The structure of an Hsp90-immunophilin complex reveals cochaperone recognition of the client maturation state.

The Hsp90 chaperone promotes folding and activation of hundreds of client proteins in the cell through an ATP-dependent conformational cycle guided by distinct cochaperone regulators. The FKBP51 immunophilin binds Hsp90 with its tetratricopeptide repeat (TPR) domain and catalyzes peptidyl-prolyl isomerase (PPIase) activity during folding of kinases, nuclear receptors, and tau. Here we determined the cryoelectron microscopy (cryo-EM) structure of the human Hsp90:FKBP51:p23 complex to 3.3 Å, which, together with mutagenesis and crosslinking analyses, reveals the basis for cochaperone binding to Hsp90 during client maturation. A helix extension in the TPR functions as a key recognition element, interacting across the Hsp90 C-terminal dimer interface presented in the closed, ATP conformation. The PPIase domain is positioned along the middle domain, adjacent to Hsp90 client binding sites, whereas a single p23 makes stabilizing interactions with the N-terminal dimer. With this architecture, FKBP51 is positioned to act on specific client residues presented during Hsp90-catalyzed remodeling.

Regulatory sites of CaM-sensitive adenylyl cyclase AC8 revealed by cryo-EM and structural proteomics.

Membrane adenylyl cyclase AC8 is regulated by G proteins and calmodulin (CaM), mediating the crosstalk between the cAMP pathway and Ca2+ signalling. Despite the importance of AC8 in physiology, the structural basis of its regulation by G proteins and CaM is not well defined. Here, we report the 3.5 Å resolution cryo-EM structure of the bovine AC8 bound to the stimulatory Gαs protein in the presence of Ca2+/CaM. The structure reveals the architecture of the ordered AC8 domains bound to Gαs and the small molecule activator forskolin. The extracellular surface of AC8 features a negatively charged pocket, a potential site for unknown interactors. Despite the well-resolved forskolin density, the captured state of AC8 does not favour tight nucleotide binding. The structural proteomics approaches, limited proteolysis and crosslinking mass spectrometry (LiP-MS and XL-MS), allowed us to identify the contact sites between AC8 and its regulators, CaM, Gαs, and Gßγ, as well as to infer the conformational changes induced by these interactions. Our results provide a framework for understanding the role of flexible regions in the mechanism of AC regulation.

Cryo-EM reconstruction of oleate hydratase bound to a phospholipid membrane bilayer.

Oleate hydratase (OhyA) is a bacterial peripheral membrane protein that catalyzes FAD-dependent water addition to membrane bilayer-embedded unsaturated fatty acids. The opportunistic pathogen Staphylococcus aureus uses OhyA to counteract the innate immune system and support colonization. Many Gram-positive and Gram-negative bacteria in the microbiome also encode OhyA. OhyA is a dimeric flavoenzyme whose carboxy terminus is identified as the membrane binding domain; however, understanding how OhyA binds to cellular membranes is not complete until the membrane-bound structure has been elucidated. All available OhyA structures depict the solution state of the protein outside its functional environment. Here, we employ liposomes to solve the cryo-electron microscopy structure of the functional unit: the OhyA•membrane complex. The protein maintains its structure upon membrane binding and slightly alters the curvature of the liposome surface. OhyA preferentially associates with 20-30 nm liposomes with multiple copies of OhyA dimers assembling on the liposome surface resulting in the formation of higher-order oligomers. Dimer assembly is cooperative and extends along a formed ridge of the liposome. We also solved an OhyA dimer of dimers structure that recapitulates the intermolecular interactions that stabilize the dimer assembly on the membrane bilayer as well as the crystal contacts in the lattice of the OhyA crystal structure. Our work enables visualization of the molecular trajectory of membrane binding for this important interfacial enzyme.

SARS-CoV-2 polyprotein substrate regulates the stepwise Mpro cleavage reaction.

The processing of the Coronavirus polyproteins pp1a and pp1ab by the main protease Mpro to produce mature proteins is a crucial event in virus replication and a promising target for antiviral drug development. Mpro cleaves polyproteins in a defined order, but how Mpro and/or the polyproteins determine the order of cleavage remains enigmatic due to a lack of structural information about polyprotein-bound Mpro. Here, we present the cryo-EM structures of SARS-CoV-2 Mpro in an apo form and in complex with the nsp7-10 region of the pp1a polyprotein. The complex structure shows that Mpro interacts with only the recognition site residues between nsp9 and nsp10, without any association with the rest of the polyprotein. Comparison between the apo form and polyprotein-bound structures of Mpro highlights the flexible nature of the active site region of Mpro, which allows it to accommodate ten recognition sites found in the polyprotein. These observations suggest that the role of Mpro in selecting a preferred cleavage site is limited and underscores the roles of the structure, conformation, and/or dynamics of the polyproteins in determining the sequence of polyprotein cleavage by Mpro.

