Structures Of Macromolecular Complexes Research Articles

P-095 Towards deciphering the molecular architecture of the human sperm connecting piece

Abstract Study question How can we make the human sperm connecting piece accessible for structural analysis by cryo-electron tomography (cryo-ET)? Summary answer Preparation of sperm by chemical thinning or production of lamellae via cryo-focused ion beam (cryo-FIB) milling allows direct imaging of the human sperm connecting piece. What is known already Previous studies from our group and others have demonstrated the versatility of cryo-ET to advance our understanding of sperm biology. Structures of macromolecular complexes have been resolved in vitro and in situ, enabling a functional assessment at molecular or even atomic level. Resolving structures at high resolution further allowed identification of proteins previously not known to have a role in mediating male fertility. Such insights are instrumental to further improve diagnosis and treatment of male infertility. Study design, size, duration In our research study we use sperm samples from patients undergoing IVF treatment or donor material, which we receive through an ongoing collaboration with the Division of Woman and Baby, University Medical Centre Utrecht (UMCU). These samples are processed in our own laboratory and subsequently imaged by cryo-ET at the Electron Microscopy Centre of Utrecht University. Participants/materials, setting, methods Due to its thickness, the connecting piece cannot be directly analyzed by cryo-ET. Thus, we designed two strategies to achieve thinning of this area and allow collection of tomograms for structural analysis. While the first approach involves incubation of sperm with a detergent, a reducing agent and heparin, the second approach utilizes cryo-FIB milling to produce thin lamellae of the connecting piece. Main results and the role of chance Both strategies developed to access the connecting piece with cryo-ET are effective and allowed us a glimpse of the molecular landscape of the link between sperm head and tail. Chemical thinning of the connecting piece enabled imaging of the proximal and distal centriole while still embedded in the segmented columns or outer dense fibers, respectively. Furthermore, electron density within the microtubules of both centrioles indicates that they are decorated by microtubule inner proteins. Data obtained from lamellae produced by cryo-FIB milling complements these findings, displaying regular densities along the microtubules extending from the proximal centriole. Analyzing tomograms collected from cryo-FIB lamellae we further found a regular protein density that appears to be connecting the nuclear envelope to the base plate. Moreover, cryo-ET of lamellae allowed a detailed view of nuclear pore complexes and the postacrosomal sheath in their native environment. Limitations, reasons for caution Altering the native state of the sperm cell by chemical preparation is a limitation of this approach. Cryo-FIB milling retains the native state of the cell but is limited in throughput. To improve our cryo-FIB milling approach, we will implement automated milling procedures. Wider implications of the findings Chemical preparation and cryo-FIB milling of sperm enable the in-depth analysis of the connecting piece by cryo-ET. These strategies can now be applied to provide structural details at the molecular level and thus advance our understanding of male infertility related to defects of the head-tail junction. Trial registration number not applicable

Characterization of Complexes and Supramolecular Structures by Electron Microscopy.

Recent advancements in cryo-electron microscopy (cryo-TEM) have enabled the determination of structures of macromolecular complexes at near-atomic resolution, establishing it as a pivotal tool in Structural Biology. This high resolution allows for the detection of ligands and substrates under physiological conditions. Enhancements in detectors and imaging devices, like phase plates, improve signal quality, facilitating the reconstruction of even smaller macromolecular complexes. The 100-kDa barrier has been surpassed, presenting new opportunities for pharmacological research and expanding the scope of crystallographic analyses in the pharmaceutical industry. Cryo-TEM produces vast data sets from minimal samples, and refined classification methods can identify different conformational states of macromolecular complexes, offering deeper insights into the functional characteristics of macromolecular systems. Additionally, cryo-TEM is paving the way for time-resolved microscopy, with rapid freezing techniques capturing snapshots of vital structural changes in biological complexes. Finally, in Structural Cell Biology, advanced cryo-TEM, through tomographic procedures, is revealing conformational changes related to the specific subcellular localization of macromolecular systems and their interactions within cells.

