cryo-EM Single-particle Analysis Research Articles

Cryo-EM Structure of the Mnx Protein Complex Reveals a Tunnel Framework for the Mechanism of Manganese Biomineralization.

The global manganese cycle relies on microbes to oxidize soluble Mn(II) to insoluble Mn(IV) oxides. Some microbes require peroxide or superoxide as oxidants, but others can use O2 directly, via multicopper oxidase (MCO) enzymes. One of these, MnxG from Bacillus sp. strain PL-12, was isolated in tight association with small accessory proteins, MnxE and MnxF. The protein complex, called Mnx, has eluded crystallization efforts, but we now report the 3D structure of a point mutant using cryo-EM single particle analysis, cross-linking mass spectrometry, and AlphaFold Multimer prediction. The β-sheet-rich complex features MnxG enzyme, capped by a heterohexameric ring of alternating MnxE and MnxF subunits, and a tunnel that runs through MnxG and its MnxE3F3 cap. The tunnel dimensions and charges can accommodate the mechanistically inferred binuclear manganese intermediates. Comparison with the Fe(II)-oxidizing MCO, ceruloplasmin, identifies likely coordinating groups for the Mn(II) substrate, at the entrance to the tunnel. Thus, the 3D structure provides a rationale for the established manganese oxidase mechanism, and a platform for further experiments to elucidate mechanistic details of manganese biomineralization.

Practices in cryo-EM single particle analysis data processing: data transfer between RELION and cryoSPARC

Practices in cryo-EM single particle analysis data processing: data transfer between RELION and cryoSPARC

The advent of preventive high-resolution structural histopathology by artificial-intelligence-powered cryogenic electron tomography.

Advances in cryogenic electron microscopy (cryoEM) single particle analysis have revolutionized structural biology by facilitating the in vitro determination of atomic- and near-atomic-resolution structures for fully hydrated macromolecular complexes exhibiting compositional and conformational heterogeneity across a wide range of sizes. Cryogenic electron tomography (cryoET) and subtomogram averaging are rapidly progressing toward delivering similar insights for macromolecular complexes in situ, without requiring tags or harsh biochemical purification. Furthermore, cryoET enables the visualization of cellular and tissue phenotypes directly at molecular, nanometric resolution without chemical fixation or staining artifacts. This forward-looking review covers recent developments in cryoEM/ET and related technologies such as cryogenic focused ion beam milling scanning electron microscopy and correlative light microscopy, increasingly enhanced and supported by artificial intelligence algorithms. Their potential application to emerging concepts is discussed, primarily the prospect of complementing medical histopathology analysis. Machine learning solutions are poised to address current challenges posed by "big data" in cryoET of tissues, cells, and macromolecules, offering the promise of enabling novel, quantitative insights into disease processes, which may translate into the clinic and lead to improved diagnostics and targeted therapeutics.

Enhancing Density Maps by Removing the Majority of Particles in Single Particle Cryogenic Electron Microscopy Final Stacks.

Over the past decade, advancements in technology and methodology within the field of cryogenic electron microscopy (cryo-EM) single-particle analysis (SPA) have substantially improved our capacity for high-resolution structural examination of biological macromolecules. This advancement has ushered in a new era of molecular insights, replacing X-ray crystallography as the dominant method and providing answers to longstanding questions in biology. Since cryo-EM does not depend on crystallization, which is a significant limitation of X-ray crystallography, it captures particles of varying quality. Consequently, the selection of particles is crucial, as the quality of the selected particles directly influences the resolution of the reconstructed density map. An innovative iterative approach for particle selection, termed CryoSieve, significantly improves the quality of reconstructed density maps by effectively reducing the number of particles in the final stack. Experimental evidence shows that this method can eliminate the majority of particles in final stacks, resulting in a notable enhancement in the quality of density maps. This article outlines the detailed workflow of this approach and showcases its application on a real-world dataset.

Structural insights into the calcium-coupled zinc export of human ZnT1.

