ScanMoment: a web server for combinatorial analysis of basic residues in nucleic acid binding sites

ScanMoment is a webserver designed to identify the presence of the basic faced α‐helix (BFAH) motif in the nucleic acid binding sites of proteins. The program calculates the ’Basic Moment‘, a parameter that quantitizes the distribution of basic residues on the surface of an α‐helix. A sliding window is used to generate a plot displaying regions of the protein sequence that possesses a high Basic Moment and hus likely to possess a BFAH motif. The user may vary the periodicity from that of an alpha‐helix (100°), to those of other secondary structures such as beta sheets and 3₁₀ helices. The program can also plot the periodicity of basic residues in a protein sequence using a Fourier transformation. The procedure has been used to characterize the presence of BFAHs in the N‐terminal extensions of the eukaryotic aminoacyl‐tRNA synthetases and to indicate the presence of a BFAH in the tRNA binding site of alanyl‐tRNA synthetase.     

Enzyme catalysis prior to aromatic residues: Reverse engineering of a dephospho‐CoA kinase

The wide variety of protein structures and functions results from the diverse properties of the 20 canonical amino acids. The generally accepted hypothesis is that early protein evolution was associated with enrichment of a primordial alphabet, thereby enabling increased protein catalytic efficiencies and functional diversification. Aromatic amino acids were likely among the last additions to genetic code. The main objective of this study was to test whether enzyme catalysis can occur without the aromatic residues (aromatics) by studying the structure and function of dephospho‐CoA kinase (DPCK) following aromatic residue depletion. We designed two variants of a putative DPCK from Aquifex aeolicus by substituting (a) Tyr, Phe and Trp or (b) all aromatics (including His). Their structural characterization indicates that substituting the aromatics does not markedly alter their secondary structures but does significantly loosen their side chain packing and increase their sizes. Both variants still possess ATPase activity, although with 150–300 times lower efficiency in comparison with the wild‐type phosphotransferase activity. The transfer of the phosphate group to the dephospho‐CoA substrate becomes heavily uncoupled and only the His‐containing variant is still able to perform the phosphotransferase reaction. These data support the hypothesis that proteins in the early stages of life could support catalytic activities, albeit with low efficiencies. An observed significant contraction upon ligand binding is likely important for appropriate organization of the active site. Formation of firm hydrophobic cores, which enable the assembly of stably structured active sites, is suggested to provide a selective advantage for adding the aromatic residues.     

Membrane‐dependent amyloid aggregation of human BAX α9 (173–192)

Mitochondrial outer membrane permeabilization, which is a critical step in apoptosis, is initiated upon transmembrane insertion of the C‐terminal α‐helix (α9) of the proapoptotic Bcl‐2 family protein BAX. The isolated α9 fragment (residues 173–192) is also competent to disrupt model membranes, and the structures of its membrane‐associated oligomers are of interest in understanding the potential roles of this sequence in apoptosis. Here, we used ultrafast two‐dimensional infrared (2D IR) spectroscopy, thioflavin T binding, and transmission electron microscopy to show that the synthetic BAX α9 peptide (α9p) forms amyloid aggregates in aqueous environments and on the surfaces of anionic small unilamellar vesicles. Its inherent amyloidogenicity was predicted by sequence analysis, and 2D IR spectra reveal that vesicles modulate the β‐sheet structures of insoluble aggregates, motivating further examination of the formation or suppression of BAX amyloids in apoptosis.     

