Structure Determination Of Macromolecular Complexes Research Articles

Improvement of cryo-EM maps by simultaneous local and non-local deep learning

Cryo-EM has emerged as the most important technique for structure determination of macromolecular complexes. However, raw cryo-EM maps often exhibit loss of contrast at high resolution and heterogeneity over the entire map. As such, various post-processing methods have been proposed to improve cryo-EM maps. Nevertheless, it is still challenging to improve both the quality and interpretability of EM maps. Addressing the challenge, we present a three-dimensional Swin-Conv-UNet-based deep learning framework to improve cryo-EM maps, named EMReady, by not only implementing both local and non-local modeling modules in a multiscale UNet architecture but also simultaneously minimizing the local smooth L1 distance and maximizing the non-local structural similarity between processed experimental and simulated target maps in the loss function. EMReady was extensively evaluated on diverse test sets of 110 primary cryo-EM maps and 25 pairs of half-maps at 3.0–6.0 Å resolutions, and compared with five state-of-the-art map post-processing methods. It is shown that EMReady can not only robustly enhance the quality of cryo-EM maps in terms of map-model correlations, but also improve the interpretability of the maps in automatic de novo model building.

A cryogenic, coincident fluorescence, electron, and ion beam microscope.

Cryogenic electron tomography (cryo-ET) combined with subtomogram averaging, allows in situ visualization and structure determination of macromolecular complexes at subnanometre resolution. Cryogenic focused ion beam (cryo-FIB) micromachining is used to prepare a thin lamella-shaped sample out of a frozen-hydrated cell for cryo-ET imaging, but standard cryo-FIB fabrication is blind to the precise location of the structure or proteins of interest. Fluorescence-guided focused ion beam (FIB) milling at target locations requires multiple sample transfers prone to contamination, and relocation and registration accuracy is often insufficient for 3D targeting. Here, we present in situ fluorescence microscopy-guided FIB fabrication of a frozen-hydrated lamella to address this problem: we built a coincident three-beam cryogenic correlative microscope by retrofitting a compact cryogenic microcooler, custom positioning stage, and an inverted widefield fluorescence microscope (FM) on an existing FIB scanning electron microscope. We show FM controlled targeting at every milling step in the lamella fabrication process, validated with transmission electron microscope tomogram reconstructions of the target regions. The ability to check the lamella during and after the milling process results in a higher success rate in the fabrication process and will increase the throughput of fabrication for lamellae suitable for high-resolution imaging.

Structure determination of an unstable macromolecular complex enabled by nanobody-peptide bridging.

Structure determination of macromolecular complexes is challenging if subunits can dissociate during crystallization or preparation of electron microscopy grids. We present an approach where a labile complex is stabilized by linking subunits though introduction of a peptide tag in one subunit that is recognized by a nanobody tethered to a second subunit. This allowed crystal structure determination at 3.9 Å resolution of the highly non‐globular 320 kDa proconvertase formed by complement components C3b, factor B, and properdin. Whereas the binding mode of properdin to C3b is preserved, an internal rearrangement occurs in the zymogen factor B von Willebrand domain type A domain compared to the proconvertase not bound to properdin. The structure emphasizes the role of two noncanonical loops in thrombospondin repeats 5 and 6 of properdin in augmenting the activity of the C3 convertase. We suggest that linking of subunits through peptide specific tethered nanobodies represents a simple alternative to approaches like affinity maturation and chemical cross‐linking for the stabilization of large macromolecular complexes. Besides applications for structural biology, nanobody bridging may become a new tool for biochemical analysis of unstable macromolecular complexes and in vitro selection of highly specific binders for such complexes.