Fabrication of Monolayer Graphene-Coated Grids for Cryoelectron Microscopy.

Cryogenic electron microscopy (cryoEM) has emerged as a powerful technique for probing the atomic structure of macromolecular complexes. Sample preparation for cryoEM requires preserving specimens in a thin layer of vitreous ice, typically suspended within the holes of a fenestrated support film. However, all commonly used sample preparation approaches for cryoEM studies expose the specimen to the air-water interface, introducing a strong hydrophobic effect on the specimen that often results in denaturation, aggregation, and complex dissociation. Further, preferred hydrophobic interactions between regions of the specimen and the air-water interface impact the orientations adopted by the macromolecules, resulting in 3D reconstructions with anisotropic directional resolution. Adsorption of cryoEM specimens to a monolayer of graphene has been shown to help mitigate interactions with the air-water interface while minimizing the introduction of background noise. Graphene supports also offer the benefit of substantially lowering the required concentration of proteins required for cryoEM imaging. Despite the advantages of these supports, graphene-coated grids are not widely used by the cryoEM community due to the prohibitive expense of commercial options and the challenges associated with large-scale in-house production. This paper describes an efficient method for preparing batches of cryoEM grids that have nearly full coverage of monolayer graphene.

Effects of cryo-EM cooling on structural ensembles of biomolecules and their solvation shell.

The recent revolution in cryo electron microscopy (cryo-EM) allows the determination of structures of macromolecular complexes at atomic resolution. Cryo-EM also provides information on structural heterogeneity and ensembles of macromolecules. To obtain cryo-EM images of macromolecules, the samples are first rapidly cooled down to liquid nitrogen temperatures. The rapid cooling prevents ice-crystal formation and preserves some information of the room-temperature ensemble. However, to what extent the ensemble of the macromolecues and of the solvation shell is perturbed by the cooling is currently unknown. To quantify the cooling effects, we first estimated the temperature drop rate by solving the heat equation which suggested rapid cooling within microseconds. Then, we started all-atom explicit-solvent molecular dynamics (MD) simulations of the ribosome from 41 snapshots taken from a room-temperature ensemble with linearly decreasing temperatures at 11 different cooling rates (simulation lengths 0.1-128 ns). In the simulations, we observed that cooling markedly decreased the structural heterogeneity of the cooled ensemble. To investigate if and how this effect depends on the cooling rate, we used Bayesian statistics to test three thermodynamic and kinetic models of the cooling process. A kinetic two-state model improves the prediction of the decrease in heterogeneity compared to the cooling-rate independent thermodynamic model suggesting that kinetic effects contribute markedly. The combination of the estimated temperature drop rate with the kinetic model suggests that small barriers between states (<10 kJ/mol) are overcome during cooling and do not contribute to the heterogeneity of the structural ensemble obtained by cryo-EM. In contrast, conformational states separated by larger barriers are expected to be trapped during plunge-freezing. The obtained parameters for the kinetic model will allow one to quantify the heterogeneity of biologically relevant room-temperature ensembles from cryo-EM structures.

Small GTPase Ran: Depicting the nucleotide-specific conformational landscape of the functionally important C-terminus.

The small GTPase Ran is the main regulator of the nucleo-cytoplasmic import and export through the nuclear pore complex. It functions as a molecular switch cycling between the GDP-bound inactive and GTP-bound active state. It consists of a globular (G) domain and a C-terminal region, which is bound to the G-domain in the inactive, GDP-bound states. Crystal structures of the GTP-bound active form complexed with Ran binding proteins (RanBP) show that the C-terminus undergoes a large conformational change, embracing Ran binding domains (RanBD). Whereas in the crystal structures of macromolecular complexes not containing RanBDs the structure of the C-terminal segment remains unresolved, indicating its large conformational flexibility. This movement could not have been followed either by experimental or simulation methods. Here, starting from the crystal structure of Ran in both GDP- and GTP-bound forms we show how rigid the C-terminal region in the inactive structure is during molecular dynamics (MD) simulations. Furthermore, we show how MD simulations of the active form are incapable of mapping the open conformations of the C-terminus. By using the MDeNM (Molecular Dynamics with excited Normal Modes) method, we were able to widely map the conformational surface of the C-terminus of Ran in the active GTP-bound form, which allows us to envisage how it can embrace RanBDs.