Cryo‐EM structures of perforin‐2 in isolation and assembled on a membrane suggest a mechanism for pore formation

Perforin‐2 (PFN2, MPEG1) is a key pore‐forming protein in mammalian innate immunity restricting intracellular bacteria proliferation. It forms a membrane‐bound pre‐pore complex that converts to a pore‐forming structure upon acidification; but its mechanism of conformational transition has been debated. Here we used cryo‐electron microscopy, tomography and subtomogram averaging to determine structures of PFN2 in pre‐pore and pore conformations in isolation and bound to liposomes. In isolation and upon acidification, the pre‐assembled complete pre‐pore rings convert to pores in both flat ring and twisted conformations. On membranes, in situ assembled PFN2 pre‐pores display various degrees of completeness; whereas PFN2 pores are mainly incomplete arc structures that follow the same subunit packing arrangements as found in isolation. Both assemblies on membranes use their P2 β‐hairpin for binding to the lipid membrane surface. Overall, these structural snapshots suggest a molecular mechanism for PFN2 pre‐pore to pore transition on a targeted membrane, potentially using the twisted pore as an intermediate or alternative state to the flat conformation, with the capacity to cause bilayer distortion during membrane insertion.     

Beyond history and “on a roll”: The list of the most well‐studied human protein structures and overall trends in the protein data bank

Of the roughly 20,000 canonical human protein sequences, as of January 20, 2021, 7,077 proteins have had their full or partial, medium‐ to high‐resolution structures determined by x‐ray crystallography or other methods. Which of these proteins dominate the protein data bank (the PDB) and why? In this paper, we list the 273 top human protein structures based on the number of their PDB entries. This set of proteins accounts for more than 40% of all available human PDB entries and represent past trends as well as current status for protein structural biology. We briefly discuss the relationship which some of the prominent protein structures have with protein research as a whole and mention their relevance to human diseases. The top‐10 soluble and membrane proteins are all well‐known (most of their first structures being deposited more than 30 years ago). Overall, there is no dramatic change in recent trends in the PDB. Remarkably, the number of structure depositions has grown nearly exponentially over the last 10 or more years (with a doubling time of 7 years for proteins, obtained from any organism). Growth in human protein structures is slightly faster (at 5.9 years). The information in this paper may be informative to senior scientists but also inspire researchers who are new to protein science, providing the year 2021 snap‐shot for the state of protein structural biology.     

Three‐dimensional structure of xylonolactonase from Caulobacter crescentus: A mononuclear iron enzyme of the 6‐bladed β‐propeller hydrolase family

Xylonolactonase Cc XylC from Caulobacter crescentus catalyzes the hydrolysis of the intramolecular ester bond of d‐xylonolactone. We have determined crystal structures of Cc XylC in complex with d‐xylonolactone isomer analogues d‐xylopyranose and (r)‐(+)‐4‐hydroxy‐2‐pyrrolidinone at high resolution. Cc XylC has a 6‐bladed β‐propeller architecture, which contains a central open channel having the active site at one end. According to our previous native mass spectrometry studies, Cc XylC is able to specifically bind Fe²⁺. The crystal structures, presented here, revealed an active site bound metal ion with an octahedral binding geometry. The side chains of three amino acid residues, Glu18, Asn146, and Asp196, which participate in binding of metal ion are located in the same plane. The solved complex structures allowed suggesting a reaction mechanism for intramolecular ester bond hydrolysis in which the major contribution for catalysis arises from the carbonyl oxygen coordination of the xylonolactone substrate to the Fe²⁺. The structure of Cc XylC was compared with eight other ester hydrolases of the β‐propeller hydrolase family. The previously published crystal structures of other β‐propeller hydrolases contain either Ca²⁺, Mg²⁺, or Zn²⁺ and show clear similarities in ligand and metal ion binding geometries to that of Cc XylC. It would be interesting to reinvestigate the metal binding specificity of these enzymes and clarify whether they are also able to use Fe²⁺ as a catalytic metal. This could further expand our understanding of utilization of Fe²⁺ not only in oxidative enzymes but also in hydrolases.     