Ice thickness monitoring for cryo-EM grids by interferometry imaging

While recent technological developments contributed to breakthrough advances in single particle cryo-electron microscopy (cryo-EM), sample preparation remains a significant bottleneck for the structure determination of macromolecular complexes. A critical time factor is sample optimization that requires the use of an electron microscope to screen grids prepared under different conditions to achieve the ideal vitreous ice thickness containing the particles. Evaluating sample quality requires access to cryo-electron microscopes and a strong expertise in EM. To facilitate and accelerate the selection procedure of probes suitable for high-resolution cryo-EM, we devised a method to assess the vitreous ice layer thickness of sample coated grids. The experimental setup comprises an optical interferometric microscope equipped with a cryogenic stage and image analysis software based on artificial neural networks (ANN) for an unbiased sample selection. We present and validate this approach for different protein complexes and grid types, and demonstrate its performance for the assessment of ice quality. This technique is moderate in cost and can be easily performed on a laboratory bench. We expect that its throughput and its versatility will contribute to facilitate the sample optimization process for structural biologists.

Preparation of RNA Polymerase Complexes for Their Analysis by Single-Particle Cryo-Electron Microscopy.

Recent technological progress revealed new prospects of high-resolution structure determination of macromolecular complexes using cryo-electron microscopy (cryo-EM) . In the field of RNA polymerase (Pol) I research, a number of cryo-EM studies contributed to understanding the highly specialized mechanisms underlying the transcription of ribosomal RNA genes . Despite a broad applicability of the cryo-EM method itself, preparation of samples for high-resolution data collection can be challenging. Here, we describe strategies for the purification and stabilization of Pol I complexes, exemplarily considering advantages and disadvantages of the methodology. We further provide an easy-to-implement protocol for the coating of EM-grids with self-made carbon support films. In sum, we present an efficient workflow for cryo-grid preparation and optimization, including early stage cryo-EM screening that can be adapted to a wide range of soluble samples for high-resolution structure determination .

Integrative structural modeling of macromolecular complexes using Assembline

Integrative modeling enables structure determination of macromolecular complexes by combining data from multiple experimental sources such as X-ray crystallography, electron microscopy or cross-linking mass spectrometry. It is particularly useful for complexes not amenable to high-resolution electron microscopy-complexes that are flexible, heterogeneous or imaged in cells with cryo-electron tomography. We have recently developed an integrative modeling protocol that allowed us to model multi-megadalton complexes as large as the nuclear pore complex. Here, we describe the Assembline software package, which combines multiple programs and libraries with our own algorithms in a streamlined modeling pipeline. Assembline builds ensembles of models satisfying data from atomic structures or homology models, electron microscopy maps and other experimental data, and provides tools for their analysis. Compared with other methods, Assembline enables efficient sampling of conformational space through a multistep procedure, provides new modeling restraints and includes a unique configuration system for setting up the modeling project. Our protocol achieves exhaustive sampling in less than 100-1,000 CPU-hours even for complexes in the megadalton range. For larger complexes, resources available in institutional or public computer clusters are needed and sufficient to run the protocol. We also provide step-by-step instructions for preparing the input, running the core modeling steps and assessing modeling performance at any stage.

Single Particle Cryo-Electron Microscopy: From Sample to Structure.

Cryo-electron microscopy (cryoEM) is a powerful technique for structure determination of macromolecular complexes, via single particle analysis (SPA). The overall process involves i) vitrifying the specimen in a thin film supported on a cryoEM grid; ii) screening the specimen to assess particle distribution and ice quality; iii) if the grid is suitable, collecting a single particle dataset for analysis; and iv) image processing to yield an EM density map. In this protocol, an overview for each of these steps is provided, with a focus on the variables which a user can modify during the workflow and the troubleshooting of common issues. With remote microscope operation becoming standard in many facilities, variations on imaging protocols to assist users in efficient operation and imaging when physical access to the microscope is limited will be described.

In Situ Studies of Mitochondrial Translation by Cryo-Electron Tomography.

