Biology subjects

Schulz, E. C.

Publications and source records attributed to Schulz, E. C..

12 recordsLinked to original sources

bioRxiv2026

Multi-state Ensemble Refinement for Occupancy Statistics (MEROS) in Time-Resolved X-ray Crystallography

Accurate determination of state occupancies is essential for interpreting the structural heterogeneity inherent in time-resolved crystallography. However, in cases of high spatial overlap between states, as commonly observed in time-resolved crystallography data, the strong correlation between occupancy and atomic displacement parameters (ADPs) can render single point estimates from standard refinement protocols unreliable. We introduce MEROS (Multi-state Ensemble Refinement for Occupancy Statistics), a pipeline that implements an ensemble refinement approach to assess the post-refinement occupancy-ADP statistics of multiple overlapping states. MEROS utilizes a Monte Carlo sampling of the parameter space, performing independent refinements from randomized starting occupancies and ADP values to empirically characterize the convergence and uncertainty of the solution. The method is implemented as a modular Python pipeline that wraps established refinement programs, ensuring compatibility with existing workflows. We demonstrate its applicability in two case studies: a two-state ligand binding model in T4 lysozyme L99A and a four-state covalent catalysis mechanism in {beta}-lactamase CTX-M-14. MEROS provides occupancy and ADP mean values with standard deviations that directly quantify the informational content of the experimental diffraction data.

biochemistry↗

bioRxiv2026

Structural basis for rifamycin recognition and sequential substrate binding by the mycobacterial ADP-ribosyltransferase ARR

The mycobacterial ADP-ribosyltransferase (ARR) mediates intrinsic antibiotic resistance by modifying the ansa-bridge of rifamycins with an ADP-ribose moiety derived from nicotinamide adenine dinucleotide (NAD+). Here, we elucidate the substrate binding mechanism of M. smegmatis ARR using X-ray crystallography and size-exclusion chromatography. We report the first apo structure of ARR, together with complexes involving rifampicin, rifabutin, and rifaximin. Biochemical data demonstrate an ordered sequential binding mechanism in which NAD+ requires prior rifamycin occupancy for recruitment to ARR. Structural comparison of apo ARR to rifamycin bound ARR, reveals that it undergoes significant conformational ordering upon rifamycin binding, characterized by a transition from highly flexible regions in the C-terminus and 2-helix to a more rigid state. Specifically, we identify a 2.9 [A] shift in the loop between {beta}-sheets 2 and 3, involving Phe39, which suggests a gating mechanism that organizes the NAD+ binding pocket. This experimental evidence is complemented by computational modelling of the NAD+ binding mode, which suggests a structurally plausible ternary arrangement. Together, these results establish that rifamycin binding is a prerequisite for NAD+ recruitment and provide a structural basis for the catalytic cycle of rifamycin ADP-ribosylation.

biochemistry↗

bioRxiv2026

Best practices for cryo-trapping time-resolvedcrystallography with the Spitrobot crystal plunger

Capturing meta-stable conformations of enzymes and ligand complexes demands structural snapshots beyond static crystal structures. While time-resolved serial crystallography at room temperature, offers a time-resolution down to the femto-second domain it requires large amounts of micro crystals, specialized beamlines and considerable experience. Moreover, as the majority of enzymes displays turnover-times in the millisecond domain or slower, simpler methods can provide meaningful structural insight into enzyme catalysis. Vitrification of protein crystals can trap reaction intermediates by rapid cooling to {inverted exclamation} 100 K, and has traditionally been used to gain insight into long lived reaction intermediates such as product complexes. However, manual vitrification procedures are limited to long delay times of at least several seconds and heavily suffer from operator variability. A solution to this problem is provided by automatic crystal plunging devices, such as the Spitrobot, that plunge loop-mounted protein crystals into liquid nitrogen within millisecond time-scales. Versatile means of reaction initiation can be achieved either by micro dispensing a ligand droplet, or via optical excitation of light-sensitive proteins, or via the photoactivation of caged compounds. In addition to the conceptual simplicity, another benefit of cryo-trapping is that data can be collected at conventional synchrotron beamlines, exploiting their robust high-throughput capabilities. Thus, compared to room-temperature time-resolved crystallography, users not only benefit from uncoupling sample-preparation and data-collection, but also from a reduction in the required technical expertise and ready access to radiation sources. However, as cryo-trapping crystallography explores dynamic structural changes that become only visible by the comparison of several samples, experiments have to be carefully planned to carry out the necessary controls and to avoid mis- or over-interpretation of the results. Here we describe a detailed protocol for cryo-trapping time-resolved crystallography using automated crystal-plungers that enables researchers to map enzymatic reaction coordinate pathways within the millisecond domain.