Solution structures of the Shewanella woodyi H‐NOX protein in the presence and absence of soluble guanylyl cyclase stimulator IWP‐051

Heme‐nitric oxide/oxygen binding (H‐NOX) domains bind gaseous ligands for signal transduction in organisms spanning prokaryotic and eukaryotic kingdoms. In the bioluminescent marine bacterium Shewanella woodyi (Sw), H‐NOX proteins regulate quorum sensing and biofilm formation. In higher animals, soluble guanylyl cyclase (sGC) binds nitric oxide with an H‐NOX domain to induce cyclase activity and regulate vascular tone, wound healing and memory formation. sGC also binds stimulator compounds targeting cardiovascular disease. The molecular details of stimulator binding to sGC remain obscure but involve a binding pocket near an interface between H‐NOX and coiled‐coil domains. Here, we report the full NMR structure for CO‐ligated Sw H‐NOX in the presence and absence of stimulator compound IWP‐051, and its backbone dynamics. Nonplanar heme geometry was retained using a semi‐empirical quantum potential energy approach. Although IWP‐051 binding is weak, a single binding conformation was found at the interface of the two H‐NOX subdomains, near but not overlapping with sites identified in sGC. Binding leads to rotation of the subdomains and closure of the binding pocket. Backbone dynamics are similar across both domains except for two helix‐connecting loops, which display increased dynamics that are further enhanced by compound binding. Structure‐based sequence analyses indicate high sequence diversity in the binding pocket, but the pocket itself appears conserved among H‐NOX proteins. The largest dynamical loop lies at the interface between Sw H‐NOX and its binding partner as well as in the interface with the coiled coil in sGC, suggesting a critical role for the loop in signal transduction.     

Tertiary motifs as building blocks for the design of protein‐binding peptides

Despite advances in protein engineering, the de novo design of small proteins or peptides that bind to a desired target remains a difficult task. Most computational methods search for binder structures in a library of candidate scaffolds, which can lead to designs with poor target complementarity and low success rates. Instead of choosing from pre‐defined scaffolds, we propose that custom peptide structures can be constructed to complement a target surface. Our method mines tertiary motifs (TERMs) from known structures to identify surface‐complementing fragments or “seeds.” We combine seeds that satisfy geometric overlap criteria to generate peptide backbones and score the backbones to identify the most likely binding structures. We found that TERM‐based seeds can describe known binding structures with high resolution: the vast majority of peptide binders from 486 peptide‐protein complexes can be covered by seeds generated from single‐chain structures. Furthermore, we demonstrate that known peptide structures can be reconstructed with high accuracy from peptide‐covering seeds. As a proof of concept, we used our method to design 100 peptide binders of TRAF6, seven of which were predicted by Rosetta to form higher‐quality interfaces than a native binder. The designed peptides interact with distinct sites on TRAF6, including the native peptide‐binding site. These results demonstrate that known peptide‐binding structures can be constructed from TERMs in single‐chain structures and suggest that TERM information can be applied to efficiently design novel target‐complementing binders.     

Crystal structure and molecular dynamics of human POLDIP2, a multifaceted adaptor protein in metabolism and genome stability

Polymerase δ‐interacting protein 2 (POLDIP2, PDIP38) is a multifaceted, “moonlighting” protein, involved in binding protein partners from many different cellular processes, including mitochondrial metabolism and DNA replication and repair. How POLDIP2 interacts with many different proteins is unknown. Towards this goal, we present the crystal structure of POLDIP2 to 2.8 Å, which exhibited a compact two‐domain β‐strand‐rich globular structure, confirmed by circular dichroism and small angle X‐ray scattering approaches. POLDIP2 comprised canonical DUF525 and YccV domains, but with a conserved domain linker packed tightly, resulting in an “extended” YccV module. A central channel was observed, which we hypothesize could influence structural changes potentially mediated by redox conditions, following observation of a modified cysteine residue in the channel. Unstructured regions were rebuilt by ab initio modelling to generate a model of full‐length POLDIP2. Molecular dynamics simulations revealed a highly dynamic N‐terminal region tethered to the YccV‐domain by an extended linker, potentially facilitating interactions with distal binding partners. Models of POLDIP2 complexed with two of its partners, PrimPol and PCNA, indicated that dynamic flexibility of the POLDIP2 N‐terminus and loop regions likely mediate protein interactions.     