Heterogeneous cryo-EM projection image classification using a two-stage spectral clustering based on novel distance measures.

Single-particle cryo-electron microscopy (cryo-EM) has become one of the mainstream technologies in the field of structural biology to determine the three-dimensional (3D) structures of biological macromolecules. Heterogeneous cryo-EM projection image classification is an effective way to discover conformational heterogeneity of biological macromolecules in different functional states. However, due to the low signal-to-noise ratio of the projection images, the classification of heterogeneous cryo-EM projection images is a very challenging task. In this paper, two novel distance measures between projection images integrating the reliability of common lines, pixel intensity and class averages are designed, and then a two-stage spectral clustering algorithm based on the two distance measures is proposed for heterogeneous cryo-EM projection image classification. In the first stage, the novel distance measure integrating common lines and pixel intensities of projection images is used to obtain preliminary classification results through spectral clustering. In the second stage, another novel distance measure integrating the first novel distance measure and class averages generated from each group of projection images is used to obtain the final classification results through spectral clustering. The proposed two-stage spectral clustering algorithm is applied on a simulated and a real cryo-EM dataset for heterogeneous reconstruction. Results show that the two novel distance measures can be used to improve the classification performance of spectral clustering, and using the proposed two-stage spectral clustering algorithm can achieve higher classification and reconstruction accuracy than using RELION and XMIPP.

The presence of uncoated gold nanoparticle aggregates may alter the phase of phosphatidylcholine lipid as evidenced by vibrational spectroscopies.

Spherical structures built from uni- and multilamellar lipid bilayers (LUV and MLV) are nowadays considered not just as nanocarriers of various kinds of therapeutics, but also as the vehicles that, when coupled with gold (Au) nanoparticles (NPs), can also serve as a tool for imaging and discriminating healthy and diseased tissues. Since the presence of Au NPs or their aggregates may affect the properties of the drug delivery vehicle, we investigated how the shape and position of Au NP aggregates adsorbed on the surface of MLV affect the arrangement and conformation of lipid molecules. By preparing MLVs constituted from 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) in the presence of uncoated Au NP aggregates found i) both within liposome core and on the surface of the outer lipid bilayer, or ii) adsorbed on the outer lipid bilayer surface only, we demonstrated the maintenance of lipid bilayer integrity by microscopic techniques (cryo-TEM, and AFM). The employment of SERS and FTIR-ATR techniques enabled us not only to elucidate the lipid interaction pattern and their orientation in regards to Au NP aggregates but also unequivocally confirmed the impact of Au NP aggregates on the persistence/breaking of van der Waals interactions between hydrocarbon chains of DPPC.

EMNUSS: a deep learning framework for secondary structure annotation in cryo-EM maps.

Cryo-electron microscopy (cryo-EM) has become one of important experimental methods in structure determination. However, despite the rapid growth in the number of deposited cryo-EM maps motivated by advances in microscopy instruments and image processing algorithms, building accurate structure models for cryo-EM maps remains a challenge. Protein secondary structure information, which can be extracted from EM maps, is beneficial for cryo-EM structure modeling. Here, we present a novel secondary structure annotation framework for cryo-EM maps at both intermediate and high resolutions, named EMNUSS. EMNUSS adopts a three-dimensional (3D) nested U-net architecture to assign secondary structures for EM maps. Tested on three diverse datasets including simulated maps, middle resolution experimental maps, and high-resolution experimental maps, EMNUSS demonstrated its accuracy and robustness in identifying the secondary structures for cyro-EM maps of various resolutions. The EMNUSS program is freely available at http://huanglab.phys.hust.edu.cn/EMNUSS.

Method for the structural analysis of Twinkle mitochondrial DNA helicase by cryo-EM.