biochemistry↗

bioRxiv2026

Structural analysis of Helicobacter pylori glutamate racemase in a monoclinic crystal form

Glutamate racemase (MurI) catalyzes the stereochemical interconversion of L-glutamate to D-glutamate, a key element of bacterial peptidoglycan biosynthesis. In this study, we present the crystal structure of Helicobacter pylori glutamate racemase at 1.43 [A] and in monoclinic symmetry, as previously reported models, but different unit-cell parameters. The present model contains a single dimer and retains the previously described head-to-head dimer arrangement. The differences between the models arise from variations in unit-cell parameters, which lead to altered crystal packing interactions rather than changes in the quaternary assembly. The monomeric fold and active-site architecture remain conserved and are consistent with the catalytic features described for bacterial glutamate racemases. This structure provides an updated, high-resolution structural model for H. pylori glutamate racemase and highlights the variability in crystal packing within the same space group.

biochemistry↗

bioRxiv2026

Tracking ligand-binding-induced structural populations in T4 lysozyme by time-resolved serial crystallography

Ligand binding has been shown to induce significant alterations in the conformational landscape of proteins. Traditional crystallography approaches have provided valuable input about the end states in ligand-binding reactions. However, dynamical relationships between ligand binding and backbone rearrangement often remain obscured by crystallographic structures. In the present study, we use time-resolved serial synchrotron crystallography (TR-SSX) to directly visualize indole binding in the cavity of T4 lysozyme L99A in microcrystals under controlled environmental conditions. By integrating fixed target crystallography with LAMA-based ligand delivery, we have been able to track the progression of ligand binding and backbone rearrangement. By utilizing an occupancy refinement protocol, we have been able to quantify structural populations. Our studies reveal that ligand binding for this protein cavity follows a diffusion-limited process that progressively rearranges the F -helix of the protein towards a dominant conformational state. These findings establish an observable link between ligand diffusion, occupancy evolution and conformational adaptation within a crystalline environment. More broadly, our work shows how TR-SSX can quantify ligand and conformational populations during binding, providing a framework to interpret structural adaptation in real time.

biochemistry↗

bioRxiv2025

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

One of the most common resistance mechanisms against antibiotics employed by Gram-negative bacteria involves the production of {beta}-lactamases, resulting in rapid hydrolysis of the antibiotic. Extensive use of the early generation cephalosporins led to the rise of extended-spectrum {beta}-lactamases (ESBLs) like CTX-Ms. Cefdinir is an extended-spectrum third-generation cephalosporin administered since the late 90s; despite this, there is no reported 3D-structure of the antibiotic bound to any {beta}-lactamase or Penicillin-Binding-Protein (PBP) in the PDB. Here we report the X-ray crystallographic structure of Cefdinir-bound CTX-M-14 E166A mutant obtained via serial cryo-crystallography (cryo-SSX). SynopsisSerial cryo-crystallography reveals the structure of the extended spectrum {beta}-lactamase CTX-M-14, in complex with the third-generation cephalosporin antibiotic Cefdinir.

biophysics↗

bioRxiv2025

Binding mode of isoxazolyl penicillins to a Class-A beta-lactamase at ambient conditions

The predominant resistance mechanism observed in Gram-negative bacteria involves the production of {beta}-lactamases, which catalyse the hydrolysis of {beta}-lactam antibiotics, thereby rendering them ineffective. Although isoxazolyl penicillins are available since the 1970s, there are currently no structures in complex with class-A {beta}-lactamases available. In order to support the rational development of new {beta}-lactamase inhibitors, we have analysed the structure of the clinically relevant {beta}-lactamase CTX-M-14 from Klebsiella pneumoniae near physiolog- ical temperatures. By utilizing serial synchrotron crystallography, we demonstrate the acyl-enzyme intermediates of the catalytically impaired CTX-M-14 mutant E166A in complex with three isoxazolyl penicillins: oxacillin, cloxacillin and dichloxacillin. While the three derivatives differ only by one and two Cl atoms, respectively, they show marked differences in their binding mode.