Cryo-electron tomography (cryo-ET) enables the three-dimensional (3D) visualization of macromolecular complexes in their native environment (in situ). The ability to visualize macromolecules in situ is in particular advantageous for complex, membrane-associated processes, such as mitochondrial translation. Mitochondrial translation occurs almost exclusively associated with the inner mitochondrial membrane, giving rise to the mitochondrial DNA-encoded subunits of oxidative phosphorylation machinery. In cryo-ET, the 3D volume is reconstructed from a set of 2D projections of a frozen-hydrated specimen, which is sequentially tilted and imaged at different angles in a transmission electron microscope. In combination with subtomogram analysis, cryo-ET enables the structure determination of macromolecular complexes and their 3D organization. In this chapter, we summarize all steps required for structural characterization of mitochondrial ribosomes in situ, ranging from data acquisition to tomogram reconstruction and subtomogram analysis.

Volta phase plate data collection facilitates image processing and cryo-EM structure determination.

A current bottleneck in structure determination of macromolecular complexes by cryo electron microscopy (cryo-EM) is the large amount of data needed to obtain high-resolution 3D reconstructions, including through sorting into different conformations and compositions with advanced image processing. Additionally, it may be difficult to visualize small ligands that bind in sub-stoichiometric levels. Volta phase plates (VPP) introduce a phase shift in the contrast transfer and drastically increase the contrast of the recorded low-dose cryo-EM images while preserving high frequency information. Here we present a comparative study to address the behavior of different data sets during image processing and quantify important parameters during structure refinement. The automated data collection was done from the same human ribosome sample either as a conventional defocus range dataset or with a Volta phase plate close to focus (cfVPP) or with a small defocus (dfVPP). The analysis of image processing parameters shows that dfVPP data behave more robustly during cryo-EM structure refinement because particle alignments, Euler angle assignments and 2D & 3D classifications behave more stably and converge faster. In particular, less particle images are required to reach the same resolution in the 3D reconstructions. Finally, we find that defocus range data collection is also applicable to VPP. This study shows that data processing and cryo-EM map interpretation, including atomic model refinement, are facilitated significantly by performing VPP cryo-EM, which will have an important impact on structural biology.

Resolving macromolecular structures from electron cryo-tomography data using subtomogram averaging in RELION.

Electron cryo-tomography (cryo-ET) is a technique that is used to produce 3D pictures (tomograms) of complex objects such as asymmetric viruses, cellular organelles or whole cells from a series of tilted electron cryo-microscopy (cryo-EM) images. Averaging of macromolecular complexes found within tomograms is known as subtomogram averaging, and this technique allows structure determination of macromolecular complexes in situ. Subtomogram averaging is also gaining in popularity for the calculation of initial models for single-particle analysis. We describe herein a protocol for subtomogram averaging from cryo-ET data using the RELION software (http://www2.mrc-lmb.cam.ac.uk/relion). RELION was originally developed for cryo-EM single-particle analysis, and the subtomogram averaging approach presented in this protocol has been implemented in the existing workflow for single-particle analysis so that users may conveniently tap into existing capabilities of the RELION software. We describe how to calculate 3D models for the contrast transfer function (CTF) that describe the transfer of information in the imaging process, and we illustrate the results of classification and subtomogram averaging refinement for cryo-ET data of purified hepatitis B capsid particles and Saccharomyces cerevisiae 80S ribosomes. Using the steps described in this protocol, along with the troubleshooting and optimization guidelines, high-resolution maps can be obtained in which secondary structure elements are resolved subtomogram.

