Delineating organizational principles of the endogenous L-A virus by cryo-EM and computational analysis of native cell extracts.

The high abundance of most viruses in infected host cells benefits their structural characterization. However, endogenous viruses are present in low copy numbers and are therefore challenging to investigate. Here, we retrieve cell extracts enriched with an endogenous virus, the yeast L-A virus. The determined cryo-EM structure discloses capsid-stabilizing cation-π stacking, widespread across viruses and within the Totiviridae, and an interplay of non-covalent interactions from ten distinct capsomere interfaces. The capsid-embedded mRNA decapping active site trench is supported by a constricting movement of two flexible opposite-facing loops. tRNA-loaded polysomes and other biomacromolecules, presumably mRNA, are found in virus proximity within the cell extract. Mature viruses participate in larger viral communities resembling their rare in-cell equivalents in terms of size, composition, and inter-virus distances. Our results collectively describe a 3D-architecture of a viral milieu, opening the door to cell-extract-based high-resolution structural virology.

Effect of Molecular Dynamics and Internal Water Contact on the Photophysical Properties of Red pH-Sensitive Proteins.

The pH dependence of the absorption and (time-resolved) fluorescence of two red-shifted fluorescent proteins, mCardinal and mNeptune, was investigated. Decay-associated spectra were measured following fluorescence excitation at 470 nm in PBS buffer with a pH that ranged from 5.5 to 8.0. The fluorescence of both proteins shows two different decay components. mCardinal exhibits an increase in the long-lived fluorescence component with acidification from 1.34 ns at pH 8.0 to 1.62 ns at pH 5.5. An additional fast decay component with 0.64 ns at pH 8.0 up to 1.1 ns at pH 5.5 was found to be blue-shifted compared to the long-lived component. The fluorescence lifetime of mNeptune is insensitive to pH. DAS of mCardinal were simulated assuming a coupled two-level system to describe the 1S state of the chromophore within two different conformations of the protein. MD simulations were conducted to correlate the experimentally observed pH-induced change in the lifetime in mCardinal with its molecular properties. While the chromophores of both protein variants are stabilized by the same number of hydrogen bonds, it was found that the chromophore in mCardinal exhibits more water contacts compared to mNeptune. In mCardinal, interaction between the chromophore and Glu-145 is reduced as compared to mNeptune, but interaction with Thr-147 which is Ser-147 in mNeptune is stronger in mCardinal. Therefore, the dynamics of the excited-state proton transfer (ESPT) might be different in mCardinal and mNeptune. The pH dependency of ESPT is suggested as a key mechanism for pH sensitivity.

Structural analysis of an endogenous 4-megadalton succinyl-CoA-generating metabolon.

The oxoglutarate dehydrogenase complex (OGDHc) participates in the tricarboxylic acid cycle and, in a multi-step reaction, decarboxylates α-ketoglutarate, transfers succinyl to CoA, and reduces NAD+. Due to its pivotal role in metabolism, OGDHc enzymatic components have been studied in isolation; however, their interactions within the endogenous OGDHc remain elusive. Here, we discern the organization of a thermophilic, eukaryotic, native OGDHc in its active state. By combining biochemical, biophysical, and bioinformatic methods, we resolve its composition, 3D architecture, and molecular function at 3.35 Å resolution. We further report the high-resolution cryo-EM structure of the OGDHc core (E2o), which displays various structural adaptations. These include hydrogen bonding patterns confining interactions of OGDHc participating enzymes (E1o-E2o-E3), electrostatic tunneling that drives inter-subunit communication, and the presence of a flexible subunit (E3BPo), connecting E2o and E3. This multi-scale analysis of a succinyl-CoA-producing native cell extract provides a blueprint for structure-function studies of complex mixtures of medical and biotechnological value.

Structural assessment of the full-length wild-type tumor suppressor protein p53 by mass spectrometry-guided computational modeling.