Cellular zinc (Zn2+) homeostasis is essential to human health and is under tight regulations. Human zinc transporter 1 (hZnT1) is a plasma membrane-localized Zn2+ exporter belonging to the ZnT family, and its functional aberration is associated with multiple diseases. Here, we show that hZnT1 works as a Zn2+/Ca2+ exchanger. We determine the structure of hZnT1 using cryo-electron microscopy (cryo-EM) single particle analysis. hZnT1 adopts a homodimeric structure, and each subunit contains a transmembrane domain consisting of six transmembrane segments, a cytosolic domain, and an extracellular domain. The transmembrane region displays an outward-facing conformation. On the basis of structural and functional analysis, we propose a model for the hZnT1-mediated Zn2+/Ca2+ exchange. Together, these results facilitate our understanding of the biological functions of hZnT1 and provide a basis for further investigations of the ZnT family transporters.

Structural and electrochemical elucidation of biocatalytic mechanisms in direct electron transfer-type D-fructose dehydrogenase

D-Fructose dehydrogenase (FDH) from Gluconobacter japonicus NBRC3260, a membrane-bound heterotrimeric flavohemoprotein capable of intense direct electron transfer (DET)-type bioelectrocatalysis, was investigated using protein engineering, electrochemistry, structural biology, and bioinformatics. DET-type reactions have led to an increase in energy efficiency, biocompatibility, and design freedom in bioelectrochemical devices, such as biofuel cells, biosensors, biosupercapacitors, and bioreactors. However, one of the greatest challenges for this reaction is the limited number of the applicable enzymes. The creation of variants with altered substrate specificities using template enzymes is a promising solution to address this problem. For creating variants using FDH as a template, it is important to understand the biocatalytic mechanisms. Recently, the structure of FDH has been analyzed using cryo-electron microscopy (cryo-EM) single particle analysis, and the three amino acid residues (N1146, H1147, and N1190) may be critical for substrate recognition by FDH. In this study, we aimed to understand the mechanisms of the biocatalytic reaction of FDH and discuss the roles of these residues. In addition, we validated the structures of the in-silico variants by comparing them to the cryo-EM structures. Furthermore, for the variants of N1146 involved in D-fructose recognition, we have verified the changes in specificity for other substrates.

Designing Rigid DNA Origami Templates for Molecular Visualization Using Cryo-EM.

DNA origami, a method for constructing nanostructures from DNA, offers potential for diverse scientific and technological applications due to its ability to integrate various molecular functionalities in a programmable manner. In this study, we examined the impact of internal crossover distribution and the compositional uniformity of staple strands on the structure of multilayer DNA origami using cryogenic electron microscopy (cryo-EM) single-particle analysis. A refined DNA object was utilized as an alignment framework in a host-guest model, where we successfully resolved an 8 kDa thrombin binding aptamer (TBA) linked to the host object. Our results broaden the spectrum of DNA in structural applications.

Molecular basis of human poly(A) polymerase recruitment by mPSF.

3' end processing of most eukaryotic precursor-mRNAs (pre-mRNAs) is a crucial cotranscriptional process that generally involves the cleavage and polyadenylation of the precursor transcripts. Within the human 3' end processing machinery, the four-subunit mammalian polyadenylation specificity factor (mPSF) recognizes the polyadenylation signal (PAS) in the pre-mRNA and recruits the poly(A) polymerase α (PAPOA) to it. To shed light on the molecular mechanisms of PAPOA recruitment to mPSF, we used a combination of cryogenic-electron microscopy (cryo-EM) single-particle analysis, computational structure prediction, and in vitro biochemistry to reveal an intricate interaction network. A short linear motif in the mPSF subunit FIP1 interacts with the structured core of human PAPOA, with a binding mode that is evolutionarily conserved from yeast to human. In higher eukaryotes, however, PAPOA contains a conserved C-terminal motif that can interact intramolecularly with the same residues of the PAPOA structured core used to bind FIP1. Interestingly, using biochemical assay and cryo-EM structural analysis, we found that the PAPOA C-terminal motif can also directly interact with mPSF at the subunit CPSF160. These results show that PAPOA recruitment to mPSF is mediated by two distinct intermolecular connections and further suggest the presence of mutually exclusive interactions in the regulation of 3' end processing.

