The Structure Dynamics in Biology (SDB) Resource focuses on developing and supporting technologies to support biomedical and structural biology research along 3 main themes:

• Structure Determination of Multi-protein Complexes and Membrane Proteins

• Accurate Active Site Structures of Macromolecular Machines

• Observing Macromolecular Dynamics

 

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Structure Determination of Multi-protein Complexes and Membrane Proteins

Most cellular processes are carried out by large molecular machines consisting of many interacting proteins or nucleic acid subunits, each with a specific function. Often, these subunits contain hydrophobic transmembrane domains or charged surfaces, which are notoriously difficult crystallization targets.

Sophisticated methods for protein expression, purification, and crystallization have enabled atomic-level structural characterization of membrane proteins and increasingly complex multicomponent machines using macromolecular crystallography. However, generating well-diffracting crystals remains a challenge.

In many cases, the best efforts result in delicate, weakly-diffracting, acutely radiation-sensitive, and/or small (< 10 µm) crystals. The extremely short and bright X-ray pulses produced at LCLS are a powerful tool to study the structure and function of molecular machines, membrane proteins, and metalloenzymes.

Examples of experiments that would be difficult to accomplish by other means:

A breakthrough at LCLS was the application of Serial Femtosecond Crystallography (SFX) to examine crystals of intrinsic membrane proteins, such as GPCRs, grown in LCP.

GPCRs represent the largest family of cell surface receptors involved in signal transduction. As a result, GPCRs are the largest target for drug development, used to treat a wide variety of illnesses (e.g., cancer, cardiovascular disease, and mental illness). GPCRs are targeted by roughly one-third of all FDA-approved drugs.

While of great medical importance, only <150 of nearly 1000 human GPCRs have been structurally characterized to date. Significantly, 25 structures of 11 distinct GPCRs (some complex with various ligands) were determined at LCLS by 2020, using an LCP injector. Among these is the first high-resolution structure of a GPCR determined at ambient temperature, which showed several differences compared to the known cryogenically cooled SR structures. SFX was used to determine the first structure of a GPCR in complex with the protein arrestin as well as providing the structure of two key melatonin receptors bound to multiple ligands.

SFX using an LCP injector is a powerful tool for the structure determination of GPCRs and consequently, the work of the Resource will support the establishment of a structure-based drug design platform targeting GPCRs and obtaining new structures in combination with a variety of ligands.

LCLS has been impactful in the study of large macromolecular machines that form small crystals with high solvent content, making them delicate, difficult to cryo-preserve, and radiation-sensitive. Improvements in the obtainable diffraction resolution from these systems by 0.5 Å or more have been consistently observed at LCLS and in many cases, the collection of useful diffraction was not possible at synchrotron sources.

Often, these machines contain acutely radiation-sensitive metals of critical importance to the mechanism of biological function, such as the Mg ions in RNA polymerase-II (Driving Biomedical Project with Guillermo Calero, University of Pittsburgh).

Researchers can obtain a more accurate structural description of these metals by conducting diffraction experiments at LCLS at room temperature. Moreover, at high resolutions, room temperature structures allow visualization of functionally relevant water networks and alternative side-chain conformations that would otherwise be disturbed by radiation damage or cryopreservation.

Obstacles that researchers will face during the structure determination process at LCLS will be addressed with the technological developments of the Resource, including challenges in crystal diffraction optimization, injector delivery of delicate micro-crystalline and solution samples, and efficient data collection and analysis.

 

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Accurate Active Site Structures of Macromolecular Machines

It is estimated that between 20% and 50% of proteins found within an organism contain a metal ion in some form.

Metalloproteins, or metalloenzymes, are critical to nearly all biological processes and as such represent a rich target space for drug development for a wide variety of diseases. High-resolution structural studies of metalloproteins are particularly challenging because the metal centers, especially those that are redox-active, are very susceptible to X-ray-induced photoreduction.

For instance, single-crystal X-ray absorption fine structure (XAFS) measurements have revealed that the Mn4Ca active site of Photosystem-II (PSII), the living machinery responsible for photosynthesis, was entirely reduced from the Mn(III2, IV2) state to the non-physiological Mn(II4) state under similar conditions that would typically be used to determine the crystal structure using synchrotron X-ray sources.

In Serial Femtosecond Crystallography (SFX), the diffraction process happens before atomic rearrangements can occur around the metal center and consequently the SFX methods are particularly advantageous for the determination of catalytically relevant structures of metalloenzymes.

Structures of the highly radiation-sensitive PSII active site in the dark-adapted and multiple intermediate states were determined using data collected at room temperature at LCLS to provide insight into the water splitting in the Mn4Ca cluster.

This work also illustrates the advantages of LCLS to collect a series of structural snapshots at different time points (a molecular movie) to describe the interactions needed for the performance of biological functions.

The technology developments of the SDB Resource will address many challenges faced by crystallographers studying metalloproteins, including verification of correct incorporation and chemical state of the metal of interest, and maintenance of the optimal environment for sample preparation and data collection, especially if the experiment requires anaerobic, low-temperature, or aphotic environments.

Since X-ray diffraction is a limited technique for determining the oxidation state of the metal, the developments of the Resource include several complementary spectroscopic methods, and some run concurrently with the diffraction experiment. These methods will also be made available before LCLS beam time.

