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.