The tetrameric tumor suppressor p53 represents a great challenge for 3D-structural analysis due to its high degree of intrinsic disorder (ca. 40%). We aim to shed light on the structural and functional roles of p53's C-terminal region in full-length, wild-type human p53 tetramer and their importance for DNA binding. For this, we employed complementary techniques of structural mass spectrometry (MS) in an integrated approach with computational modeling. Our results show no major conformational differences in p53 between DNA-bound and DNA-free states, but reveal a substantial compaction of p53's C-terminal region. This supports the proposed mechanism of unspecific DNA binding to the C-terminal region of p53 prior to transcription initiation by specific DNA binding to the core domain of p53. The synergies between complementary structural MS techniques and computational modeling as pursued in our integrative approach is envisioned to serve as general strategy for studying intrinsically disordered proteins (IDPs) and intrinsically disordered region (IDRs).

Novel exotic alleles of EARLY FLOWERING 3 determine plant development in barley.

EARLY FLOWERING 3 (ELF3) is an important regulator of various physiological and developmental processes and hence may serve to improve plant adaptation which will be essential for future plant breeding. To expand the limited knowledge on barley ELF3 in determining agronomic traits, we conducted field studies with heterogeneous inbred families (HIFs) derived from selected lines of the wild barley nested association mapping population HEB-25. During two growing seasons, phenotypes of nearly isogenic HIF sister lines, segregating for exotic and cultivated alleles at the ELF3 locus, were compared for 10 developmental and yield-related traits. We determine novel exotic ELF3 alleles and show that HIF lines, carrying the exotic ELF3 allele, accelerated plant development compared with the cultivated ELF3 allele, depending on the genetic background. Remarkably, the most extreme effects on phenology could be attributed to one exotic ELF3 allele differing from the cultivated Barke ELF3 allele in only one single nucleotide polymorphism (SNP). This SNP causes an amino acid substitution (W669G), which as predicted has an impact on the protein structure of ELF3. Consequently, it may affect phase separation behaviour and nano-compartment formation of ELF3 and, potentially, also its local cellular interactions causing significant trait differences between HIF sister lines.

Enabling cryo-EM density interpretation from yeast native cell extracts by proteomics data and AlphaFold structures.

In the cellular context, proteins participate in communities to perform their function. The detection and identification of these communities as well as in-community interactions has long been the subject of investigation, mainly through proteomics analysis with mass spectrometry. With the advent of cryogenic electron microscopy and the "resolution revolution," their visualization has recently been made possible, even in complex, native samples. The advances in both fields have resulted in the generation of large amounts of data, whose analysis requires advanced computation, often employing machine learning approaches to reach the desired outcome. In this work, we first performed a robust proteomics analysis of mass spectrometry (MS) data derived from a yeast native cell extract and used this information to identify protein communities and inter-protein interactions. Cryo-EM analysis of the cell extract provided a reconstruction of a biomolecule at medium resolution (∼8 Å (FSC = 0.143)). Utilizing MS-derived proteomics data and systematic fitting of AlphaFold-predicted atomic models, this density was assigned to the 2.6 MDa complex of yeast fatty acid synthase. Our proposed workflow identifies protein complexes in native cell extracts from Saccharomyces cerevisiae by combining proteomics, cryo-EM, and AI-guided protein structure prediction.

Cryo-Electron Microscopy Snapshots of Eukaryotic Membrane Proteins in Native Lipid-Bilayer Nanodiscs.

New technologies for purifying membrane-bound protein complexes in combination with cryo-electron microscopy (EM) have recently allowed the exploration of such complexes under near-native conditions. In particular, polymer-encapsulated nanodiscs enable the study of membrane proteins at high resolution while retaining protein-protein and protein-lipid interactions within a lipid bilayer. However, this powerful technology has not been exploited to address the important question of how endogenous─as opposed to overexpressed─membrane proteins are organized within a lipid environment. In this work, we demonstrate that biochemical enrichment protocols for native membrane-protein complexes from Chaetomium thermophilum in combination with polymer-based lipid-bilayer nanodiscs provide a substantial improvement in the quality of recovered endogenous membrane-protein complexes. Mass spectrometry results revealed ∼1123 proteins, while multiple 2D class averages and two 3D reconstructions from cryo-EM data furnished prominent structural signatures. This integrated methodological approach to enriching endogenous membrane-protein complexes provides unprecedented opportunities for a deeper understanding of eukaryotic membrane proteomes.