The mitochondrial replisome replicates the 16.6 kb mitochondria DNA (mtDNA). The proper functioning of this multicomponent protein complex is vital for the integrity of the mitochondrial genome. One of the critical protein components of the mitochondrial replisome is the Twinkle helicase, a member of the Superfamily 4 (SF4) helicases. Decades of research has uncovered common themes among SF4 helicases including self-assembly, ATP-dependent translocation, and formation of protein-protein complexes. Some of the molecular details of these processes are still unknown for the mitochondria SF4 helicase, Twinkle. Here, we describe a protocol for expression, purification, and single-particle cryo-electron microscopy of the Twinkle helicase clinical variant, W315L, which resulted in the first high-resolution structure of Twinkle helicase. The methods described here serve as an adaptable protocol to support future high-resolution studies of Twinkle helicase or other SF4 helicases.

Handling Difficult Cryo-ET Samples: A Study with Primary Neurons from Drosophila melanogaster.

Cellular neurobiology has benefited from recent advances in the field of cryo-electron tomography (cryo-ET). Numerous structural and ultrastructural insights have been obtained from plunge-frozen primary neurons cultured on electron microscopy grids. With most primary neurons having been derived from rodent sources, we sought to expand the breadth of sample availability by using primary neurons derived from 3rd instar Drosophila melanogaster larval brains. Ultrastructural abnormalities were encountered while establishing this model system for cryo-ET, which were exemplified by excessive membrane blebbing and cellular fragmentation. To optimize neuronal samples, we integrated substrate selection, micropatterning, montage data collection, and chemical fixation. Efforts to address difficulties in establishing Drosophila neurons for future cryo-ET studies in cellular neurobiology also provided insights that future practitioners can use when attempting to establish other cell-based model systems.

Identifying Heterozipper ß-Sheet in Twisted Amyloid Aggregation.

Amyloid peptide (AP) self-assembly is a hierarchical process. However, the mechanistic rule of guiding peptides to organize well-ordered nanostructure in a clear and precise manner remains poorly understood. Herein we explored the molecular insight of AP motif aggregates underlying hierarchical process with helical fibrillar structure by atomic force microscope, cryo-electron microscopy (cryo-EM), and molecular dynamics simulation. AP assembly encompasses well-ordered twisted fibrils with uniform morphology, size, and periodicity. More importantly, a heterozipper ß-sheet was identified in a protofilament of AP assembly determined by cryo-EM with a high resolution of 3.5 Å. Each peptide heterozipper was further composed of two antiparallel ß strands and arranged by an alternative manner in a protofilament. The hydrophobic core and hydrophilic area in each zipper played the significant role for peptide assembling. This work proposed and verified the rule facilitating the basic building unit to form twisted fibrils and gave the explanation of peptide hierarchical assembling.

Interaction of Vanadium Complexes with Proteins: Revisiting the Reported Structures in the Protein Data Bank (PDB) since 2015.

The structural determination and characterization of molecules, namely proteins and enzymes, is crucial to gaining a better understanding of their role in different chemical and biological processes. The continuous technical developments in the experimental and computational resources of X-ray diffraction (XRD) and, more recently, cryogenic Electron Microscopy (cryo-EM) led to an enormous growth in the number of structures deposited in the Protein Data Bank (PDB). Bioinorganic chemistry arose as a relevant discipline in biology and therapeutics, with a massive number of studies reporting the effects of metal complexes on biological systems, with vanadium complexes being one of the relevant systems addressed. In this review, we focus on the interactions of vanadium compounds (VCs) with proteins. Several types of binding are established between VCs and proteins/enzymes. Considering that the V-species that bind may differ from those initially added, the mentioned structural techniques are pivotal to clarifying the nature and variety of interactions of VCs with proteins and to proposing the mechanisms involved either in enzymatic inhibition or catalysis. As such, we provide an account of the available structural information of VCs bound to proteins obtained by both XRD and/or cryo-EM, mainly exploring the more recent structures, particularly those containing organic-based vanadium complexes.

Integrated multidisciplinarity in the natural sciences.

The integration of multiple perspectives in both the arts and natural sciences is tremendously powerful and arguably necessary for capturing relevant features of complex phenomena. Individual methods and models comprise abstractions from and idealizations of nature, and only the integration of multiple models, methods, and representations provides a means to reach more accurate results than relying on any single approach. In my Mildred Cohn Award Lecture at the 2019 ASBMB meeting, I illustrated the power of such multidisciplinary work by highlighting the successful integration of data and multiple views afforded by NMR spectroscopy, cryo-electron microscopy, cryo-electron tomography, X-ray crystallography, computation, and functional assays made possible through collaborative efforts by members of the Pittsburgh Center for HIV Protein Interactions. This approach permitted us to generate the first all-atom model of a native HIV-1 capsid core.