Sub-tomogram Averaging in RELION

Electron cryo-tomography (cryo-ET) is a technique that is used to produce 3D pictures (tomograms) of complex objects such as asymmetric viruses, cellular organelles or whole cells from a series of tilted electron cryo-microscopy (cryo-EM) images. Averaging of macromolecular complexes found within tomograms is known as subtomogram averaging, and this technique allows structure determination of macromolecular complexes in situ. Subtomogram averaging is also gaining in popularity for the calculation of initial models for single-particle analysis. We describe herein a protocol for subtomogram averaging from cryo-ET data using the RELION software (http://www2.mrc-lmb.cam.ac.uk/relion). RELION was originally developed for cryo-EM single-particle analysis, and the subtomogram averaging approach presented in this protocol has been implemented in the existing workflow for single-particle analysis so that users may conveniently tap into existing capabilities of the RELION software. We describe how to calculate 3D models for the contrast transfer function (CTF) that describe the transfer of information in the imaging process, and we illustrate the results of classification and subtomogram averaging refinement for cryo-ET data of purified hepatitis B capsid particles and Saccharomyces cerevisiae 80S ribosomes. Using the steps described in this protocol, along with the troubleshooting and optimization guidelines, high-resolution maps can be obtained in which secondary structure elements are resolved subtomogram.

Combining dehydration, construct optimization and improved data collection to solve the crystal structure of a CRM1-RanGTP-SPN1-Nup214 quaternary nuclear export complex.

High conformational flexibility is an intrinsic and indispensable property of nuclear transport receptors, which makes crystallization and structure determination of macromolecular complexes containing exportins or importins particularly challenging. Here, the crystallization and structure determination of a quaternary nuclear export complex consisting of the exportin CRM1, the small GTPase Ran in its GTP-bound form, the export cargo SPN1 and an FG repeat-containing fragment of the nuclear pore complex component nucleoporin Nup214 fused to maltose-binding protein is reported. Optimization of constructs, seeding and the development of a sophisticated protocol including successive PEG-mediated crystal dehydration as well as additional post-mounting steps were essential to obtain well diffracting crystals.

Structure of the E. coli ribosome-EF-Tu complex at

Single particle electron cryomicroscopy (cryo-EM) has recently made significant progress in high-resolution structure determination of macromolecular complexes due to improvements in electron microscopic instrumentation and computational image analysis. However, cryo-EM structures can be highly non-uniform in local resolution and all structures available to date have been limited to resolutions above 3Å. Here we present the cryo-EM structure of the 70S ribosome from Escherichia coli in complex with elongation factor Tu, aminoacyl-tRNA and the antibiotic kirromycin at 2.65-2.9Å resolution using spherical aberration (Cs)-corrected cryo-EM. Overall, the cryo-EM reconstruction at 2.9Å resolution is comparable to the best-resolved X-ray structure of the E. coli 70S ribosome (2.8Å), but provides more detailed information (2.65Å) at the functionally important ribosomal core. The cryo-EM map elucidates for the first time the structure of all 35 rRNA modifications in the bacterial ribosome, explaining their roles in fine-tuning ribosome structure and function and modulating the action of antibiotics. We also obtained atomic models for flexible parts of the ribosome such as ribosomal proteins L9 and L31. The refined cryo-EM-based model presents the currently most complete high-resolution structure of the E. coli ribosome, which demonstrates the power of cryo-EM in structure determination of large and dynamic macromolecular complexes.

Structure Sorting of Multiple Macromolecular States in Heterogeneous Cryo-EM Samples by 3D Multivariate Statistical Analysis

Heterogeneity of biological samples is usually considered a major obstacle for three-dimensional (3D) structure determination of macromolecular complexes. Heterogeneity may occur at the level of composition or conformational variability of complexes and affects most 3D structure determination methods that rely on signal averaging. Here, an approach is described that allows sorting structural states based on a 3D statistical approach, the 3D sampling and classification (3D-SC) of 3D structures derived from single particles imaged by cryo electron microscopy (cryo-EM). The method is based on jackknifing & bootstrapping of 3D sub-ensembles and 3D multivariate statistical analysis followed by 3D classification. The robustness of the statistical sorting procedure is corroborated using model data from an RNA polymerase structure and experimental data from a ribosome complex. It allows resolving multiple states within heterogeneous complexes that thus become amendable for a structural analysis despite of their highly flexible nature. The method has important implications for high-resolution structural studies and allows describing structure ensembles to provide insights into the dynamics of multi-component macromolecular assemblies.