An Electrostatically-steered Conformational Selection Mechanism Promotes SARS-CoV-2 Spike Protein Variation.

After two years since the outbreak, the COVID-19 pandemic remains a global public health emergency. SARS-CoV-2 variants with substitutions on the spike (S) protein emerge increasing the risk of immune evasion and cross-species transmission. Here, we analyzed the evolution of the S protein as recorded in 276,712 samples collected before the start of vaccination efforts. Our analysis shows that most variants destabilize the S protein trimer, increase its conformational heterogeneity and improve the odds of the recognition by the host cell receptor. Most frequent substitutions promote overall hydrophobicity by replacing charged amino acids, reducing stabilizing local interactions in the unbound S protein trimer. Moreover, our results identify "forbidden" regions that rarely show any sequence variation, and which are related to conformational changes occurring upon fusion. These results are significant for understanding the structure and function of SARS-CoV-2 related proteins which is a critical step in vaccine development and epidemiological surveillance.

Reconstitution of 3' end processing of mammalian pre-mRNA reveals a central role of RBBP6.

The 3' ends of almost all eukaryotic mRNAs are generated in an essential two-step processing reaction: endonucleolytic cleavage of an extended precursor followed by the addition of a poly(A) tail. By reconstituting the reaction from overproduced and purified proteins, we provide a minimal list of 14 polypeptides that are essential and two that are stimulatory for RNA processing. In a reaction depending on the polyadenylation signal AAUAAA, the reconstituted system cleaves pre-mRNA at a single preferred site corresponding to the one used in vivo. Among the proteins, cleavage factor I stimulates cleavage but is not essential, consistent with its prominent role in alternative polyadenylation. RBBP6 is required, with structural data showing it to contact and presumably activate the endonuclease CPSF73 through its DWNN domain. The C-terminal domain of RNA polymerase II is dispensable. ATP, but not its hydrolysis, supports RNA cleavage by binding to the hClp1 subunit of cleavage factor II with submicromolar affinity.

Cryo-EM and artificial intelligence visualize endogenous protein community members.

Cellular function is underlined by megadalton assemblies organizing in proximity, forming communities. Metabolons are protein communities involving metabolic pathways such as protein, fatty acid, and thioesters of coenzyme-A synthesis. Metabolons are highly heterogeneous due to their function, making their analysis particularly challenging. Here, we simultaneously characterize metabolon-embedded architectures of a 60S pre-ribosome, fatty acid synthase, and pyruvate/oxoglutarate dehydrogenase complex E2 cores de novo. Cryo-electron microscopy (cryo-EM) 3D reconstructions are resolved at 3.84-4.52 Å resolution by collecting <3,000 micrographs of a single cellular fraction. After combining cryo-EM with artificial intelligence-based atomic modeling and de novo sequence identification methods, at this resolution range, polypeptide hydrogen bonding patterns are discernible. Residing molecular components resemble their purified counterparts from other eukaryotes but also exhibit substantial conformational variation with potential functional implications. Our results propose an integrated tool, boosted by machine learning, that opens doors for structural systems biology spearheaded by cryo-EM characterization of native cell extracts.

Cryo-EM snapshots of a native lysate provide structural insights into a metabolon-embedded transacetylase reaction.

Found across all kingdoms of life, 2-keto acid dehydrogenase complexes possess prominent metabolic roles and form major regulatory sites. Although their component structures are known, their higher-order organization is highly heterogeneous, not only across species or tissues but also even within a single cell. Here, we report a cryo-EM structure of the fully active Chaetomium thermophilum pyruvate dehydrogenase complex (PDHc) core scaffold at 3.85 Å resolution (FSC = 0.143) from native cell extracts. By combining cryo-EM with macromolecular docking and molecular dynamics simulations, we resolve all PDHc core scaffold interfaces and dissect the residing transacetylase reaction. Electrostatics attract the lipoyl domain to the transacetylase active site and stabilize the coenzyme A, while apolar interactions position the lipoate in its binding cleft. Our results have direct implications on the structural determinants of the transacetylase reaction and the role of flexible regions in the context of the overall 10 MDa PDHc metabolon architecture.

