Label-free visual proteomics: Coupling MS- and EM-based approaches in structural biology.

Combining diverse experimental structural and interactomic methods allows for the construction of comprehensible molecular encyclopedias of biological systems. Typically, this involves merging several independent approaches that provide complementary structural and functional information from multiple perspectives and at different resolution ranges. A particularly potent combination lies in coupling structural information from cryoelectron microscopy or tomography (cryo-EM or cryo-ET) with interactomic and structural information from mass spectrometry (MS)-based structural proteomics. Cryo-EM/ET allows for sub-nanometer visualization of biological specimens in purified and near-native states, while MS provides bioanalytical information for proteins and protein complexes without introducing additional labels. Here we highlight recent achievements in protein structure and interactome determination using cryo-EM/ET that benefit from additional MS analysis. We also give our perspective on how combining cryo-EM/ET and MS will continue bridging gaps between molecular and cellular studies by capturing and describing 3D snapshots of proteomes and interactomes.

Square beams for optimal tiling in transmission electron microscopy.

Imaging large fields of view at a high magnification requires tiling. Transmission electron microscopes typically have round beam profiles; therefore, tiling across a large area is either imperfect or results in uneven exposures, a problem for dose-sensitive samples. Here, we introduce a square electron beam that can easily be retrofitted in existing microscopes, and demonstrate its application, showing that it can tile nearly perfectly and deliver cryo-electron microscopy imaging with a resolution comparable to conventional set-ups.

Broadening access to cryoEM through centralized facilities.

Cryogenic electron microscopy (cryoEM) uses images of frozen hydrated biological specimens to produce macromolecular structures, opening up previously inaccessible levels of biological organization to high-resolution structural analysis. CryoEM has the potential for broad impact in biomedical research, including basic cell, molecular, and structural biology, and increasingly in drug discovery and vaccine development. Recent advances have led to the expansion of molecular and cellular structure determination at an exponential rate. National and regional centers have emerged to support this growth by increasing the accessibility of cryoEM throughout the biomedical research community. Through cooperation and synergy, these centers form a network of resources that accelerate the adoption of best practices for access and training and establish sustainable workflows to build future research capacity.

Time-resolved cryo-EM using Spotiton.

We present an approach for preparing cryo-electron microscopy (cryo-EM) grids to study short-lived molecular states. Using piezoelectric dispensing, two independent streams of ~50-pl droplets of sample are deposited within 10 ms of each other onto the surface of a nanowire EM grid, and the mixing reaction stops when the grid is vitrified in liquid ethane ~100 ms later. We demonstrate this approach for four biological systems where short-lived states are of high interest.

Mimotope discovery as a tool to design a vaccine against Zika and dengue viruses.

Vaccine development against dengue virus is challenging because of the antibody-dependent enhancement of infection (ADE), which causes severe disease. Consecutive infections by Zika (ZIKV) and/or dengue viruses (DENV), or vaccination can predispose to ADE. Current vaccines and vaccine candidates contain the complete envelope viral protein, with epitopes that can raise antibodies causing ADE. We used the envelope dimer epitope (EDE), which induces neutralizing antibodies that do not elicit ADE, to design a vaccine against both flaviviruses. However, EDE is a discontinuous quaternary epitope that cannot be isolated from the E protein without other epitopes. Utilizing phage display, we selected three peptides that mimic the EDE. Free mimotopes were disordered and did not elicit an immune response. After their display on adeno-associated virus (AAV) capsids (VLP), they recovered their structure and were recognized by an EDE-specific antibody. Characterization by cryo-EM and enzyme-linked immunosorbent assay confirmed the correct display of a mimotope on the surface of the AAV VLP and its recognition by the specific antibody. Immunization with the AAV VLP displaying one of the mimotopes induced antibodies that recognized ZIKV and DENV. This work provides the basis for developing a Zika and dengue virus vaccine candidate that will not induce ADE.

Ribosome Dimerization Protects the Small Subunit.