 

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Observing Macromolecular Dynamics

Many proteins in crystals, including many enzymes, can retain the conformational flexibility needed to perform biological processes, making crystallographic studies of protein dynamics possible.

These studies provide details of the atomic positions and motions involved in molecular recognition, transition state stabilization, and other aspects of the catalytic process. While time-resolved studies of this sort had been challenging in the past, the extremely short pulse length of 5–50 fs provided by LCLS enables dynamic crystallography on shorter time scales and using smaller, more radiation-sensitive samples, expanding these methods to investigate a wider variety of scientific questions.

X-rays produced by LCLS are nominally 40 fs long, allowing for a greater time resolution of biomolecules than any other source in the world.

The proceeding figure shows the time scales accessible to LCLS and SSRL for room temperature and near-physiological studies. The time scales are relevant for light-activated processes (Optical pump-probe experiment) as well as for rapid mixing studies serial crystallography (MISC) for SSRL and LCLS that can combine to provide capabilities spanning much of the biomedically relevant time scales of interest.

Successes at LCLS using photoexcitation at LCLS include molecular movies of conformation changes in photoactive yellow protein, CO dissociation from myoglobin, retinal isomerization in bacteriorhodopsin, and chromophore dynamics in a switchable fluorescent protein.

The SDB Resource builds on these results to develop more generalized time-resolved approaches applicable to non-photoactive systems, such as those involved in cell signaling and transport.

These methods include the use of photo-released caged compounds (Driving Biomedical Projects on RNA polymerase II with Guillermo Calero, University of Pittsburgh, and GPCRs with Vadim Cherezov, University of Southern California) and laser-pump temperature-jump techniques (Driving Biomedical Projects with Michael Thompson, University of California Merced).

The Resource will also further develop Mix and Inject Serial Crystallography (MISC) to support work on cytochrome c oxidase dynamics (Driving Biomedical Project with Denis Rousseau, Albert Einstein College of Medicine), riboswitches (Driving Biomedical Project with Yun-Xing Wang, National Cancer Institute), and enzymology (Driving Biomedical Project with Marius Schmidt, University of Wisconsin-Milwaukee).

MISC enables the examination of interaction dynamics between a molecule and a substrate and has strong implications for drug development, understanding disease mechanisms, cellular messaging, signaling, and transport.

MISC has been used to study large domain motions in an RNA riboswitch and the ring cleavage of an antibiotic by a protein involved in antibiotic resistance in bacteria. TR-SFX experiments using either MISC or photoexcitation pose numerous challenges in sample handling, injector operation, and data collection that will be addressed by the Resource developments.

Many developments for TR-SFX also benefit TR-Small and Wide-Angle X-ray Scattering (SAXS/WAXS) experiments, including experiments to follow antibiotic binding and to follow the dynamics of molecules after a temperature jump. SAXS/WAXS at LCLS can monitor transient protein structures to detect structural intermediates along reaction pathways at timescales not possible at synchrotron beamlines.

For example, the structural response of CO-bound myoglobin after photolysis was recorded at multiple time points up to 100 ps. Within 0.8 ps, clear difference signals were observed, with most structural changes completed within 10 ps.

This type of ultra-fast TR-SAXS/WAXS experiment enables observed structural entities to be correlated with markers obtained through optical spectroscopy methods of similar time resolution. Furthermore, coupled with rapid mixers, the LCLS source can mitigate radiation-induced artifacts during TR-SAXS/WAXS studies based on parameters such as temperature, pH, and ligand binding at a range of timescales.

TR-SAXS/WAXS can track both reversible and irreversible reactions, such as protein folding induced by ligand dissociation and electron transfer, expanding its applicability to many protein reactions such as drug binding to target proteins. The short X-ray pulses combined with Fluctuation X-ray Scattering (FXS) analysis offer enhanced resolution by extracting correlations in the SAXS/WAXS patterns to provide finer detail to the reconstructed objects.

The work of the Resource will drive technology and methodology developments to support all these scientific goals.

 

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Maximizing Experimental Throughput

Beam time at X-ray FELs is generally limited by the small number of simultaneous X-ray sources afforded by the linear configuration of an X-ray FEL facility. Compared to synchrotron facilities, which can house dozens of beamlines, each operating simultaneously, X-ray FELs typically have fewer than five experiments that can run simultaneously.

In the case of LCLS, the only X-ray FEL facility in the United States, only two X-ray sources can feed experiments at a given time. Further optical beam sharing possibilities can deliver X-rays to a 3rd or 4th X-ray experiment at the most. The sparsity of beam time makes it imperative to maximize scientific delivery efficiency at LCLS.

As efficient use of the LCLS resource is paramount to providing access to as many experiments as possible, the developments of the Resource also aim to ensure that LCLS beam time applied to biomedical problems is used as effectively as possible. To ensure that their samples and experimental setups are well-characterized and optimized prior to LCLS beam time, we will support users utilizing offline laboratory resources and, when appropriate, SSRL capabilities.

The ease of use, reliability, and throughput of existing sample delivery technologies and features to make full use of the LCLS source will be expanded and improved. Automated methods will also be developed to reduce the cycle time between runs and the amount of beam time required for sample screening, structure solution, and TR experiments, including new real-time data analysis and feedback automation.

 

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