First 3D-Structural Data of Full-Length Guanylyl Cyclase 1 in Rod-Outer-Segment Preparations of Bovine Retina by Cross-Linking/Mass Spectrometry.

The rod-outer-segment guanylyl cyclase 1 (ROS-GC1) is a key transmembrane protein for retinal phototransduction. Mutations of ROS-GC1 correlate with different retinal diseases that often lead to blindness. No structural data are available for ROS-GC1 so far. We performed a 3D-structural analysis of native ROS-GC1 from bovine retina by cross-linking/mass spectrometry (XL-MS) and computational modeling. Absolute quantification and activity measurements of native ROS-GC1 were performed by MS-based assays directly in bovine retina samples. Our data present the first 3D-structural analysis of active, full-length ROS-GC1 derived from bovine retina. We propose a novel domain organization for the intracellular domain ROS-GC1. Our XL-MS data of native ROS-GC1 from rod-outer-segment preparations of bovine retina agree with a dimeric architecture. Our integrated approach can serve as a blueprint for conducting 3D-structural studies of membrane proteins in their native environment.

Integrative structure of a 10-megadalton eukaryotic pyruvate dehydrogenase complex from native cell extracts.

The pyruvate dehydrogenase complex (PDHc) is a giant enzymatic assembly involved in pyruvate oxidation. PDHc components have been characterized in isolation, but the complex's quaternary structure has remained elusive due to sheer size, heterogeneity, and plasticity. Here, we identify fully assembled Chaetomium thermophilum α-keto acid dehydrogenase complexes in native cell extracts and characterize their domain arrangements utilizing mass spectrometry, activity assays, crosslinking, electron microscopy (EM), and computational modeling. We report the cryo-EM structure of the PDHc core and observe unique features of the previously unknown native state. The asymmetric reconstruction of the 10-MDa PDHc resolves spatial proximity of its components, agrees with stoichiometric data (60 E2p:12 E3BP:∼20 E1p: ≤ 12 E3), and proposes a minimum reaction path among component enzymes. The PDHc shows the presence of a dynamic pyruvate oxidation compartment, organized by core and peripheral protein species. Our data provide a framework for further understanding PDHc and α-keto acid dehydrogenase complex structure and function.

Unstructured regions of large enzymatic complexes control the availability of metabolites with signaling functions.

Metabolites produced via traditional biochemical processes affect intracellular communication, inflammation, and malignancy. Unexpectedly, acetyl-CoA, α-ketoglutarate and palmitic acid, which are chemical species of reactions catalyzed by highly abundant, gigantic enzymatic complexes, dubbed as "metabolons", have broad "nonmetabolic" signaling functions. Conserved unstructured regions within metabolons determine the yield of these metabolites. Unstructured regions tether functional protein domains, act as spatial constraints to confine constituent enzyme communication, and, in the case of acetyl-CoA production, tend to be regulated by intricate phosphorylation patterns. This review presents the multifaceted roles of these three significant metabolites and describes how their perturbation leads to altered or transformed cellular function. Their dedicated enzymatic systems are then introduced, namely, the pyruvate dehydrogenase (PDH) and oxoglutarate dehydrogenase (OGDH) complexes, and the fatty acid synthase (FAS), with a particular focus on their structural characterization and the localization of unstructured regions. Finally, upstream metabolite regulation, in which spatial occupancy of unstructured regions within dedicated metabolons may affect metabolite availability and subsequently alter cell functions, is discussed. Video abstract.

Structural analysis of 70S ribosomes by cross-linking/mass spectrometry reveals conformational plasticity.

