/pdf/rapid-and-efficient-room-temperature-serial-synchrotron-1jur8pbo.pdf

Rapid and efficient room-temperature serial synchrotron crystallography using the CFEL TapeDrive

Antimicrobial resistance: new insights and therapeutic implications

Macromolecular crystallography is a well-established method in the field of structure biology and has led to the majority of known protein structures to date. After focusing on static structures, the method is now developing towards the investigation of protein dynamics through time-resolved methods. These experiments often require multiple handling steps of the sensitive protein crystals, e.g. for ligand soaking and cryo-protection. These handling steps can cause significant crystal damage, causing a decrease in data quality. Furthermore, in time-resolved experiments based on serial crystallography that use micron-sized crystals for short diffusion times of ligands, certain crystal morphologies with small solvent channels can prevent sufficient ligand diffusion. Described here is a method combining protein crystallization and data collection in a novel one-step-process. Corresponding experiments were successfully performed as a proof-of-principle using hen egg white lysozyme and crystallization times of only a few seconds. This method called JINXED (Just in time crystallization for easy structure determination) promises to result in high-quality data due the avoidance of crystal handling and has the potential to enable time-resolved experiments with crystals containing small solvent channels by adding potential ligands to the crystallization buffer, simulating traditional co-crystallization approaches.

/pdf/jinxed-just-in-time-crystallization-for-easy-structure-qij98zdf.pdf

JINXED: just in time crystallization for easy structure determination of biological macromolecules

Serial crystallography, born from groundbreaking experiments at the Linac Coherent Light Source in 2009, has evolved into a pivotal technique in structural biology. Initially pioneered at X-ray free-electron laser facilities, it has now expanded to synchrotron-radiation facilities globally, with dedicated experimental stations enhancing its accessibility. This review gives an overview of current developments in serial crystallography, emphasizing recent results in time-resolved crystallography, and discussing challenges and shortcomings. 

A snapshot love story: what serial crystallography has done and will do for us

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

β-lactamases are a major cause of rapidly emerging and spreading antibiotic resistance. Currently β-lactamase inhibitors (BLIs) in clinical use act only on Ambler Class A, C and some class D lactamases. The urgent need to identify new BLIs recently lead to FDA approval of boron-based compounds BLIs, e.g. Vaborbactam. The boron-based proteasome inhibitors Bortezomib and Ixazomib are used in cancer therapy as multiple myeloma drugs but they also bind to Ser-/Thr- proteases. In this study we show the crystal structures of the β-lactamase CTX-M-14 with covalently bound Bortezomib and Ixazomib at high resolutions of 1.3 and 1.1 Å, respectively. Ixazomib is well defined in electron density whereas Bortezomib show some disorder which corresponds to weaker inhibition efficiency observed for Ixazomib. Both inhibitors mimic the deacylation transition state of β-lactam hydrolysis, because they replace the deacylating water molecule. We further investigate differences in binding of Bortezomib/Ixazomib to CTX-M-14 and its target proteases as well as known β-lactamase drugs. Our findings can help to use Bortezomib/Ixazomib as lead compounds for development of new BLIs. 

/pdf/structural-basis-to-repurpose-boron-based-proteasome-1v9rsd8e.pdf

Structural basis to repurpose boron-based proteasome inhibitors Bortezomib and Ixazomib as β-lactamase inhibitors

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

Cefdinir binding to a class-A β-lactamase revealed by serial cryo-crystallography

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

Antibiotic resistance is a growing global health threat that risks the lives of millions. Among the resistance mechanisms, that mediated by metallo-β-lactamases is of particular concern as these bacterial enzymes dismantle most β-lactam antibiotics, which are our widest applied and cheapest to produce antibiotic agents. So far, no clinically applicable metallo-β-lactamase inhibitors are available. Aiming to adapt to structural variations, we introduce the inhibitor concept: dynamically chiral phosphonic acids. We demonstrate that they are straightforward to synthesize, penetrate bacterial membranes, inhibit the metallo-β-lactamase enzymes NDM-1, VIM-2 and GIM-1, and are non-toxic to human cells. Mimicking the transition state of β-lactam hydrolysis, they target the Zn ions of the metallo-β-lactamase active site. As a unique feature, both of their stereoisomers bind metallo-β-lactamases, which provides them unparalleled adaptability to the structural diversity of these enzymes, and may allow them to hamper bacteria’s ability for resistance development. Metallo-β-lactamases dismantle most β-lactam antibiotics and cause antibiotic resistance, however, no metallo-β-lactamase inhibitors are clinically available. Here, the authors report stereodynamically chiral phosphonic acids as potential metallo-β-lactamase inhibitors, showing unparalleled adaptability to the structural diversity of metallo-β-lactamases.

Dynamically chiral phosphonic acid-type metallo-β-lactamase inhibitors

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