Cryo-electron Microscopy Analysis of Structurally Heterogeneous Macromolecular Complexes

Structural studies of biocomplexes using single-particle cryo-electron microscopy (cryo-EM) is now a well-established technique in structural biology and has become competitive with X-ray crystallography. The latest advances in EM enable us to determine structures of protein complexes at 3-5 Å resolution for an extremely broad range of sizes from ~200 kDa up to hundreds of megadaltons (Bartesaghi et al., Science 348(6239):1147-1151, 2051; Bai et al., Nature 525(7568):212-217, 2015; Vinothkumar et al., Nature 515(7525):80-84, 2014; Grigorieff and Harrison, Curr Opin Struct Biol 21(2):265-273, 2011). The majority of biocomplexes comprise a number of different components and are not amenable to crystallisation. Secretion systems are typical examples of such multi-protein complexes, and structural studies of them are extremely challenging. The only feasible approach to revealing their spatial organisation and functional modification is cryo-EM. The development of systems for digital registration of images and algorithms for the fast and efficient processing of recorded images and subsequent analysis facilitated the determination of structures at near-atomic resolution. In this review we will describe sample preparation for cryo-EM, how data are collected by new detectors, and the logistics of image analysis through the basic steps required for reconstructions of both small and large biological complexes and their refinement to nearly atomic resolution. The processing workflow is illustrated using examples of EM analysis of a Type IV Secretion System.

Hybrid structure fitting methods combine data from cryo-electron microscopy and X-ray crystallography with molecular dynamics simulations for the determination of all-atom structures of large biomolecular complexes. Evaluating the quality-of-fit obtained from hybrid fitting is computationally demanding, particularly in the context of a multiplicity of structural conformations that must be evaluated. Existing tools for quality-of-fit analysis and visualization have previously targeted small structures and are too slow to be used interactively for large biomolecular complexes of particular interest today such as viruses or for long molecular dynamics trajectories as they arise in protein folding. We present new data-parallel and GPU-accelerated algorithms for rapid interactive computation of quality-of-fit metrics linking all-atom structures and molecular dynamics trajectories to experimentally-determined density maps obtained from cryo-electron microscopy or X-ray crystallography. We evaluate the performance and accuracy of the new quality-of-fit analysis algorithms vis-à-vis existing tools, examine algorithm performance on GPU-accelerated desktop workstations and supercomputers, and describe new visualization techniques for results of hybrid structure fitting methods.

Using EM to Understand the Structure and Function of Molecular Machines

In modern structural biology, there are three major methods for structural biologists to obtain structural information of macromolecules: cryo-electron microscopy (cryo-EM), nuclear magnetic resonance (NMR), and X-ray crystallography. Cryo-EM, in comparison with the other two methods, allows structural biologists to obtain the structures of various macromolecules in a more native and less perturbed system. Over the past decade, cryo-EM has enabled scientists to determine the structures of protein complexes at atomic resolution and made a profound impact in molecular bioscience and pharmaceutical sectors. Along with cryo-EM, another emerging technique called cryo-electron tomography (cryo-ET) has gained increasing importance in structural biology. It has the potential to visualize macromolecular complexes and assemblies in their native environments at high resolution, but there are still some challenges for small, sparse subjects and in approaching atomic resolution in situ. This chapter summarizes the major steps involved in structure determination using cryo-EM and cryo-ET and highlights the major challenges for in situ cryo-ET. We also present a few examples of near-atomic resolution structure determination of macromolecular assemblies both in purified systems in vitro and in native contexts in situ. Future perspectives are discussed as well.

Progressive Stochastic Reconstruction Technique (PSRT) for cryo electron tomography.

Decision letter: Dynamic Na+/H+ exchanger 1 (NHE1) – calmodulin complexes of varying stoichiometry and structure regulate Ca2+-dependent NHE1 activation

BackgroundProteins in their majority act rarely as single entities. Multisubunit macromolecular complexes are the actors in most of the cellular processes. These nanomachines are hold together by weak protein-protein interactions and undergo functionally important conformational changes. TFIID is such a multiprotein complex acting in eukaryotic transcription initiation. This complex is first to be recruited to the promoter of the genes and triggers the formation of the transcription preinitiation complex involving RNA polymerase II which leads to gene transcription. The exact role of TFIID in this process is not yet understood.MethodsLast generation electron microscopes, improved data collection and new image analysis tools made it possible to obtain structural information of biological molecules at atomic resolution. Cryo-electron microscopy of vitrified samples visualizes proteins in a fully hydrated, close to native state. Molecular images are recorded at liquid nitrogen temperature in low electron dose conditions to reduce radiation damage. Digital image analysis of these noisy images aims at improving the signal-to-noise ratio, at separating distinct molecular views and at reconstructing a three-dimensional model of the biological particle.ResultsUsing these methods we showed the early events of an activated transcription initiation process. We explored the interaction of the TFIID coactivator with the yeast Rap1 activator, the transcription factor TFIIA and the promoter DNA. We demonstrated that TFIID serves as an assembly platform for transient protein-protein interactions, which are essential for transcription initiation.ConclusionsRecent developments in electron microscopy have provided new insights into the structural organization and the dynamic reorganization of large macromolecular complexes. Examples of near-atomic resolutions exist but the molecular flexibility of macromolecular complexes remains the limiting factor in most case. Electron microscopy has the potential to provide both structural and dynamic information of biological assemblies in order to understand the molecular mechanisms of their functions.

