Engineering topics

Kupitz, Christopher

Publications and source records attributed to Kupitz, Christopher.

8 records

DOE OSTI2024

Time-resolved cryogenic electron tomography for the study of transient cellular processes

Cryogenic electron tomography (cryo-ET) is the highest resolution imaging technique applicable to the life sciences, enabling sub-nanometer visualization of specimens preserved in their near native states. The rapid plunge freezing process used to prepare samples lends itself to time-resolved studies, which researchers have pursued for in vitro samples for decades. Here, we focus on developing a freezing apparatus for time-resolved studies in situ. The device mixes cellular samples with solution-phase stimulants before spraying them directly onto an electron microscopy grid that is transiting into cryogenic liquid ethane. By varying the flow rates of cell and stimulant solutions within the device, we can control the reaction time from tens of milliseconds to over a second prior to freezing. In a proof-of-principle demonstration, the freezing method is applied to a model bacterium, Caulobacter crescentus, mixed with an acidic buffer. Through cryo-ET we resolved structural changes throughout the cell, including surface-layer protein dissolution, outer membrane deformation, and cytosolic rearrangement, all within 1.5 seconds of reaction time. This new approach, Time-Resolved cryo-ET (TR-cryo-ET), enhances the capabilities of cryo-ET by incorporating a sub-second temporal axis and enables the visualization of induced structural changes at the molecular, organelle, or cellular level.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2024

Changes in an enzyme ensemble during catalysis observed by high-resolution XFEL crystallography

Enzymes populate ensembles of structures necessary for catalysis that are difficult to experimentally characterize. We use time-resolved mix-and-inject serial crystallography at an x-ray free electron laser to observe catalysis in a designed mutant isocyanide hydratase (ICH) enzyme that enhances sampling of important minor conformations. The active site exists in a mixture of conformations, and formation of the thioimidate intermediate selects for catalytically competent substates. The influence of cysteine ionization on the ICH ensemble is validated by determining structures of the enzyme at multiple pH values. Large molecular dynamics simulations in crystallo and time-resolved electron density maps show that Asp 17 ionizes during catalysis and causes conformational changes that propagate across the dimer, permitting water to enter the active site for intermediate hydrolysis. ICH exhibits a tight coupling between ionization of active site residues and catalysis-activated protein motions, exemplifying a mechanism of electrostatic control of enzyme dynamics.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2023

RNA structures and dynamics with Å resolution revealed by x-ray free-electron lasers

RNA macromolecules, like proteins, fold to assume shapes that are intimately connected to their broadly recognized biological functions; however, because of their high charge and dynamic nature, RNA structures are far more challenging to determine. We introduce an approach that exploits the high brilliance of x-ray free-electron laser sources to reveal the formation and ready identification of angstrom-scale features in structured and unstructured RNAs. Previously unrecognized structural signatures of RNA secondary and tertiary structures are identified through wide-angle solution scattering experiments. With millisecond time resolution, we observe an RNA fold from a dynamically varying single strand through a base-paired intermediate to assume a triple-helix conformation. While the backbone orchestrates the folding, the final structure is locked in by base stacking. This method may help to rapidly characterize and identify structural elements in nucleic acids in both equilibrium and time-resolved experiments.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2023

A user-friendly plug-and-play cyclic olefin copolymer-based microfluidic chip for room-temperature, fixed-target serial crystallography

Over the past two decades, serial X-ray crystallography has enabled the structure determination of a wide range of proteins. With the advent of X-ray free-electron lasers (XFELs), ever-smaller crystals have yielded high-resolution diffraction and structure determination. A crucial need to continue advancement is the efficient delivery of fragile and micrometre-sized crystals to the X-ray beam intersection. This paper presents an improved design of an all-polymer microfluidic `chip' for room-temperature fixed-target serial crystallography that can be tailored to broadly meet the needs of users at either synchrotron or XFEL light sources. The chips are designed to be customized around different types of crystals and offer users a friendly, quick, convenient, ultra-low-cost and robust sample-delivery platform. Compared with the previous iteration of the chip [Gilbile et al. (2021), Lab Chip , 21 , 4831–4845], the new design eliminates cleanroom fabrication. It has a larger imaging area to volume, while maintaining crystal hydration stability for both in situ crystallization or direct crystal slurry loading. Crystals of two model proteins, lysozyme and thaumatin, were used to validate the effectiveness of the design at both synchrotron (lysozyme and thaumatin) and XFEL (lysozyme only) facilities, yielding complete data sets with resolutions of 1.42, 1.48 and 1.70 Å, respectively. Overall, the improved chip design, ease of fabrication and high modifiability create a powerful, all-around sample-delivery tool that structural biologists can quickly adopt, especially in cases of limited sample volume and small, fragile crystals.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗

DOE OSTI2023

Heterogeneity in M. tuberculosis β-lactamase inhibition by Sulbactam

Abstract For decades, researchers have elucidated essential enzymatic functions on the atomic length scale by tracing atomic positions in real-time. Our work builds on possibilities unleashed by mix-and-inject serial crystallography (MISC) at X-ray free electron laser facilities. In this approach, enzymatic reactions are triggered by mixing substrate or ligand solutions with enzyme microcrystals. Here, we report in atomic detail (between 2.2 and 2.7 Å resolution) by room-temperature, time-resolved crystallography with millisecond time-resolution (with timepoints between 3 ms and 700 ms) how the Mycobacterium tuberculosis enzyme BlaC is inhibited by sulbactam (SUB). Our results reveal ligand binding heterogeneity, ligand gating, cooperativity, induced fit, and conformational selection all from the same set of MISC data, detailing how SUB approaches the catalytic clefts and binds to the enzyme noncovalently before reacting to a trans- enamine. This was made possible in part by the application of singular value decomposition to the MISC data using a program that remains functional even if unit cell parameters change up to 3 Å during the reaction.

74 ATOMIC AND MOLECULAR PHYSICS↗

DOE OSTI2023

Room-temperature structural studies of SARS-CoV-2 protein NendoU with an X-ray free-electron laser

NendoU from SARS-CoV-2 is responsible for the virus’s ability to evade the innate immune system by cleaving the polyuridine leader sequence of antisense viral RNA. Here we report the room-temperature structure of NendoU, solved by serial femtosecond crystallography at an X-ray free-electron laser to 2.6 Å resolution. The room-temperature structure provides insight into the flexibility, dynamics, and other intrinsic properties of NendoU, with indications that the enzyme functions as an allosteric switch. Functional studies examining cleavage specificity in solution and in crystals support the uridine-purine cleavage preference, and we demonstrate that enzyme activity is fully maintained in crystal form. Optimizing the purification of NendoU and identifying suitable crystallization conditions set the benchmark for future time-resolved serial femtosecond crystallography studies. This could advance the design of antivirals with higher efficacy in treating coronaviral infections, since drugs that block allosteric conformational changes are less prone to drug resistance.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2022

Cooperative allostery and structural dynamics of streptavidin at cryogenic- and ambient-temperature

Multimeric protein assemblies are abundant in nature. Streptavidin is an attractive protein that provides a paradigm system to investigate the intra- and intermolecular interactions of multimeric protein complexes. Also, it offers a versatile tool for biotechnological applications. Here, we present two apo-streptavidin structures, the first one is an ambient temperature Serial Femtosecond X-ray crystal (Apo-SFX) structure at 1.7 Å resolution and the second one is a cryogenic crystal structure (Apo-Cryo) at 1.1 Å resolution. These structures are mostly in agreement with previous structural data. Combined with computational analysis, these structures provide invaluable information about structural dynamics of apo streptavidin. Collectively, these data further reveal a novel cooperative allostery of streptavidin which binds to substrate via water molecules that provide a polar interaction network and mimics the substrate biotin which displays one of the strongest affinities found in nature.

60 APPLIED LIFE SCIENCES↗

DOE OSTI2021

Near-physiological-temperature serial crystallography reveals conformations of SARS-CoV-2 main protease active site for improved drug repurposing

The COVID-19 pandemic has resulted in 198 million reported infections and more than 4 million deaths as of July 2021 (covid19.who.int). Research to identify effective therapies for COVID-19 includes: (1) designing a vaccine as future protection; (2) de novo drug discovery; and (3) identifying existing drugs to repurpose them as effective and immediate treatments. To assist in drug repurposing and design, we determine two apo structures of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) main protease at ambient temperature by serial femtosecond X-ray crystallography. We employ detailed molecular simulations of selected known main protease inhibitors with the structures and compare binding modes and energies. The combined structural and molecular modeling studies not only reveal the dynamics of small molecules targeting the main protease but also provide invaluable opportunities for drug repurposing and structure-based drug design strategies against SARS-CoV-2.

59 BASIC BIOLOGICAL SCIENCES↗