BCCIP is required for nucleolar recruitment of eIF6 and 12S pre-rRNA production during 60S ribosome biogenesis

Ribosome biogenesis is a fundamental process required for cell proliferation. Although evolutionally conserved, the mammalian ribosome assembly system is more complex than in yeasts. BCCIP was originally identified as a BRCA2 and p21 interacting protein. A partial loss of BCCIP function was sufficient to trigger genomic instability and tumorigenesis. However, a complete deletion of BCCIP arrested cell growth and was lethal in mice. Here, we report that a fraction of mammalian BCCIP localizes in the nucleolus and regulates 60S ribosome biogenesis. Both abrogation of BCCIP nucleolar localization and impaired BCCIP–eIF6 interaction can compromise eIF6 recruitment to the nucleolus and 60S ribosome biogenesis. BCCIP is vital for a pre-rRNA processing step that produces 12S pre-rRNA, a precursor to the 5.8S rRNA. However, a heterozygous Bccip loss was insufficient to impair 60S biogenesis in mouse embryo fibroblasts, but a profound reduction of BCCIP was required to abrogate its function in 60S biogenesis. These results suggest that BCCIP is a critical factor for mammalian pre-rRNA processing and 60S generation and offer an explanation as to why a subtle dysfunction of BCCIP can be tumorigenic but a complete depletion of BCCIP is lethal.     

CTD of SARS‐CoV‐2 N protein is a cryptic domain for binding ATP and nucleic acid that interplay in modulating phase separation

SARS‐CoV‐2 nucleocapsid (N) protein plays essential roles in many steps of the viral life cycle, thus representing a key drug target. N protein contains the folded N‐/C‐terminal domains (NTD/CTD) and three intrinsically disordered regions, while its functions including liquid–liquid phase separation (LLPS) depend on the capacity in binding various viral/host‐cell RNA/DNA of diverse sequences. Previously NTD was established to bind various RNA/DNA while CTD to dimerize/oligomerize for forming high‐order structures. By NMR, here for the first time we decrypt that CTD is not only capable of binding S2m, a specific probe derived from SARS‐CoV‐2 gRNA but with the affinity even higher than that of NTD. Very unexpectedly, ATP, the universal energy currency for all living cells with high cellular concentrations (2–16 mM), specifically binds CTD with Kd of 1.49 ± 0.28 mM. Strikingly, the ATP‐binding residues of NTD/CTD are identical in the SARS‐CoV‐2 variants while ATP and S2m interplay in binding NTD/CTD, as well as in modulating LLPS critical for the viral life cycle. Results together not only define CTD as a novel binding domain for ATP and nucleic acid, but enforce our previous proposal that ATP has been evolutionarily exploited by SARS‐CoV‐2 to complete its life cycle in the host cell. Most importantly, the unique ATP‐binding pockets on NTD/CTD may offer promising targets for design of specific anti‐SARS‐CoV‐2 molecules to fight the pandemic. Fundamentally, ATP emerges to act at mM as a cellular factor to control the interface between the host cell and virus lacking the ability to generate ATP.     

Architecture of the active post‐translational Sec translocon

In eukaryotes, most secretory and membrane proteins are targeted by an N‐terminal signal sequence to the endoplasmic reticulum, where the trimeric Sec61 complex serves as protein‐conducting channel (PCC). In the post‐translational mode, fully synthesized proteins are recognized by a specialized channel additionally containing the Sec62, Sec63, Sec71, and Sec72 subunits. Recent structures of this Sec complex in the idle state revealed the overall architecture in a pre‐opened state. Here, we present a cryo‐EM structure of the yeast Sec complex bound to a substrate, and a crystal structure of the Sec62 cytosolic domain. The signal sequence is inserted into the lateral gate of Sec61α similar to previous structures, yet, with the gate adopting an even more open conformation. The signal sequence is flanked by two Sec62 transmembrane helices, the cytoplasmic N‐terminal domain of Sec62 is more rigidly positioned, and the plug domain is relocated. We crystallized the Sec62 domain and mapped its interaction with the C‐terminus of Sec63. Together, we obtained a near‐complete and integrated model of the active Sec complex.     