Lysate-to-grid: Rapid Isolation of Native Complexes from Budding Yeast for Cryo-EM Imaging.

Single-particle electron cryo-microscopy (cryo-EM) is an effective tool to determine high-resolution structures of macromolecular complexes. Its lower requirements for sample concentration and purity make it an accessible method to determine structures of low-abundant protein complexes, such as those isolated from native sources. While there are many approaches to protein purification for cryo-EM, attaining suitable particle quality and abundance is generally the major bottleneck to the typical single-particle project workflow. Here, we present a protocol using budding yeast ( S. cerevisiae ), in which a tractable immunoprecipitation tag (3xFLAG) is appended at the endogenous locus of a gene of interest (GOI). The modified gene is expressed under its endogenous promoter, and cells are grown and harvested using standard procedures. Our protocol describes the steps in which the tagged proteins and their associated complexes are isolated within three hours of thawing cell lysates, after which the recovered proteins are used directly for cryo-EM specimen preparation. The prioritization of speed maximizes the ability to recover intact, scarce complexes. The protocol is generalizable to soluble yeast proteins that tolerate C-terminal epitope tags. Graphical abstract Overview of lysate-to-grid workflow. Yeast cells are transformed to express a tractable tag on a gene of interest. Following cell culture and lysis, particles of interest are rapidly isolated by co-immunoprecipitation and prepared for cryo-EM imaging (created with BioRender.com).

Building Protein Atomic Models from Cryo-EM Density Maps and Residue Co-Evolution.

Electron cryo-microscopy (cryo-EM) has emerged as a powerful method by which to obtain three-dimensional (3D) structures of macromolecular complexes at atomic or near-atomic resolution. However, de novo building of atomic models from near-atomic resolution (3–5 Å) cryo-EM density maps is a challenging task, in particular because poorly resolved side-chain densities hamper sequence assignment by automatic procedures at a lower resolution. Furthermore, segmentation of EM density maps into individual subunits remains a difficult problem when the structure of the subunits is not known, or when significant conformational rearrangement occurs between the isolated and associated form of the subunits. To tackle these issues, we have developed a graph-based method to thread most of the C- trace of the protein backbone into the EM density map. The EM density is described as a weighted graph such that the resulting minimum spanning tree encompasses the high-density regions of the map. A pruning algorithm cleans the tree and finds the most probable positions of the C- atoms, by using side-chain density when available, as a collection of C- trace fragments. By complementing experimental EM maps with contact predictions from sequence co-evolutionary information, we demonstrate that this approach can correctly segment EM maps into individual subunits and assign amino acid sequences to backbone traces to generate atomic models.

Artificial Intelligence in Cryo-Electron Microscopy.

Cryo-electron microscopy (cryo-EM) has become an unrivaled tool for determining the structure of macromolecular complexes. The biological function of macromolecular complexes is inextricably tied to the flexibility of these complexes. Single particle cryo-EM can reveal the conformational heterogeneity of a biochemically pure sample, leading to well-founded mechanistic hypotheses about the roles these complexes play in biology. However, the processing of increasingly large, complex datasets using traditional data processing strategies is exceedingly expensive in both user time and computational resources. Current innovations in data processing capitalize on artificial intelligence (AI) to improve the efficiency of data analysis and validation. Here, we review new tools that use AI to automate the data analysis steps of particle picking, 3D map reconstruction, and local resolution determination. We discuss how the application of AI moves the field forward, and what obstacles remain. We also introduce potential future applications of AI to use cryo-EM in understanding protein communities in cells.

