemical mixing devices for time-resolved serial femtosecond crystallography

Macromolecular crystallography has historically provided the atomic structures of proteins fundamental to cellular functions. However, the advent of cryo-electron microscopy for structure determination of large and increasingly smaller and flexible proteins signaled a paradigm shift in structural biology. The extensive structural and sequence data from crystallography and advanced sequencing techniques have been pivotal for training computational models for accurate structure prediction, unveiling the general fold of most proteins. Here, we present a perspective on the rise of time-resolved crystallography as the new frontier of macromolecular structure determination. We trace the evolution from the pioneering time-resolved crystallography methods to modern serial crystallography, highlighting the synergy between rapid detection technologies and state-of-the-art x-ray sources. These innovations are redefining our exploration of protein dynamics, with high-resolution crystallography uniquely positioned to elucidate rapid dynamic processes at ambient temperatures, thus deepening our understanding of protein functionality. We propose that the integration of dynamic structural data with machine learning advancements will unlock predictive capabilities for protein kinetics, revolutionizing dynamics like macromolecular crystallography revolutionized structural biology.

The time revolution in macromolecular crystallography

Spitrobot-2 advances time-resolved cryo-trapping crystallography to under 25 ms

The authors introduce the spitrobot, a crystal plunger, enabling cryo-trapping with millisecond time-resolution via the liquid application method (LAMA). Ligand binding and reaction intermediates are demonstrated in three different enzymes. We introduce the spitrobot , a protein crystal plunger, enabling reaction quenching via cryo-trapping with a time-resolution in the millisecond range. Protein crystals are mounted on canonical micromeshes on an electropneumatic piston, where the crystals are kept in a humidity and temperature-controlled environment, then reactions are initiated via the liquid application method (LAMA) and plunging into liquid nitrogen is initiated after an electronically set delay time to cryo-trap intermediate states. High-magnification images are automatically recorded before and after droplet deposition, prior to plunging. The SPINE-standard sample holder is directly plunged into a storage puck, enabling compatibility with high-throughput infrastructure. Here we demonstrate binding of glucose and 2,3-butanediol in microcrystals of xylose isomerase, and of avibactam and ampicillin in microcrystals of the extended spectrum beta-lactamase CTX-M-14. We also trap reaction intermediates and conformational changes in macroscopic crystals of tryptophan synthase to demonstrate that the spitrobot enables insight into catalytic events.

/pdf/millisecond-cryo-trapping-by-the-spitrobot-crystal-plunger-3as3n8iz.pdf

Millisecond cryo-trapping by the spitrobot crystal plunger simplifies time-resolved crystallography

OaPAC is a recently discovered blue-light using flavin adenosine dinucleotide (BLUF) photoactivated adenylate cyclase from the cyanobacterium Oscillatoria acuminata that uses adenosine triphosphate and translates the light signal into the production of cyclic adenosine monophosphate. Here, we report the crystal structures of the enzyme in the absence of its natural substrate determined from room temperature serial crystallography data collected at both an X-ray free electron laser and a synchrotron and we compare them with the cryo macromolecular crystallography structures obtained at a synchrotron by us and others. These results reveal slight differences in the structure of the enzyme due to data collection at different temperatures and X-ray sources. We further investigate the effect of the Y6 mutation in the blue-light using flavin adenosine dinucleotide domain, a mutation which results in a rearrangement of the hydrogen-bond network around the flavin and a notable rotation of the side-chain of the critical Q48 residue. These studies pave the way for ps - ms time-resolved serial crystallography experiments at X-ray free electron lasers and synchrotrons in order to determine the early structural intermediates and correlate them with the well-studied ps - ms spectroscopic intermediates. Synopsis Structures of the dark-adapted state of a photoactivated adenylate cyclase are determined from serial crystallography (SX) data collected at room temperature at an X-ray free electron laser (XFEL) and a synchrotron and are compared with cryo macromolecular crystallography (MX) synchrotron structures obtained by us and others. These structures of the wild-type enzyme in combination with the cryo MX synchrotron structure of a light-sensor domain mutant provide insight into the hydrogen bond network rearrangement upon blue-light illumination and pave the way for the determination of structural intermediates of the enzyme by time-resolved SX.

Crystal structure of a bacterial photoactivated adenylate cyclase determined at room temperature by serial femtosecond crystallography

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Spitrobot-2 advances time-resolved cryo-trapping crystallography to under 25 ms

Millisecond cryo-trapping by the spitrobot crystal plunger simplifies time-resolved crystallography

Crystal structure of a bacterial photoactivated adenylate cyclase determined at room temperature by serial femtosecond crystallography