Adenosine triphosphate energy‐independently controls protein homeostasis with unique structure and diverse mechanisms

Proteins function in the crowded cellular environments with high salt concentrations, thus facing tremendous challenges of misfolding/aggregation which represents a pathological hallmark of aging and an increasing spectrum of human diseases. Recently, intrinsically disordered regions (IDRs) were recognized to drive liquid–liquid phase separation (LLPS), a common principle for organizing cellular membraneless organelles (MLOs). ATP, the universal energy currency for all living cells, mysteriously has concentrations of 2–12 mM, much higher than required for its previously‐known functions. Only recently, ATP was decoded to behave as a biological hydrotrope to inhibit protein LLPS and aggregation at mM. We further revealed that ATP also acts as a bivalent binder, which not only biphasically modulates LLPS driven by IDRs of human and viral proteins, but also bind to the conserved nucleic‐acid‐binding surfaces of the folded proteins. Most unexpectedly, ATP appears to act as a hydration mediator to antagonize the crowding‐induced destabilization as well as to enhance folding of proteins without significant binding. Here, this review focuses on summarizing the results of these biophysical studies and discussing their implications in an evolutionary context. By linking triphosphate with unique hydration property to adenosine, ATP appears to couple the ability for establishing hydrophobic, π‐π, π‐cation and electrostatic interactions to the capacity in mediating hydration of proteins, which is at the heart of folding, dynamics, stability, phase separation and aggregation. Consequently, ATP acquired a category of functions at ~mM to energy‐independently control protein homeostasis with diverse mechanisms, thus implying a link between cellular ATP concentrations and protein‐aggregation diseases.     

Cryo‐EM structures of pentameric autoinducer‐2 exporter from Escherichia coli reveal its transport mechanism

Bacteria utilize small extracellular molecules to communicate in order to collectively coordinate their behaviors in response to the population density. Autoinducer‐2 (AI‐2), a universal molecule for both intra‐ and inter‐species communication, is involved in the regulation of biofilm formation, virulence, motility, chemotaxis, and antibiotic resistance. While many studies have been devoted to understanding the biosynthesis and sensing of AI‐2, very little information is available on its export. The protein TqsA from Escherichia coli, which belongs to the AI‐2 exporter superfamily, has been shown to export AI‐2. Here, we report the cryogenic electron microscopic structures of two AI‐2 exporters (TqsA and YdiK) from E. coli at 3.35 Å and 2.80 Å resolutions, respectively. Our structures suggest that the AI‐2 exporter exists as a homo‐pentameric complex. In silico molecular docking and native mass spectrometry experiments were employed to demonstrate the interaction between AI‐2 and TqsA, and the results highlight the functional importance of two helical hairpins in substrate binding. We propose that each monomer works as an independent functional unit utilizing an elevator‐type transport mechanism.     