Effects of cryo-EM cooling on structural ensembles

The recent revolution in cryo electron microscopy (cryo-EM) allows the determination of structures of macromolecular complexes at atomic resolution. Cryo-EM also provides information on structural heterogeneity and ensembles of macromolecules. To obtain cryo-EM images of macromolecules, the samples are first rapidly cooled down to liquid nitrogen temperatures. The rapid cooling preserves some information of the room temperature ensemble. However, to what extent the structural ensemble is perturbed by the cooling is currently unknown.

High-resolution in situ structure determination by cryo-electron tomography and subtomogram averaging using emClarity.

Cryo-electron tomography and subtomogram averaging (STA) has developed rapidly in recent years. It provides structures of macromolecular complexes in situ and in cellular context at or below subnanometer resolution and has led to unprecedented insights into the inner working of molecular machines in their native environment, as well as their functional relevant conformations and spatial distribution within biological cells or tissues. Given the tremendous potential of cryo-electron tomography STA in in situ structural cell biology, we previously developed emClarity, a graphics processing unit-accelerated image-processing software that offers STA and classification of macromolecular complexes at high resolution. However, the workflow remains challenging, especially for newcomers to the field. In this protocol, we describe a detailed workflow, processing and parameters associated with each step, from initial tomography tilt-series data to the final 3D density map, with several features unique to emClarity. We use four different samples, including human immunodeficiency virus type 1 Gag assemblies, ribosome and apoferritin, to illustrate the procedure and results of STA and classification. Following the processing steps described in this protocol, along with a comprehensive tutorial and guidelines for troubleshooting and parameter optimization, one can obtain density maps up to 2.8 Å resolution from six tilt series by cryo-electron tomography STA.

Perspective: Emerging strategies for determining atomic-resolution structures of macromolecular complexes within cells

In principle, electron cryo-tomography (cryo-ET) of thin portions of cells provides high-resolution images of the three-dimensional spatial arrangement of all members of the proteome. In practice, however, radiation damage creates a tension between recording images at many different tilt angles, but at correspondingly reduced exposure levels, versus limiting the number of tilt angles in order to improve the signal-to-noise ratio (SNR). Either way, it is challenging to read the available information out at the level of atomic structure. Here, we first review work that explores the optimal strategy for data collection, which currently seems to favor the use of a limited angular range for tilting the sample or even the use of a single image to record the high-resolution information. Looking then to the future, we point to the alternative of so-called “deconvolution microscopy”, which may be applied to tilt-series or optically-sectioned, focal series data. Recording data as a focal series has the advantage that little or no translational alignment of frames might be needed, and a three-dimensional reconstruction might require only 2/3 the number of images as does standard tomography. We also point to the unexploited potential of phase plates to increase the contrast, and thus to reduce the electron exposure levels while retaining the ability align and merge the data. In turn, using much lower exposures per image could have the advantage that high-resolution information is retained throughout the full data-set, whether recorded as a tilt series or a focal series of images.

Step-by-step guide to efficient subtomogram averaging of virus-like particles with Dynamo.

Subtomogram averaging (STA) is a powerful image processing technique in electron tomography used to determine the 3D structure of macromolecular complexes in their native environments. It is a fast growing technique with increasing importance in structural biology. The computational aspect of STA is very complex and depends on a large number of variables. We noticed a lack of detailed guides for STA processing. Also, current publications in this field often lack a documentation that is practical enough to reproduce the results with reasonable effort, which is necessary for the scientific community to grow. We therefore provide a complete, detailed, and fully reproducible processing protocol that covers all aspects of particle picking and particle alignment in STA. The command line–based workflow is fully based on the popular Dynamo software for STA. Within this workflow, we also demonstrate how large parts of the processing pipeline can be streamlined and automatized for increased throughput. This protocol is aimed at users on all levels. It can be used for training purposes, or it can serve as basis to design user-specific projects by taking advantage of the flexibility of Dynamo by modifying and expanding the given pipeline. The protocol is successfully validated using the Electron Microscopy Public Image Archive (EMPIAR) database entry 10164 from immature HIV-1 virus-like particles (VLPs) that describe a geometry often seen in electron tomography.