When nutrients become scarce, bacteria can enter an extended state of quiescence. A major challenge of this state is how to preserve ribosomes for the return to favorable conditions. Here, we show that the ribosome dimerization protein hibernation-promoting factor (HPF) functions to protect essential ribosomal proteins. Ribosomes isolated from strains lacking HPF (Δhpf) or encoding a mutant allele of HPF that binds the ribosome but does not mediate dimerization were substantially depleted of the small subunit proteins S2 and S3. Strikingly, these proteins are located directly at the ribosome dimer interface. We used single-particle cryo-electron microscopy (cryo-EM) to further characterize these ribosomes and observed that a high percentage of ribosomes were missing S2, S3, or both. These data support a model in which the ribosome dimerization activity of HPF evolved to protect labile proteins that are essential for ribosome function. HPF is almost universally conserved in bacteria, and HPF deletions in diverse species exhibit decreased viability during starvation. Our data provide mechanistic insight into this phenotype and establish a mechanism for how HPF protects ribosomes during quiescence.IMPORTANCE The formation of ribosome dimers during periods of dormancy is widespread among bacteria. Dimerization is typically mediated by a single protein, hibernation-promoting factor (HPF). Bacteria lacking HPF exhibit strong defects in viability and pathogenesis and, in some species, extreme loss of rRNA. The mechanistic basis of these phenotypes has not been determined. Here, we report that HPF from the Gram-positive bacterium Bacillus subtilis preserves ribosomes by preventing the loss of essential ribosomal proteins at the dimer interface. This protection may explain phenotypes associated with the loss of HPF, since ribosome protection would aid survival during nutrient limitation and impart a strong selective advantage when the bacterial cell rapidly reinitiates growth in the presence of sufficient nutrients.

Reducing cryoEM file storage using lossy image formats.

Recent advances in instrumentation and software for cryoEM have increased the applicability and utility of this method. High levels of automation and faster data acquisition rates require hard decisions to be made regarding data retention. Here we investigate the efficacy of data compression applied to aligned summed movie files. Surprisingly, these images can be compressed using a standard lossy method that reduces file storage by 90-95% and yet can still be processed to provide sub-2 Šreconstructed maps. We do not advocate this as an archival method, but it may provide a useful means for retaining images as an historical record, especially at large facilities.

Bulged and Canonical G-Quadruplex Conformations Determine NDPK Binding Specificity.

Guanine-rich DNA strands can adopt tertiary structures known as G-quadruplexes (G4s) that form when Hoogsteen base-paired guanines assemble as planar stacks, stabilized by a central cation like K+. In this study, we investigated the conformational heterogeneity of a G-rich sequence from the 5' untranslated region of the Zea mays hexokinase4 gene. This sequence adopted an extensively polymorphic G-quadruplex, including non-canonical bulged G-quadruplex folds that co-existed in solution. The nature of this polymorphism depended, in part, on the incorporation of different sets of adjacent guanines into a quadruplex core, which permitted the formation of the different conformations. Additionally, we showed that the maize homolog of the human nucleoside diphosphate kinase (NDPK) NM23-H2 protein-ZmNDPK1-specifically recognizes and promotes formation of a subset of these conformations. Heteromorphic G-quadruplexes play a role in microorganisms' ability to evade the host immune system, so we also discuss how the underlying properties that determine heterogeneity of this sequence could apply to microorganism G4s.

Biosynthesis of cell envelope-associated phenolic glycolipids in Mycobacterium marinum.

Phenolic glycolipids (PGLs) are polyketide synthase-derived glycolipids unique to pathogenic mycobacteria. PGLs are found in several clinically relevant species, including various Mycobacterium tuberculosis strains, Mycobacterium leprae, and several nontuberculous mycobacterial pathogens, such as M. marinum. Multiple lines of investigation implicate PGLs in virulence, thus underscoring the relevance of a deep understanding of PGL biosynthesis. We report mutational and biochemical studies that interrogate the mechanism by which PGL biosynthetic intermediates (p-hydroxyphenylalkanoates) synthesized by the iterative polyketide synthase Pks15/1 are transferred to the noniterative polyketide synthase PpsA for acyl chain extension in M. marinum. Our findings support a model in which the transfer of the intermediates is dependent on a p-hydroxyphenylalkanoyl-AMP ligase (FadD29) acting as an intermediary between the iterative and the noniterative synthase systems. Our results also establish the p-hydroxyphenylalkanoate extension ability of PpsA, the first-acting enzyme of a multisubunit noniterative polyketide synthase system. Notably, this noniterative system is also loaded with fatty acids by a specific fatty acyl-AMP ligase (FadD26) for biosynthesis of phthiocerol dimycocerosates (PDIMs), which are nonglycosylated lipids structurally related to PGLs. To our knowledge, the partially overlapping PGL and PDIM biosynthetic pathways provide the first example of two distinct, pathway-dedicated acyl-AMP ligases loading the same type I polyketide synthase system with two alternate starter units to produce two structurally different families of metabolites. The studies reported here advance our understanding of the biosynthesis of an important group of mycobacterial glycolipids.