NMR hawk‐eyed view of AlphaFold2 structures

The prediction of the three‐dimensional (3D) structure of proteins from the amino acid sequence made a stunning breakthrough reaching atomic accuracy. Using the neural network‐based method AlphaFold2, 3D structures of almost the entire human proteome have been predicted and made available (https://www.alphafold.ebi.ac.uk). To gain insight into how well AlphaFold2 structures represent the conformation of proteins in solution, I here compare the AlphaFold2 structures of selected small proteins with their 3D structures that were determined by nuclear magnetic resonance (NMR) spectroscopy. Proteins were selected for which the 3D solution structures were determined on the basis of a very large number of distance restraints and residual dipolar couplings and are thus some of the best‐resolved solution structures of proteins to date. The quality of the backbone conformation of the AlphaFold2 structures is assessed by fitting a large set of experimental residual dipolar couplings (RDCs). The analysis shows that experimental RDCs fit extremely well to the AlphaFold2 structures predicted for GB3, DinI, and ubiquitin. In the case of GB3, the accuracy of the AlphaFold2 structure even surpasses that of a 1.1 Å crystal structure. Fitting of experimental RDCs furthermore allows identification of AlphaFold2 structures that are best representative of the protein''s conformation in solution as seen for the EF hands of the N‐terminal domain of Ca²⁺‐ligated calmodulin. Taken together, the analysis shows that structures predicted by AlphaFold2 can be highly representative of the solution conformation of proteins. The combination of AlphaFold2 structures with RDCs promises to be a powerful approach to study structural changes in proteins.     

The free energy folding penalty accompanying binding of intrinsically disordered α‐helical motifs

Intrinsically disordered proteins (IDPs) are abundant in eukaryotic proteomes and preform critical roles in many cellular processes, most often through the association with globular proteins. Despite lacking a stable three‐dimensional structure by themselves, they may acquire a defined conformation upon binding globular targets. The most common type of secondary structure acquired by these binding motifs entails formation of an α‐helix. It has been hypothesized that such disorder‐to‐order transitions are associated with a significant free energy penalty due to IDP folding, which reduces the overall IDP‐target affinity. However, the exact magnitude of IDP folding penalty in α‐helical binding motifs has not been systematically estimated. Here, we report the folding penalty contributions for 30 IDPs undergoing folding‐upon‐binding and find that the average IDP folding penalty is +2.0 kcal/mol and ranges from 0.7 to 3.5 kcal/mol. We observe that the folding penalty scales approximately linearly with the change in IDP helicity upon binding, which provides a simple empirical way to estimate folding penalty. We analyze to what extent do pre‐structuring and target‐bound IDP dynamics (fuzziness) reduce the folding penalty and find that these effects combined, on average, reduce the folding cost by around half. Taken together, the presented analysis provides a quantitative basis for understanding the role of folding penalty in IDP‐target interactions and introduces a method estimate this quantity. Estimation and reduction of IDP folding penalty may prove useful in the rational design of helix‐stabilized inhibitors of IDP‐target interactions.StatementThe α‐helical binding motifs are ubiquitous among the intrinsically disordered proteins (IDPs). Upon binding their targets, they undergo a disorder‐to‐order transition, which is accompanied by a significant folding penalty whose magnitude is generally not known. Here, we use recently developed statistical‐thermodynamic model to estimate the folding penalties for 30 IDPs and clarify the roles of IDP pre‐folding and bound‐state dynamics in reducing the folding penalty.     

Salinomycin biosynthesis reversely regulates the β‐oxidation pathway in Streptomyces albus by carrying a 3‐hydroxyacyl‐CoA dehydrogenase gene in its biosynthetic gene cluster

Streptomyces is well known for synthesis of many biologically active secondary metabolites, such as polyketides and non‐ribosomal peptides. Understanding the coupling mechanisms of primary and secondary metabolism can help develop strategies to improve secondary metabolite production in Streptomyces. In this work, Streptomyces albus ZD11, an oil‐preferring industrial Streptomyces strain, was proved to have a remarkable capability to generate abundant acyl‐CoA precursors for salinomycin biosynthesis with the aid of its enhanced β‐oxidation pathway. It was found that the salinomycin biosynthetic gene cluster contains a predicted 3‐hydroxyacyl‐CoA dehydrogenase (FadB3), which is the third enzyme of β‐oxidation cycle. Deletion of fadB3 significantly reduced the production of salinomycin. A variety of experimental evidences showed that FadB3 was mainly involved in the β‐oxidation pathway rather than ethylmalonyl‐CoA biosynthesis and played a very important role in regulating the rate of β‐oxidation in S. albus ZD11. Our findings elucidate an interesting coupling mechanism by which a PKS biosynthetic gene cluster could regulate the β‐oxidation pathway by carrying β‐oxidation genes, enabling Streptomyces to efficiently synthesize target polyketides and economically utilize environmental nutrients.