Editorial overview: New protein production tools for structural biology

<p xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" class="first" dir="auto" id="d20682e98">Cryo-electron microscopy (Cryo-EM) has emerged as a powerful tool for determining the structures of biomolecules, especially in cases where traditional methods like X-ray crystallography and nuclear magnetic resonance (NMR) fall short [ <a class="xref-link" href="#r1">1</a>]. Its ability to visualize biomolecules in near-native states at cryogenic temperatures has revolutionized structural biology, leading to the resolution of complex molecular machines such as the ribosome and membrane proteins, which are critical targets for drug development. The recent “resolution revolution” [ <a class="xref-link" href="#r2">2</a>] in Cryo-EM has made it possible to achieve atomic or near-atomic resolution for many large macromolecular assemblies. <p xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" dir="auto" id="d20682e106">In Cryo-EM, a substantial volume of particle data is often excluded due to inadequate phase contrast, which impedes full utilization of these particles. This limited insight into discarded structures constrains our capacity to attain the utmost resolution. Furthermore, the 2D classification task in single-particle reconstruction proves especially challenging for heterogeneous samples, low-concentration or low-symmetry structures, and small (&lt;250 kDa) or flexible molecules. To enhance phase contrast, a conventional approach involves integrating a phase-shifting device—such as Zernike or Volta phase plates—within the back focal plane of the objective lens. However, these devices are prone to high-frequency signal attenuation, variability in fabrication quality, reliability issues, and shortened operational lifespans due to electrostatic charging. In recent years, computational phase retrieval algorithms have advanced markedly and are now widely applied in coherent diffraction imaging, holographic imaging, and Fourier ptychographic microscopy. Some studies in Cryo-EM have also begun investigating the potential of computational phase retrieval to extend imaging contrast to the limits allowable by our understanding of the physics of contrast formation [ <a class="xref-link" href="#r3">3</a>].

Role of artificial intelligence in advancing cryo electron microscopy.

Membrane proteins are still heavily under-represented in the protein data bank (PDB), owing to multiple bottlenecks. The typical low abundance of membrane proteins in their natural hosts makes it necessary to overexpress these proteins either in heterologous systems or through in vitro translation/cell-free expression. Heterologous expression of proteins, in turn, leads to multiple obstacles, owing to the unpredictability of compatibility of the target protein for expression in a given host. The highly hydrophobic and (or) amphipathic nature of membrane proteins also leads to challenges in producing a homogeneous, stable, and pure sample for structural studies. Circumventing these hurdles has become possible through the introduction of novel protein production protocols; efficient protein isolation and sample preparation methods; and, improvement in hardware and software for structural characterization. Combined, these advances have made the past 10-15 years very exciting and eventful for the field of membrane protein structural biology, with an exponential growth in the number of solved membrane protein structures. In this review, we focus on both the advances and diversity of protein production and purification methods that have allowed this growth in structural knowledge of membrane proteins through X-ray crystallography, nuclear magnetic resonance (NMR) spectroscopy, and cryo-electron microscopy (cryo-EM).