The maize (Zea mays L.) nucleoside diphosphate kinase1 (ZmNDPK1) gene encodes a human NM23-H2 homologue that binds and stabilizes G-quadruplex DNA.

Noncanonical forms of DNA like the guanine quadruplex (G4) play important roles in regulating transcription and translation through interactions with their protein partners. Although potential G4 elements have been identified in or near genes from species diverse as bacteria, mammals, and plants, little is known about how they might function as cis-regulatory elements or as binding sites for trans-acting protein partners. In fact, until now no G4 binding partners have been identified in the plant kingdom. Here, we report on the cloning and characterization of the first plant-kingdom gene known to encode a G4-binding protein, maize (Zea mays L.) nucleoside diphosphate kinase1 (ZmNDPK1). Structural characterization by X-ray crystallography reveals that it is a homohexamer, akin to other known NDPKs like the human homologue NM23-H2. Further probing into the G4-binding properties of both NDPK homologues suggests that ZmNDPK1 possesses properties distinct from that of NM23-H2, which is known to interact with a G-rich sequence element upstream of the c-myc gene and, in doing so, modulate its expression. Indeed, ZmNDPK1 binds the folded G4 with low nanomolar affinity but corresponding unfolded G-rich DNA more weakly, whereas NM23-H2 binds both folded and unfolded G4 with low nanomolar affinities; nonetheless, both homologues appear to stabilize folded DNAs whether they were prefolded or not. We also demonstrate that the G4-binding activity of ZmNDPK1 is independent of nucleotide binding and kinase activity, suggesting that the G4-binding region and the enzyme active sites are separate. Together, these findings establish a broad evolutionary conservation of some NDPKs as G4-DNA binding enzymes, but with potentially distinct biochemical properties that may reflect divergent evolution or species-specific deployment of these elements in gene regulatory processes.

Multi-species cryoEM calibration and workflow verification standard.

Cryogenic electron microscopy (cryoEM) is a rapidly growing structural biology modality that has been successful in revealing molecular details of biological systems. However, unlike established biophysical and analytical techniques with calibration standards, cryoEM has lacked comprehensive biological test samples. We introduce a cryoEM calibration sample that is a mixture of compatible macromolecules that can be used not only for resolution optimization but also provides multiple reference points for evaluating instrument performance, data quality, and image processing workflows in a single experiment. This combined test specimen provides researchers a reference point for validating their cryoEM pipeline, benchmarking their methodologies, and testing new algorithms.

Cryo-ET reveals the in situ architecture of the polar tube invasion apparatus from microsporidian parasites.

Microsporidia are divergent fungal pathogens that employ a harpoon-like apparatus called the polar tube (PT) to invade host cells. The PT architecture and its association with neighboring organelles remain poorly understood. Here, we use cryo-electron tomography to investigate the structural cell biology of the PT in dormant spores from the human-infecting microsporidian species, Encephalitozoon intestinalis . Segmentation and subtomogram averaging of the PT reveal at least four layers: two protein-based layers surrounded by a membrane, and filled with a dense core. Regularly spaced protein filaments form the structural skeleton of the PT. Combining cryo-electron tomography with cellular modeling, we propose a model for the 3-dimensional organization of the polaroplast, an organelle that is continuous with the membrane layer that envelops the PT. Our results reveal the ultrastructure of the microsporidian invasion apparatus in situ , laying the foundation for understanding infection mechanisms.

Heterologous Prime-Boost with Immunologically Orthogonal Protein Nanoparticles for Peptide Immunofocusing.