Optimizing cryo-EM structural analysis of Gi-coupling receptors via engineered Gt and Nb35 application

Cryo-EM single particle analysis has recently facilitated the high-resolution structural determination of numerous GPCR-G complexes. Diverse methodologies have been devised with this trend, and in the case of GPCR-Gi complexes, scFv16, an antibody that recognizes the intricate interface of the complex, has been mainly implemented to stabilize the complex. However, owing to their flexibility and heterogeneity, structural determinations of GPCR-Gi complexes remain both challenging and resource-intensive. By employing eGαt, which exhibits binding affinity to modified nanobody Nb35, the cryo-EM structure of Rhodopsin-eGαt complex was previously reported. Using this modified G protein, we determined the structure of the ETB-eGt complex bound to the modified Nb35. The determined structure of ETB receptor was the same as the previously reported ETB-Gi complex, and the resulting dataset demonstrated significantly improved anisotropy. This modified G protein will be utilized for the structural determination of other GPCR-Gi complexes.

Studies of Protein Disulfide Isomerase (PDI) Binding to Platelets and Production of a Murine Monoclonal Antibody (mAb) to Integrin αIIbβ3 That Inhibits PDI Binding and Platelet Aggregation

L.W. and J.W. contributed equally Introduction: The platelet integrin αIIbβ3 plays a pivotal role in both hemostasis and thrombosis, and is an established target for antiplatelet therapy. Upon activation, αIIbβ3 undergoes a conformational change from a low-affinity, bent-closed conformation to a high-affinity, extended-open conformation, resulting in the binding of fibrinogen and von Willebrand factor and platelet aggregation. αIIbβ3 activation is regulated by protein disulfide isomerase (PDI), an enzyme that catalyzes disulfide bond formation, reduction, and isomerization. One of us (LW) previously showed that PDI binds to thrombin-activated platelets, but the αIIb or β3 conformation(s) and domain(s) to which PDI binds is unknown. Methods: PDI was produced in E. coli and purified and labeled with Alexa-488. Human platelets and Alexa-488-PDI were stimulated with a variety of agonists either in the absence or presence of unlabeled PDI (background control) and then analyzed by flow cytometry. For monoclonal antibody (mAb) production, BALB/cAnN mice were immunized with human platelets and their spleen cells isolated and fused with a non-secretory myeloma cell line. Hybridomas were isolated by colony picking in semi-solid media and culture supernatants were assayed for their ability to inhibit PDI binding to platelets activated with the PAR1 thrombin receptor activating peptide SFLLRN (T6). One mAb that inhibited PDI binding (21D10) was assessed for its effects on platelet aggregation. The epitope of 21D10 on αIIbβ3 was explored by comparing the binding properties of 21D10 to that of previously characterized anti-αIIbβ3 mAbs. Using cryo-electron microscopy (cryo-EM) single particle analysis, we determined the structure of the Fab fragment of 21D10 in complex with full-length αIIbβ3 purified from human platelets. Results: T6, the PAR4 thrombin receptor activating peptide AYPGKF, and collagen-related peptide induced PDI binding to washed platelets [mean fluorescence intensity (MFI) of 547 ± 21 compared with 229 ± 12 for background control, p < 0.001; 576 ± 111 vs 215 ± 27 for background control, p < 0.05; and 934 ± 99 vs 167 ± 14 for background control, p < 0.001, respectively]. The reducing agent dithiothreitol (DTT) also enhanced PDI binding (MFI of 595 ± 147 vs 178 ± 24 for background control, p < 0.05). mAb 21D10 and its Fab fragment (40 μg/ml) inhibited PDI binding to platelets [MFI of 357 ± 25 vs 567 ± 60 for buffer control, p < 0.01 (figure); and 286 ± 38 vs 507 ± 86 for buffer control; p < 0.05)], fibrinogen binding (6,493 ± 815 vs 15,058 ± 1,935 for buffer control, p < 0.001; 1,714 ± 276 vs 14,917 ± 2,129 for buffer control, p < 0.0001), and maximum platelet aggregation (mean ± SD of 32 ± 8 vs 67 ± 2% for buffer control, p < 0.01; and 30 ± 8 vs 69 ± 3% for buffer control; p < 0.01). For comparison, the PDI inhibitor rutin at 45 μM produced similar inhibition. 21D10 did not inhibit the binding of mAbs 10E5 or 7E3, both of which inhibit fibrinogen binding to αIIbβ3, to platelets, and vice versa. EDTA treatment of platelets at either 22°C, pH 7.4, or 37°C, pH 8.0, had minimal effect on 21D10 binding, while both conditions inhibited 7E3 binding and the latter inhibited 10E5 binding. 21D10 reacted with the nonreduced but not reduced β3 subunit by immunoblotting. With chymotrypsin digested platelets, both 21D10 and the mAb 7H2, which binds to the PSI domain of β3, bound to bands extending down to 66 kDa, but 21D10 also bound to the bands below 66 kDa, whereas 7H2 did not. The cryo-EM structure of the 21D10 Fab-αIIbβ3 complex revealed that 21D10 binds to two different αIIbβ3 conformations that differ in the orientation of the leg domains (in one the legs are nearly bent and in the other the legs are partially extended). In both conformations, 21D10 binds to the PSI, I-EGF-1 and I-EGF-2 domains of β3 (figure). Conclusion: We have shown that in addition to thrombin, T6, a PAR4 activating peptide, collagen-related peptide, and DTT can induce PDI binding to platelets. We produced a mAb to human platelets (21D10) that can inhibit T6-induced PDI binding to platelets. Immunoblotting and cryo-EM analysis shows that 21D10 binds to the PSI, I-EGF1, and I-EGF2 regions of β3. If 21D10 prevents PDI binding by steric hindrance, it is likely that PDI binds to β3 at a site distant from the RGD ligand binding pocket. The functional effects of 21D10 may reflect its inhibition of PDI to platelets, but it may also reflect inhibition of other related proteins, in particular, ERp57.