Background: Beyond phenotypic efficacy and safety categorization, high resolution profiling of drug-protein interactions and binding mechanisms remains a major hurdle during lead selection and optimization. A key milestone in structure-based drug design is compound binding site identification and characterization. Structure-activity relationship (SAR) studies utilize techniques such as nuclear magnetic resonance (NMR), x-ray crystallography (X-ray), cryo-electron microscopy (cryo-EM) and the mass spectrometry-based hydrogen-deuterium exchange (HDX) to address these hurdles but they are labor, time and cost intensive. Further, SAR studies are often complicated by protein size (i.e. large proteins) and location (i.e. membrane proteins), which can lead to protocol adaptations (e.g. recombinant protein usage and/or protein truncation) that can introduce artifacts. Using limited proteolysis (LiP) coupled to next-generation mass spectrometry we have developed a high-throughput, high-resolution approach (HR-LiP) that utilizes peptide-level resolution to characterize drug-protein interactions including for proteins that hindered by the previously mentioned limitations. Methods: To boost protein abundance in their native environment, proteins of interest were overexpressed in HEK293 cells using a simple plasmid construct. Cell lysates were incubated with the compounds of interest at increasing concentrations. Samples were then subjected to a limited digest using proteinase K and further processed for data independent acquisition (DIA)-MS analysis using trypsin. Data was analyzed using a directDIA workflow in Spectronaut. Results: Two well-characterized drug target proteins, bromodomain-containing protein 4 (BRD4) and epidermal growth factor receptor (EGFR), were selected for analysis. Using HR-LiP we identify the binding site of the BRD4 inhibitor JQ1 in the full-length protein, which is typically too large to be used directly in with conventional methods. Further, we map the intracellular binding location of both gefitinib and afatinib, two inhibitors of the membrane protein EGFR. Our data for both proteins are in good accordance with orthogonal data obtained by HDX-MS, NMR and X-ray studies. Conclusions: We demonstrate that HR-LiP can be used to dissect small molecule-protein binding events, including compound binding site prediction for protein targets classically considered to be difficult. Given its biological power, broad applicability and ease of implementation, we envision the use of HR-LiP as a routine approach for target validation and lead optimization in small molecule drug discovery pipelines. Citation Format: Nigel Beaton, Jagat Adhikari, Roland Bruderer, Ron Tomlinson, Yuehan Feng, Ivan Cornella-Taracido, Lukas Reiter. Prediction of small molecule-protein binding events for BRD4 and EGFR inhibitors using HR-LiP, a novel structural proteomics approach [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2136.

Analysis of RNA structure using small-angle X-ray scattering

Many functions in living cells are governed by macromolecular complexes. To fully describe the underlying mechanisms, they have to be understood at atomic level. The present study combines data obtained by X-ray crystallography and cryo-electron microscopy (cryo-EM) with molecular dynamics (MD) simulations. Two functions of macromolecular complexes, the downregulation of neurotransmitter release by the SNARE protein complex under oxidative stress and the translocation of transfer RNAs (tRNAs) through the ribosome during protein synthesis, were investigated. First, the hypothesis that oxidation of two cysteines on linker of the SNARE protein SNAP-25B and consequent disulfide bond formation shortens this linker sufficiently to hinder complex formation was tested. For this purpose, MD simulations of the SNARE complex with and without the disulfide bond were compared. Disulfide bond formation lead to conformational changes of the linker and of three central hydrophobic layers necessary to form the SNARE complex. Previously, mutations of residues contributing to these layers have been shown to reduce neurotransmitter release, suggesting that the stability of these layers is crucial for complex formation. The results from the simulations agree with the hypothesis that disulfide bond formation leads to a destabilization of the SNARE complex thus rendering it dysfunctional. This mechanism is interpreted as a chemomechanical regulation to shut down neurotransmitter release under oxidative stress, which has been linked to neurodegenerative diseases. In a second part I investigated the ribosome, where after peptide bond formation, bound tRNAs translocate by more than 7 nm to adjacent binding sites (A and P to P and E), accompanied by large-scale conformational motions (L1-stalk, intersubunit rotation) of the ribosome. By combining existing cryo-EM reconstructions of translocation intermediates with high resolution crystal structures, we obtained 13 near-atomic resolution structures. Subsequently, MD simulations of were carried out for each intermediate state. The obtained dynamics within these states allowed to estimate transition rates between states for motions of the L1-stalk, tRNAs and intersubunit rotations. Rapid motions of the L1-stalk and the small (30S) subunit on sub-microsecond timescales were revealed, whereas tRNA motions were seen to be rate-limiting for most transitions. By calculating the interaction free energy between L1-stalk and tRNA, molecular forces were derived showing that the L1-stalk is actively pulling the tRNA from P to E binding site, thereby overcoming barriers for the tRNA motion. Further, ribosomal proteins L5 and L16 guide the tRNAs by 'sliding' and 'stepping' mechanisms involving key protein-tRNA contacts. This explains how tRNA binding affinity is kept sufficiently constant to allow rapid translocation despite large-scale displacements. Translocation is accompanied by rotations of the 30S ribosomal subunit of more than 20 degrees relative to the large (50S) subunit. For each translocation intermediate, the affinity of the two subunits with each other must be finely tuned enabling such conformational flexibility while maintaining integrity of the ribosomal complex. Analyzing the trajectories at residue level reveals two classes of intersubunit contact interactions: i) persistent residue contacts which are independent of 30S rotation and primarily located close to the axis of rotation. ii) contacts that are formed and ruptured depending on the rotation angle, seen mainly on the periphery. Strikingly, also these rotation specific contacts substantially contribute to the overall stability of the ribosomal assembly and are expected to maintain a constant interaction energy with low barriers for rotation. The simulations reveal that upon removal of tRNAs peripheral contacts are weakened and, in turn, intersubunit rotation angles decrease, in agreement with cryo-EM analysis of tRNA depleted ribosomes. The identified mechanisms for lowering free energy barriers and for fine-tuning affinities might have developed similarly in other macromolecular complexes.

SAXS Ensemble Refinement of ESCRT-III CHMP3 Conformational Transitions