Protein nanoparticles are effective platforms for antigen presentation and targeting effector immune cells in vaccine development. Encapsulins are a class of protein-based microbial nanocompartments that self-assemble into icosahedral structures with external diameters ranging from 24 to 42 nm. Encapsulins from Mxyococcus xanthus were designed to package bacterial RNA when produced in E. coli and were shown to have immunogenic and self-adjuvanting properties enhanced by this RNA. We genetically incorporated a 20-mer peptide derived from a mutant strain of the SARS-CoV-2 receptor binding domain (RBD) into the encapsulin protomeric coat protein for presentation on the exterior surface of the particle. This immunogen elicited conformationally-relevant humoral responses to the SARS-CoV-2 RBD. Immunological recognition was enhanced when the same peptide was presented in a heterologous prime/boost vaccination strategy using the engineered encapsulin and a previously reported variant of the PP7 virus-like particle, leading to the development of a selective antibody response against a SARS-CoV-2 RBD point mutant. While generating epitope-focused antibody responses is an interplay between inherent vaccine properties and B/T cells, here we demonstrate the use of orthogonal nanoparticles to fine-tune the control of epitope focusing.

Heterologous Prime-Boost with Immunologically Orthogonal Protein Nanoparticles for Peptide Immunofocusing.

Protein nanoparticles are effective platforms for antigen presentation and targeting effector immune cells in vaccine development. Encapsulins are a class of protein-based microbial nanocompartments that self-assemble into icosahedral structures with external diameters ranging from 24 to 42 nm. Encapsulins from Myxococcus xanthus were designed to package bacterial RNA when produced in E. coli and were shown to have immunogenic and self-adjuvanting properties enhanced by this RNA. We genetically incorporated a 20-mer peptide derived from a mutant strain of the SARS-CoV-2 receptor binding domain (RBD) into the encapsulin protomeric coat protein for presentation on the exterior surface of the particle, inducing the formation of several nonicosahedral structures that were characterized by cryogenic electron microscopy. This immunogen elicited conformationally relevant humoral responses to the SARS-CoV-2 RBD. Immunological recognition was enhanced when the same peptide was presented in a heterologous prime/boost vaccination strategy using the engineered encapsulin and a previously reported variant of the PP7 virus-like particle, leading to the development of a selective antibody response against a SARS-CoV-2 RBD point mutant. While generating epitope-focused antibody responses is an interplay between inherent vaccine properties and B/T cells, here we demonstrate the use of orthogonal nanoparticles to fine-tune the control of epitope focusing.

Square beams for optimal tiling in TEM.

Imaging large fields-of-view at a high magnification requires tiling. Transmission electron microscopes typically have round beam profiles; therefore, tiling across a large field-of-view is either imperfect or results in uneven exposures, which is a problem on dose-sensitive samples. Here we introduce a square electron beam that can be easily retrofitted in existing microscopes and demonstrate its application showing it can tile nearly perfectly and deliver cryo-EM imaging with resolution comparable to conventional setups.

Square beams for optimal tiling in TEM.

Imaging large fields of view at a high magnification requires tiling. Transmission electron microscopes typically have round beam profiles; therefore, tiling across a large area is either imperfect or results in uneven exposures, a problem on dose-sensitive samples. Here, we introduce a square electron beam that can be easily retrofitted in existing microscopes and demonstrate its application, showing it can tile nearly perfectly and deliver cryo-EM imaging with a resolution comparable to conventional setups.

Exploring the Landscape of the PP7 Virus-like Particle for Peptide Display.

Self-assembling virus-like particles (VLPs) can tolerate a wide degree of genetic and chemical manipulation to their capsid protein to display a foreign molecule polyvalently. We previously reported the successful incorporation of foreign peptide sequences in the junction loop and onto the C-terminus of PP7 dimer VLPs, as these regions are accessible for surface display on assembled capsids. Here, we report the implementation of a library-based approach to test the assembly tolerance of PP7 dimer capsid proteins to insertions or terminal extensions of randomized 15-mer peptide sequences. By performing two iterative rounds of assembly-based selection, we evaluated the degree of favorability of all 20 amino acids at each of the 15 randomized positions. Deep sequencing analysis revealed a distinct preference for the inclusion of hydrophilic peptides and negatively charged amino acids (Asp and Glu) and the exclusion of positively charged peptides and bulky and hydrophobic amino acid residues (Trp, Phe, Tyr, and Cys). Within the libraries tested here, we identified 4000 to 22,000 unique 15-mer peptide sequences that can successfully be displayed on the surface of the PP7 dimer capsid. Overall, the use of small initial libraries consisting of no more than a few million members yielded a significantly larger number of unique and assembly-competent VLP sequences than have been previously characterized for this class of nucleoprotein particle.