OPUS-DSD: deep structural disentanglement for cryo-EM single-particle analysis

Cryo-electron microscopy (cryo-EM) captures snapshots of dynamic macromolecules, collectively illustrating the involved structural landscapes. This provides an exciting opportunity to explore the structural variations of macromolecules under study. However, traditional cryo-EM single-particle analysis often yields static structures. Here we describe OPUS-DSD, an algorithm capable of efficiently reconstructing the structural landscape embedded in cryo-EM data. OPUS-DSD uses a three-dimensional convolutional encoder–decoder architecture trained with cryo-EM images, thereby encoding structural variations into a smooth and easily analyzable low-dimension space. This space can be traversed to reconstruct continuous dynamics or clustered to identify distinct conformations. OPUS-DSD can offer meaningful insights into the structural variations of macromolecules, filling in the gaps left by traditional cryo-EM structural determination, and potentially improves the reconstruction resolution by reliably clustering similar particles within the dataset. These functionalities are especially relevant to the study of highly dynamic biological systems. OPUS-DSD is available at https://github.com/alncat/opusDSD.

Particle fusion of super-resolution data reveals the unit structure of Nup96 in Nuclear Pore Complex

Single molecule localization microscopy offers resolution nearly down to the molecular level with specific molecular labelling, and is thereby a promising tool for structural biology. In practice, however, the actual value to this field is limited primarily by incomplete fluorescent labelling of the structure. This missing information can be completed by merging information from many structurally identical particles in a particle fusion approach similar to cryo-EM single-particle analysis. In this paper, we present a data analysis of particle fusion results of fluorescently labelled Nup96 nucleoporins in the Nuclear Pore Complex to show that Nup96 occurs in a spatial arrangement of two rings of 8 units with two Nup96 copies per unit giving a total of 32 Nup96 copies per pore. We use Artificial Intelligence assisted modeling in Alphafold to extend the existing cryo-EM model of Nup96 to accurately pinpoint the positions of the fluorescent labels and show the accuracy of the match between fluorescent and cryo-EM data to be better than 3 nm in-plane and 5 nm out-of-plane.