Endocytosed nanogold fiducials for improved in-situ cryo–electron tomography tilt-series alignment

Cryo–electron tomography (CET) on cryo–focused ion beam (FIB)–milled lamellae is becoming a powerful technique for determining the structure of macromolecular complexes in their native cellular environment. Prior to tomogram reconstruction, CET tilt-series recorded on FIB lamellae need to be aligned. Traditionally, CET tilt-series alignment is performed with 5–20 nm gold fiducials, but it has thus far proven difficult to apply this to FIB lamellae of eukaryotic cells. In here, we describe a simple method to allow uptake of bovine serum albumin (BSA)-gold fiducials into mammalian cells via endocytosis, which can subsequently be used as fiducials for tilt-series alignment of cryo-FIB lamellae. We compare the alignment of tilt-series with BSA-gold fiducials to fiducial-less patch-tracking, and find better alignment results with BSA-gold. This technique can contribute to understand cells at a structural and ultrastructural level with both cryo- and room-temperature electron tomography. Furthermore, fluorescently labeled BSA-gold has the potential to be used as fiducials for correlative light and electron microscopy studies.

Effects of cryo-EM Freezing on Structural Ensembles

The recent revolution in cryo-electron microscopy (cryo-EM) allows the determination of structures of macromolecular complexes at atomic resolution. Cryo-EM also provides information on structural heterogeneity and ensembles of macromolecules. To obtain cryo-EM images of macromolecules, the samples are first rapidly cooled down to liquid nitrogen temperatures. The rapid cooling preserves some information of the room temperature ensemble. However, to what extent the structural ensemble is perturbed by the cooling is currently unknown.

A Pipeline for Integrative Structural Modelling of Very Complex Molecular Complexes

Although electron microscopy (EM) allows determining the structure of macromolecular complexes at near-atomic resolution, high resolution is still difficult to achieve for complexes that are flexible, heterogenous, or imaged in cells with cryo-electron tomography. A state-of-the-art methodology to obtain structures of such complexes is integrative structural modelling, which combines data from complementary techniques, such as X-ray crystallography, EM, NMR, SAXS or cross-linking mass spectrometry. The current integrative modelling software, however, is either limited to complexes of simple architectures or requires custom modifications, and thus a major expertise, when applied to more complex architectures. Thus, we developed a versatile modelling pipeline for integrative modelling of protein complexes with very complex architecture in an accessible and efficient way. Our pipeline is implemented using Integrative Modeling Platform library, on top of which it implements custom algorithms for efficient sampling of the conformational space, versatile configuration language and graphical interface for input preparation, modelling and analysis, and additional restraints. Flexible and symmetrical modeling is also supported. The pipeline enables modeling of very complex architectures, for example, with multiple symmetries or with EM maps for alternative conformational states. I will present the pipeline and how we applied it to model complexes ranging from the 1 MDa Elongator to the 110 MDa human nuclear pore complex (NPC). For the Elongator complex, I will present how our published model agrees with the subsequent high-resolution cryo-EM structure. For the NPC, I will show our most recent results of how the pipeline allowed building structural models of NPCs from yeast S. cerevisiae and S. pombe, including a novel multi-state modelling protocol for modelling based on multiple EM maps simultaneously. The new NPC models reveal surprisingly different architectures and depict conformational changes of NPCs in response to cellular states.

Cryo-EM is a powerful tool, but helical applications can have pitfalls.