The ribosome is not only a highly complex molecular machine that translates the genetic information into proteins, but also an exceptional specimen for testing and optimizing cross-linking/mass spectrometry (XL-MS) workflows. Due to its high abundance, ribosomal proteins are frequently identified in proteome-wide XL-MS studies of cells or cell extracts. Here, we performed in-depth cross-linking of the E. coli ribosome using the amine-reactive cross-linker disuccinimidyl diacetic urea (DSAU). We analyzed 143 E. coli ribosomal structures, mapping a total of 10,771 intramolecular distances for 126 cross-link-pairs and 3,405 intermolecular distances for 97 protein pairs. Remarkably, 44% of intermolecular cross-links covered regions that have not been resolved in any high-resolution E. coli ribosome structure and point to a plasticity of cross-linked regions. We systematically characterized all cross-links and discovered flexible regions, conformational changes, and stoichiometric variations in bound ribosomal proteins, and ultimately remodeled 2,057 residues (15,794 atoms) in total. Our working model explains more than 95% of all cross-links, resulting in an optimized E. coli ribosome structure based on the cross-linking data obtained. Our study might serve as benchmark for conducting biochemical experiments on newly modeled protein regions, guided by XL-MS. Data are available via ProteomeXchange with identifier PXD018935.

2.7 Å cryo-EM structure of vitrified M. musculus H-chain apoferritin from a compact 200 keV cryo-microscope.

Here we present the structure of mouse H-chain apoferritin at 2.7 Å (FSC = 0.143) solved by single particle cryogenic electron microscopy (cryo-EM) using a 200 kV device, the Thermo Fisher Glacios®. This is a compact, two-lens illumination system with a constant power objective lens, without any energy filters or aberration correctors, often thought of as a "screening cryo-microscope". Coulomb potential maps reveal clear densities for main chain carbonyl oxygens, residue side chains (including alternative conformations) and bound solvent molecules. We used a quasi-crystallographic reciprocal space approach to fit model coordinates to the experimental cryo-EM map. We argue that the advantages offered by (a) the high electronic and mechanical stability of the microscope, (b) the high emission stability and low beam energy spread of the high brightness Field Emission Gun (X-FEG), (c) direct electron detection technology and (d) particle-based Contrast Transfer Function (CTF) refinement have contributed to achieving high resolution. Overall, we show that basic electron optical settings for automated cryo-electron microscopy imaging can be used to determine structures approaching atomic resolution.

Reconstitution of mammalian cleavage factor II involved in 3' processing of mRNA precursors.

Cleavage factor II (CF II) is a poorly characterized component of the multiprotein complex catalyzing 3' cleavage and polyadenylation of mammalian mRNA precursors. We have reconstituted CF II as a heterodimer of hPcf11 and hClp1. The heterodimer is active in partially reconstituted cleavage reactions, whereas hClp1 by itself is not. Pcf11 moderately stimulates the RNA 5' kinase activity of hClp1; the kinase activity is dispensable for RNA cleavage. CF II binds RNA with nanomolar affinity. Binding is mediated mostly by the two zinc fingers in the C-terminal region of hPcf11. RNA is bound without pronounced sequence-specificity, but extended G-rich sequences appear to be preferred. We discuss the possibility that CF II contributes to the recognition of cleavage/polyadenylation substrates through interaction with G-rich far-downstream sequence elements.

A Novel Continuous Assay for the Deacylase Sirtuin 5 and Other Deacetylases.

Sirtuins are NAD(+) dependent lysine deacylases involved in many regulatory processes like control of metabolic pathways, DNA repair, and stress response. Modulators of sirtuin activity are needed as tools for uncovering the biological function of these enzymes and as potential therapeutics. Systematic discovery of such modulators is hampered by the lack of efficient and simple continuous activity assays running at low sirtuin concentrations in microtiter plates. Here we describe an improved continuous sirtuin 5 assay based on the coupling of the sirtuin reaction to a proteolytic cleavage using internally fluorescence-quenched substrates. Systematic optimization of a carbamoyl phosphate synthetase 1 derived, glutarylated peptide yielded a Sirt5 substrate with k(cat)/K(M) value of 337,000 M(-1) s(-1), which represents the best sirtuin substrate described so far. These extraordinary substrate properties allowed reliable determination of Ki values for different inhibitors in the presence of only 10 nM sirtuin in microtiter plate format. Assay conditions could be transferred effectively to other lysine deacetylases, like sirtuin 2 and sirtuin 3, which now enables more efficient development of sirtuin targeting drugs.