Ten antenna proteins are associated with the core in the supramolecular organization of the photosystem I supercomplex in Chlamydomonas reinhardtii.

Photosystem I (PSI) is a large pigment-protein complex mediating light-driven charge separation and generating a highly negative redox potential, which is eventually utilized to produce organic matter. In plants and algae, PSI possesses outer antennae, termed light-harvesting complex I (LHCI), which increase the energy flux to the reaction center. The number of outer antennae for PSI in the green alga Chlamydomonas reinhardtii is known to be larger than that of land plants. However, their exact number and location remain to be elucidated. Here, applying a newly established sample purification procedure, we isolated a highly pure PSI-LHCI supercomplex containing all nine LHCA gene products under state 1 conditions. Single-particle cryo-EM revealed the 3D structure of this supercomplex at 6.9 Å resolution, in which the densities near the PsaF and PsaJ subunits were assigned to two layers of LHCI belts containing eight LHCIs, whereas the densities between the PsaG and PsaH subunits on the opposite side of the LHCI belt were assigned to two extra LHCIs. Using single-particle cryo-EM, we also determined the 2D projection map of the lhca2 mutant, which confirmed the assignment of LHCA2 and LHCA9 to the densities between PsaG and PsaH. Spectroscopic measurements of the PSI-LHCI supercomplex suggested that the bound LHCA2 and LHCA9 proteins have the ability to increase the light-harvesting energy for PSI. We conclude that the PSI in C. reinhardtii has a larger and more distinct outer-antenna organization and higher light-harvesting capability than that in land plants.

Cryo-EM structure of the native rhodopsin dimer in nanodiscs.

Imaging of rod photoreceptor outer-segment disc membranes by atomic force microscopy and cryo-electron tomography has revealed that the visual pigment rhodopsin, a prototypical class A G protein-coupled receptor (GPCR), can organize as rows of dimers. GPCR dimerization and oligomerization offer possibilities for allosteric regulation of GPCR activity, but the detailed structures and mechanism remain elusive. In this investigation, we made use of the high rhodopsin density in the native disc membranes and of a bifunctional cross-linker that preserves the native rhodopsin arrangement by covalently tethering rhodopsins via Lys residue side chains. We purified cross-linked rhodopsin dimers and reconstituted them into nanodiscs for cryo-EM analysis. We present cryo-EM structures of the cross-linked rhodopsin dimer as well as a rhodopsin dimer reconstituted into nanodiscs from purified monomers. We demonstrate the presence of a preferential 2-fold symmetrical dimerization interface mediated by transmembrane helix 1 and the cytoplasmic helix 8 of rhodopsin. We confirmed this dimer interface by double electron-electron resonance measurements of spin-labeled rhodopsin. We propose that this interface and the arrangement of two protomers is a prerequisite for the formation of the observed rows of dimers. We anticipate that the approach outlined here could be extended to other GPCRs or membrane receptors to better understand specific receptor dimerization mechanisms.

Biological Applications at the Cutting Edge of Cryo-Electron Microscopy.

Cryo-electron microscopy (cryo-EM) is a powerful tool for macromolecular to near-atomic resolution structure determination in the biological sciences. The specimen is maintained in a near-native environment within a thin film of vitreous ice and imaged in a transmission electron microscope. The images can then be processed by a number of computational methods to produce three-dimensional information. Recent advances in sample preparation, imaging, and data processing have led to tremendous growth in the field of cryo-EM by providing higher resolution structures and the ability to investigate macromolecules within the context of the cell. Here, we review developments in sample preparation methods and substrates, detectors, phase plates, and cryo-correlative light and electron microscopy that have contributed to this expansion. We also have included specific biological applications.

The Methods of Choice for Extracellular Vesicles (EVs) Characterization.

In recent years, extracellular vesicles (EVs) have become a subject of intense study. These membrane-enclosed spherical structures are secreted by almost every cell type and are engaged in the transport of cellular content (cargo) from parental to target cells. The impact of EVs transfer has been observed in many vital cellular processes including cell-to-cell communication and immune response modulation; thus, a fast and precise characterization of EVs may be relevant for both scientific and diagnostic purposes. In this review, the most popular analytical techniques used in EVs studies are presented with the emphasis on exosomes and microvesicles characterization.

Practical Experience with Hole-Free Phase Plates for Cryo Electron Microscopy.