A strategy combining denoising and cryo-EM single particle analysis

In cryogenic electron microscopy (cryo-EM) single particle analysis (SPA), high-resolution three-dimensional structures of biological macromolecules are determined by iteratively aligning and averaging a large number of two-dimensional projections of molecules. Since the correlation measures are sensitive to the signal-to-noise ratio, various parameter estimation steps in SPA will be disturbed by the high-intensity noise in cryo-EM. However, denoising algorithms tend to damage high frequencies and suppress mid- and high-frequency contrast of micrographs, which exactly the precise parameter estimation relies on, therefore, limiting their application in SPA. In this study, we suggest combining a cryo-EM image processing pipeline with denoising and maximizing the signal's contribution in various parameter estimation steps. To solve the inherent flaws of denoising algorithms, we design an algorithm named MScale to correct the amplitude distortion caused by denoising and propose a new orientation determination strategy to compensate for the high-frequency loss. In the experiments on several real datasets, the denoised particles are successfully applied in the class assignment estimation and orientation determination tasks, ultimately enhancing the quality of biomacromolecule reconstruction. The case study on classification indicates that our strategy not only improves the resolution of difficult classes (up to 5Å) but also resolves an additional class. In the case study on orientation determination, our strategy improves the resolution of the final reconstructed density map by 0.34Å compared with conventional strategy. The code is available at https://github.com/zhanghui186/Mscale.

ZART: A Novel Multiresolution Reconstruction Algorithm with Motion-blur Correction for Single Particle Analysis

One of the main purposes of CryoEM Single Particle Analysis is to reconstruct the three-dimensional structure of a macromolecule thanks to the acquisition of many particle images representing different poses of the sample. By estimating the orientation of each projected particle, it is possible to recover the underlying 3D volume by multiple 3D reconstruction methods, usually working either in Fourier or in real space. However, the reconstruction from the projected images works under the assumption that all particles in the dataset correspond to the same conformation of the macromolecule. Although this requisite holds for some macromolecules, it is not true for flexible specimens, leading to motion-induced artefacts in the reconstructed CryoEM maps. In this work, we introduce a new Algebraic Reconstruction Technique called ZART, which is able to include continuous flexibility information during the reconstruction process to improve local resolution and reduce motion blurring. The conformational changes are modelled through Zernike3D polynomials. Our implementation allows for a multiresolution description of the macromolecule adapting itself to the local resolution of the reconstructed map. In addition, ZART has also proven to be a useful algorithm in cases where flexibility is not so dominant, as it improves the overall aspect of the reconstructed maps by improving their local and global resolution.

Near-physiological in vitro assembly of 50S ribosomes involves parallel pathways.

Understanding the assembly principles of biological macromolecular complexes remains a significant challenge, due to the complexity of the systems and the difficulties in developing experimental approaches. As a ribonucleoprotein complex, the ribosome serves as a model system for the profiling of macromolecular complex assembly. In this work, we report an ensemble of large ribosomal subunit intermediate structures that accumulate during synthesis in a near-physiological and co-transcriptional in vitro reconstitution system. Thirteen pre-50S intermediate maps covering the entire assembly process were resolved using cryo-EM single-particle analysis and heterogeneous subclassification. Segmentation of the set of density maps reveals that the 50S ribosome intermediates assemble based on fourteen cooperative assembly blocks, including the smallest assembly core reported to date, which is composed of a 600-nucleotide-long folded rRNA and three ribosomal proteins. The cooperative blocks assemble onto the assembly core following defined dependencies, revealing the parallel pathways at both early and late assembly stages of the 50S subunit.

The Function of X-ray Crystallography in Modern Structural Biology

In modern life science research, structural information on proteins is indispensable for understanding the mechanisms of their biological functions. Many protein structures have been determined by X-ray crystallography, which has been developed in conjunction with molecular biology experiments and synchrotron radiation development. Since the Nobel Prize in Chemistry in 2017, the rapid progress of cryo-EM hardware and analysis software has led to the use of cryo-EM single-particle analysis for structural analysis. In this article, we compare the features of protein crystallography and cryo-EM single-particle analysis and discuss the best method for protein crystallography.

Capsid structure of a fungal dsRNA megabirnavirus reveals its previously unidentified surface architecture.