In structural biology, cryo-electron microscopy (cryo-EM) has emerged as the main technique for determining the atomic structures of macromolecular complexes. This has largely been due to the introduction of direct electron detectors, which have allowed for routinely reaching a near-atomic resolution when imaging such complexes. In chemistry and materials science, the applications of cryo-EM have been much more limited. A recent paper (Z. Li et al., Chemically Controlled Helical Polymorphism In Protein Tubes By Selective Modulation Of Supramolecular Interactions, J. Am. Chem. Soc. 2019, 141, 19448-19457) has used low resolution cryo-EM to analyze polymorphic helical tubes formed by a tetrameric protein, and has made detailed models for the interfaces between the tetramers in these assemblies. Due to intrinsic ambiguities in determining the correct helical symmetry, we show that many of the models are likely to be wrong. This note highlights both the enormous potential for using cryo-EM, and also the pitfalls possible for helical assemblies when a near-atomic level of resolution is not reached.

Preparation of Doublet Microtubule Fraction for Single ParticleCryo-electron Microscopy.

Over the years, studying the ultrastructure of the eukaryotic cilia/flagella using electron microscopy (EM) has contributed significantly toward our understanding of ciliary function. Major complexes in the cilia, such as inner and outer dynein arms, radial spokes, and dynein regulatory complexes, were originally discovered by EM. Classical resin-embedding EM or cryo-electron tomography can be performed directly on the isolated cilia or in some cases, cilia directly attached to the cell body. Recently, single particle cryo-EM has emerged as a powerful structural technique to elucidate high-resolution structures of macromolecular complexes; however, single particle cryo-EM requires non-overlapping complexes, i.e., the doublet microtubule of the cilia. Here, we present a protocol to separate the doublet microtubule from the isolated cilia bundle of two species, Tetrahymena thermophila and Chlamydomonas reinhardtii, using ATP reactivation and sonication. Our approach produces good distribution and random orientation of the doublet microtubule fragments, which is suitable for single particle cryo-EM analysis.

From phenotype to structure

Systems Biology Much insight has come from structures of macromolecular complexes determined by methods such as crystallography or cryo–electron microscopy. However, looking at transient complexes remains challenging, as does determining structures in the context of the cellular environment. Braberg et al. used an integrative approach in which they mapped the phenotypic profiles of a comprehensive set of mutants in a protein complex in the context of gene deletions or environmental perturbations (see the Perspective by Wang). By associating the similarity between phenotypic profiles with the distance between residues, they determined structures for the yeast histone H3-H4 complex, subunits Rpb1-Rpb2 of yeast RNA polymerase II, and subunits RpoB-RpoC of bacterial RNA polymerase. Comparison with known structures shows that the accuracy is comparable to structures determined based on chemical cross-links. Science , this issue p. [eaaz4910][1]; see also p. [1269][2] [1]: /lookup/doi/10.1126/science.aaz4910 [2]: /lookup/doi/10.1126/science.abf3863

Genetic interaction mapping informs integrative structure determination of protein complexes.

Determining structures of protein complexes is crucial for understanding cellular functions. Here, we describe an integrative structure determination approach that relies on in vivo measurements of genetic interactions. We construct phenotypic profiles for point mutations crossed against gene deletions or exposed to environmental perturbations, followed by converting similarities between two profiles into an upper bound on the distance between the mutated residues. We determine the structure of the yeast histone H3-H4 complex based on ~500,000 genetic interactions of 350 mutants. We then apply the method to subunits Rpb1-Rpb2 of yeast RNA polymerase II and subunits RpoB-RpoC of bacterial RNA polymerase. The accuracy is comparable to that based on chemical cross-links; using restraints from both genetic interactions and cross-links further improves model accuracy and precision. The approach provides an efficient means to augment integrative structure determination with in vivo observations.

Cryo-Electron Tomography Studies of Cell Systems

Cryo-electron tomography is a powerful tool for determining the three-dimensional structures of macromolecular complexes in their natural environment. The minimization of external impacts on the investigated objects, possibility of conducting in vitro and in cellulo experiments under close-to-native conditions, and high spatial resolution of the obtained three-dimensional reconstructions make cryo-electron tomography one of the most promising methods for studying a large class of objects in the fields of structural biology and visual proteomics. The main aspects of cryo-electron tomography are discussed as applied to studies of cell systems.