biochemistry↗

bioRxiv2025

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

We previously introduced the SPITROBOT, a protein crystal plunging system that enables reaction quenching via cryo-trapping with a time resolution in the millisecond range. Here we present the next generation, SPITROBOT-2, as an integrated benchtop device, condensed to approximately an A4 footprint. The user experience has been enhanced by the integration of a guiding beam and a sample-switch dial, to optimise sample exchange operations. This is complemented by a light-indicated liquid nitrogen level sensor, ensuring enhanced reliability. Moreover, a fully automated shutter shields the liquid nitrogen from the humidified environment, improving sample integrity. These improvements reduce the net sample preparation time to approximately three minutes per sample. Most importantly, the cryo-trapping delay time has been reduced to 23 milliseconds, making SPITROBOT-2 twice as fast as the previous generation. This further expands the number of target systems that can be addressed by cryo-trapping time-resolved crystallography. We demonstrate successful cryo-trapping via ligand binding and conformational changes using 12 crystal structures of three independent model systems: xylose isomerase, human insulin and bacteriophage T4 lysozyme. Taken together, these improvements increase the convenient access to cryo-trapping, time-resolved X-ray crystallography empowering the MX community with efficient tools to advance research in structural biology.

biochemistry↗

bioRxiv2022

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

We introduce the spitrobot, a protein crystal plunger, enabling reaction quenching via cryo-trapping with millisecond time-resolution. Canonical micromesh loops are mounted on an electropneumatic piston, reactions are initiated via the liquid application method (LAMA), and finally intermediate states are cryo-trapped in liquid nitrogen. We demonstrate binding of several ligands in microcrystals of three enzymes, and trapping of reaction intermediates and conformational changes in macroscopic crystals of tryptophan synthase.

biophysics↗

bioRxiv2021

An environmental control box for serial crystallography enables multi-dimensional experiments

We present an environmental enclosure for fixed-target serial crystallography, enabling X-ray diffraction experiments in a temperature window from below 10 {degrees}C to above 70 {degrees}C - a universal parameter of protein function. Via 5D-SSX time-resolved experiments can now be carried out at physiological temperatures, providing fundamentally new insights into protein function. We show temperature-dependent modulation of turnover kinetics for the mesophilic {beta}-lactamase CTX-M-14 and for the hyperthermophilic enzyme xylose isomerase.

biophysics↗

bioRxiv2020

Serial femtosecond and serial synchrotron crystallography yield data of equivalent quality: a systematic comparison

For the two proteins myoglobin (MB) and fluoroacetate dehalogenase (FAcD), we present a systematic comparison of crystallographic diffraction data collected by serial femtosecond (SFX) and serial synchrotron crystallography (SSX). To maximize comparability, we used the same batch of crystals, the same sample delivery device, as well as the same data analysis software. Overall figures of merit indicate that the data of both radiation sources are of equivalent quality. For both proteins reasonable data statistics can be obtained with approximately 5000 room temperature diffraction images irrespective of the radiation source. The direct comparability of SSX and SFX data indicates that diffraction quality is rather linked to the properties of the crystals than to the radiation source. Time-resolved experiments can therefore be conducted at the source that best matches the desired time-resolution.

biophysics↗

bioRxiv2019

Serial electron diffraction for high-throughput protein nano-crystallography

Serial X-ray crystallography at free-electron lasers allows to solve biomolecular structures from sub-micron-sized crystals. However, beam time at these facilities is scarce, and involved sample delivery techniques are required. On the other hand, rotation electron diffraction (MicroED) has shown great potential as an alternative means for protein nano-crystallography. Here, we present a method for serial electron diffraction of protein nanocrystals combining the benefits of both approaches. In a scanning transmission electron microscope, crystals randomly dispersed on a sample grid are automatically mapped, and a diffraction pattern at fixed orientation is recorded from each at a high acquisition rate. Dose fractionation ensures minimal radiation damage effects. We demonstrate the method by solving the structure of granulovirus occlusion bodies and lysozyme to resolutions of 1.55 [A] and 1.80 [A], respectively. Our method promises to provide rapid structure determination for many classes of materials with minimal sample consumption, using readily available instrumentation.

molecular biology↗