Rosellinia necatrix megabirnavirus 1-W779 (RnMBV1) is a non-enveloped icosahedral double-stranded (ds)RNA virus that infects the ascomycete fungus Rosellinia necatrix, a causative agent that induces a lethal plant disease white root rot. Herein, we have first resolved the atomic structure of the RnMBV1 capsid at 3.2 Å resolution using cryo-electron microscopy (cryo-EM) single-particle analysis. Compared with other non-enveloped icosahedral dsRNA viruses, the RnMBV1 capsid protein structure exhibits an extra-long C-terminal arm and a surface protrusion domain. In addition, the previously unrecognized crown proteins are identified in a symmetry-expanded cryo-EM model and are present over the 3-fold axes. These exclusive structural features of the RnMBV1 capsid could have been acquired for playing essential roles in transmission and/or particle assembly of the megabirnaviruses. Our findings, therefore, will reinforce the understanding of how the structural and molecular machineries of the megabirnaviruses influence the virulence of the disease-related ascomycete fungus.

Structural insight into the mitochondrial TOM holo complex.

The Translocase of the Outer Membrane (TOM) complex is the main entry gate into the mitochondria for most precursor proteins. These proteins have mitochondrial targeting sequences that contain information about their distribution throughout the organelle. The TOM core complex is a heterodimer formed by Tom40, the translocation pore, Tom22, the main presequence receptor, and three small Tom subunits. However, two other receptor subunits, Tom20 and Tom70, are associated with the complex and have been a difficult target in structural biology. In this study we obtained the high-resolution structure of the TOM core complex from Neurospora crassa, obtained through Cryo-EM Single Particle Analysis, as well as a lower-resolution structure of the TOM core complex in association with Tom20. Furthermore, we present the multiple subcomplexes formed by the TOM subunits obtained by native mass spectrometry, bringing insight to the interaction of Tom20 and Tom70 with the TOM core complex. We propose that Tom20 is the dynamic gatekeeper guiding precursor proteins in the first steps of translocation, as it guides them into the Tom40 pores. Our study sheds new light into the so far elusive TOM holo complex and offers an integral translocation mechanism.

Near-physiological in vitro reconstitution of 50S subunit reveals parallel assembly pathway.

The ribosome is one of the largest and the most complicated enzymes in cells, and is responsible for protein synthesis in all living organisms on earth. During the bacterial ribosome biogenesis, the ∼5-kb transcriptional products are cleaved into three component ribosomal RNAs, undergo post-transcriptional chemical modifications by a set of enzymes, and fold into ∼150 secondary helices structures, facilitated by the chaperoning and controlling of more than 100 ribosome biogenesis co-factors. Despite its complexity, the bacterial ribosome assembly process takes less than 2 minutes in cells, so the intermediates during the step-wised ribosome assembly process are incredibly short-lived, making it difficult to directly separate and investigate the incomplete ribosomes under native physiological environments. In this work, we report a time-course structure library of ribosome large subunit intermediates accumulated from a near-physiological and co-transcriptional in vitro reconstitution system. Thirteen pre-50S intermediate structures covering the whole assembly process were resolved by cryo-EM single particle analysis using heterogeneous subclassification. Segment behavior analysis reveals that the ribosome intermediates assemble based on fourteen cooperative assembly blocks, including a smallest assembly core reported up to now, which is composed of a 600-nucleotide-long folded rRNA and three ribosomal proteins. The cooperative assembly blocks are found to fold onto the assembly core following a defined set of dependencies. Some of these dependencies are parallel to each other, highlighting the parallel assembly pathways at both early and late assembly stages of the 50S subunit.

Cryo-EM image alignment: From pair-wise to joint with deep unsupervised difference learning

Cryo-electron microscopy (cryo-EM) single-particle analysis is a revolutionary imaging technique to resolve and visualize biomacromolecules. Image alignment in cryo-EM is an important and basic step to improve the precision of the image distance calculation. However, it is a very challenging task due to high noise and low signal-to-noise ratio. Therefore, we propose a new deep unsupervised difference learning (UDL) strategy with novel pseudo-label guided learning network architecture and apply it to pair-wise image alignment in cryo-EM. The training framework is fully unsupervised. Furthermore, a variant of UDL called joint UDL (JUDL), is also proposed, which is capable of utilizing the similarity information of the whole dataset and thus further increase the alignment precision. Assessments on both real-world and synthetic cryo-EM single-particle image datasets suggest the new unsupervised joint alignment method can achieve more accurate alignment results. Our method is highly efficient by taking advantages of GPU devices. The source code of our methods is publicly available at “http://www.csbio.sjtu.edu.cn/bioinf/JointUDL/” for academic use.