Comprehensive understanding the relationship between primary metabolism and secondary metabolism is important for high‐efficiency production of antibiotics in Streptomyces species. In contrast to the well‐known pattern that primary metabolic processes regulate secondary metabolism, this study reports that a secondary biosynthetic pathway can reversely regulate primary metabolic process by carrying a key gene of the primary metabolic pathway.     

The leucine zipper EF‐hand containing transmembrane protein‐1 EF‐hand is a tripartite calcium,temperature, and pH sensor

Leucine Zipper EF‐hand containing transmembrane protein‐1 (LETM1) is an inner mitochondrial membrane protein that mediates mitochondrial calcium (Ca²⁺)/proton exchange. The matrix residing carboxyl (C)‐terminal domain contains a sequence identifiable EF‐hand motif (EF1) that is highly conserved among orthologues. Deletion of EF1 abrogates LETM1 mediated mitochondrial Ca²⁺ flux, highlighting the requirement of EF1 for LETM1 function. To understand the mechanistic role of this EF‐hand in LETM1 function, we characterized the biophysical properties of EF1 in isolation. Our data show that EF1 exhibits α‐helical secondary structure that is augmented in the presence of Ca²⁺. Unexpectedly, EF1 features a weak (~mM), but specific, apparent Ca²⁺‐binding affinity, consistent with the canonical Ca²⁺ coordination geometry, suggested by our solution NMR. The low affinity is, at least in part, due to an Asp at position 12 of the binding loop, where mutation to Glu increases the affinity by ~4‐fold. Further, the binding affinity is sensitive to pH changes within the physiological range experienced by mitochondria. Remarkably, EF1 unfolds at high and low temperatures. Despite these unique EF‐hand properties, Ca²⁺ binding increases the exposure of hydrophobic regions, typical of EF‐hands; however, this Ca²⁺‐induced conformational change shifts EF1 from a monomer to higher order oligomers. Finally, we showed that a second, putative EF‐hand within LETM1 is unreactive to Ca²⁺ either in isolation or tandem with EF1. Collectively, our data reveal that EF1 is structurally and biophysically responsive to pH, Ca²⁺ and temperature, suggesting a role as a multipartite environmental sensor within LETM1.     

Improving integrative 3D modeling into low‐ to medium‐resolution electron microscopy structures with evolutionary couplings

Electron microscopy (EM) continues to provide near‐atomic resolution structures for well‐behaved proteins and protein complexes. Unfortunately, structures of some complexes are limited to low‐ to medium‐resolution due to biochemical or conformational heterogeneity. Thus, the application of unbiased systematic methods for fitting individual structures into EM maps is important. A method that employs co‐evolutionary information obtained solely from sequence data could prove invaluable for quick, confident localization of subunits within these structures. Here, we incorporate the co‐evolution of intermolecular amino acids as a new type of distance restraint in the integrative modeling platform in order to build three‐dimensional models of atomic structures into EM maps ranging from 10–14 Å in resolution. We validate this method using four complexes of known structure, where we highlight the conservation of intermolecular couplings despite dynamic conformational changes using the BAM complex. Finally, we use this method to assemble the subunits of the bacterial holo‐translocon into a model that agrees with previous biochemical data. The use of evolutionary couplings in integrative modeling improves systematic, unbiased fitting of atomic models into medium‐ to low‐resolution EM maps, providing additional information to integrative models lacking in spatial data.