Phase plate (PP) imaging has proven to be valuable in transmission cryo electron microscopy of unstained, native-state biological specimens. Many PP types have been described, however until the recent implementation of the "hole-free" phase plate (HFPP), imaging has been challenging. We found the HFPP to be simple to construct and to set up in the transmission electron microscopy, but care in implementing automated data collection is needed. Performance may be variable, both initially and over time, thus it is important to monitor and evaluate image quality by observing the power spectrum. We found that while some HFPPs gave transfer to high resolution without CTF oscillation, most reached high resolution when operated with modest defocus.

Hsc70-induced changes in clathrin-auxilin cage structure suggest a role for clathrin light chains in cage disassembly.

The molecular chaperone, Hsc70, together with its co-factor, auxilin, facilitates the ATP-dependent removal of clathrin during clathrin-mediated endocytosis in cells. We have used cryo-electron microscopy to determine the 3D structure of a complex of clathrin, auxilin(401-910) and Hsc70 at pH 6 in the presence of ATP, frozen within 20 seconds of adding Hsc70 in order to visualize events that follow the binding of Hsc70 to clathrin and auxilin before clathrin disassembly. In this map, we observe density beneath the vertex of the cage that we attribute to bound Hsc70. This density emerges asymmetrically from the clathrin vertex, suggesting preferential binding by Hsc70 for one of the three possible sites at the vertex. Statistical comparison with a map of whole auxilin and clathrin previously published by us reveals the location of statistically significant differences which implicate involvement of clathrin light chains in structural rearrangements which occur after Hsc70 is recruited. Clathrin disassembly assays using light scattering suggest that loss of clathrin light chains reduces the efficiency with which auxilin facilitates this reaction. These data support a regulatory role for clathrin light chains in clathrin disassembly in addition to their established role in regulating clathrin assembly.

Quantitation and characterization of serotype 6A activation for pneumococcal conjugate vaccine by cryo-EM and SEC methods.

Pneumococcal conjugate vaccines (PCV) typically consist of capsular polysaccharides from different S. pneumoniae serotypes which are covalently attached to carrier protein. A well-established process to manufacture PCV is through activating polysaccharide by oxidation of vicinal diols to aldehydes, followed by protein conjugation via reductive amination. Polysaccharide activation is a crucial step that affects vaccine product critical attributes including conjugate size and structure. Therefore, it is highly desired to have robust analytical methods to well characterize this activation process. In this study, using pneumococcal serotype 6A as the model, we present two complimentary analytical methods for characterization of activated polysaccharide. First, a size exclusion chromatography (SEC) method was developed for quantitative measurement of polysaccharide activation levels. This SEC method demonstrated good assay characteristics on accuracy, precision and linearity. Second, a gold nanoparticle labeled cryo-electron microscopy (Cryo-EM) technique was developed to visualize activation site distribution along polysaccharide chain and provide information on activation heterogeneity. These two complimentary methods can be utilized to control polysaccharide activation process and ensure consistent delivery of conjugate vaccine products.

Conformational landscape of soluble α-klotho revealed by cryogenic electron microscopy.

α-Klotho (KLA) is a type-1 membranous protein that can associate with fibroblast growth factor receptor (FGFR) to form co-receptor for FGF23. The ectodomain of unassociated KLA is shed as soluble KLA (sKLA) to exert FGFR/FGF23-independent pleiotropic functions. The previously determined X-ray crystal structure of the extracellular region of sKLA in complex with FGF23 and FGFR1c suggests that sKLA functions solely as an on-demand coreceptor for FGF23. To understand the FGFR/FGF23-independent pleiotropic functions of sKLA, we investigated biophysical properties and structure of apo-sKLA. Mass photometry revealed that sKLA can form a stable structure with FGFR and/or FGF23 as well as sKLA dimer in solution. Single particle cryogenic electron microscopy (cryo-EM) supported the dimeric structure of sKLA. Cryo-EM further revealed a 3.3Å resolution structure of apo-sKLA that overlays well with its counterpart in the ternary complex with several distinct features. Compared to the ternary complex, the KL2 domain of apo-sKLA is more flexible. 3D variability analysis revealed that apo-sKLA adopts conformations with different KL1-KL2 interdomain bending and rotational angles. The potential multiple forms and shapes of sKLA support its role as FGFR-independent hormone with pleiotropic functions. A comprehensive understanding of the sKLA conformational landscape will provide the foundation for developing klotho-related therapies for diseases.