From cryo-EM, multiple protein structures in one shot

A new maximum likelihood–based algorithm allows cryo-EM structure determination of macromolecular complexes even when conformational heterogeneity is present.

Defining and solving the essential protein–protein interactions in HIV infection

The structure determination of macromolecular complexes is entering a new era. The methods of optical microscopy, electron microscopy, X-ray crystallography, and nuclear magnetic resonance increasingly are being combined in hybrid method approaches to achieve an integrated view of macromolecular complexes that span from cellular context to atomic detail. A particularly important application of these hybrid method approaches is the structural analysis of the Human Immunodeficiency Virus (HIV) proteins with their cellular binding partners. High resolution structure determination of essential HIV – host cell protein complexes and correlative analysis of these complexes in the live cell can serve as critical guides in the design of a broad, new class of therapeutics that function by disrupting such complexes. Here, with the hope of stimulating some discussion, we will briefly review some of the literature in the context of what could be done to further apply structural methods to HIV research. We have chosen to focus our attention on certain aspects of the HIV replication cycle where we think that structural information would contribute substantially to the development of new therapeutic and vaccine targets for HIV.

Structural Characterization of Assemblies from Overall Shape and Subcomplex Compositions

We suggest structure characterization of macromolecular assemblies by combining assembly shape determined by electron cyromicroscopy with information about subunit proximity determined by affinity purification. To achieve this aim, structure characterization is expressed as a problem in satisfaction of spatial restraints that (1) represents subunits as spheres, (2) encodes information about the subunit excluded volume, assembly shape, and pulldowns in a scoring function, and (3) finds subunit configurations that satisfy the input restraints by an optimization of the scoring function. Testing of the approach with model systems suggests its feasibility.

Crystallographic Analysis of Thermus aquaticus RNA Polymerase Holoenzyme and a Holoenzyme⧸Promoter DNA Complex

Publisher Summary This chapter reviews the RNA in all cellular organisms that are synthesized by a complex molecular machine, the DNA-dependent RNA polymerase (RNAP). The catalytically competent bacterial core RNAP is conserved evolutionarily in sequence, structure, and function from bacteria to humans. It discusses that in both prokaryotes and eukaryotes, promoter-specific initiation of transcription requires protein factors in addition to the catalytic core RNAP, ranging from an 750-kD collection of more than a dozen basal initiation factors for messenger RNA (mRNA) synthesis in eukaryotes to a single polypeptide in bacteria, the σ subunit, which binds core RNAP to form the holoenzyme. The holoenzyme locates specific DNA sequences called “promoters”, forms the open complex by melting the DNA surrounding the transcription start site, and initiates the synthesis of an RNA chain. To understand the molecular mechanism of transcription initiation, it is necessary to solve the structures of the holoenzyme and holoenzyme–DNA complex. The chapter reports on details of the purification, crystallization, and structure determination of macromolecular complexes.

IMIRS: a high-resolution 3D reconstruction package integrated with a relational image database

Recent advances in electron cryomicroscopy instrumentation and single particle reconstruction have created opportunities for high-throughput and high-resolution three-dimensional (3D) structure determination of macromolecular complexes. However, it has become impractical and inefficient to rely on conventional text file data management and command-line programs to organize and process the increasing numbers of image data required in high-resolution studies. Here, we present a distributed relational database for managing complex datasets and its integration into our high-resolution software package IMIRS (Image Management and Icosahedral Reconstruction System). IMIRS consists of a complete set of modular programs for icosahedral reconstruction organized under a graphical user interface and provides options for user-friendly, step-by-step data processing as well as automatic reconstruction. We show that the integration of data management with processing in IMIRS automates the tedious tasks of data management, enables data coherence, and facilitates information sharing in a distributed computer and user environment without significantly increasing the time of program execution. We demonstrate the applicability of IMIRS in icosahedral reconstruction toward high resolution by using it to obtain an 8-Å 3D structure of an intermediate-sized dsRNA virus.