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The recent revolution in cryo-electron microscopy has made it possible to determine macromolecular structures directly from cell extracts. However, identifying the correct protein from the cryo-EM map is still challenging and often needs additional sequence information from other techniques, such as tandem mass spectrometry and/or bioinformatics. Here, we present DeepTracer-ID, a server-based approach to identify the candidate protein in a user-provided organism de novo from a cryo-EM map, without the need for additional information. Our method first uses DeepTracer to generate a protein backbone model that best represents the cryo-EM map, and this model is then searched against the library of AlphaFold2 predictions for all proteins in the given organism. This method is highly accurate and robust: in all 13 experimental maps tested blindly, DeepTracer-ID identified the correct proteins as the top candidates. Eight of the maps were of known structures, while the other five unpublished maps were validated by prior protein annotation and careful inspection of the model refined into the map. The program also showed promising results for both homomeric and heteromeric protein complexes. This platform is possible because of the recent breakthroughs in large-scale protein 3D structure prediction.Statement of SignificanceWhile it has now become routine for cryo-EM maps of proteins to reach a near-atomic resolution, potentially allowing for reliable atomic models to be built, there are a growing number of instances where the protein identity may not be known. Without knowing the protein sequence, it is impossible to build an atomic model. DeepTracer-ID is a server-based approach to surmount this problem by identifying the proteins in a given organism that are found in the cryo-EM map. A free web service for global academic access is provided.

Summary The self-assembly of designed peptides into filaments and other higher-order structures has been the focus of intense interest because of the potential for creating new biomaterials and biomedical devices. These peptide assemblies have also been used as models for understanding biological processes, such as the pathological formation of amyloid. We investigate the assembly of an octapeptide sequence, Ac-FKFEFKFE-NH2, motivated by prior studies that demonstrated that this amphipathic β strand peptide self-assembled into fibrils and biocompatible hydrogels. Using high-resolution cryoelectron microscopy (cryo-EM), we are able to determine the atomic structure for two different coexisting forms of the fibrils, containing four and five β sandwich protofilaments, respectively. Surprisingly, the inner walls in both forms are parallel β sheets, while the outer walls are antiparallel β sheets. Our results demonstrate the chaotic nature of peptide self-assembly and illustrate the importance of cryo-EM structural analysis to understand the complex phase behavior of these materials at near-atomic resolution.

The exquisite structure-function correlations observed in filamentous protein assemblies provide a paradigm for the design of synthetic peptide-based nanomaterials. However, the plasticity of quaternary structure in sequence-space and the lability of helical symmetry present significant challenges to the de novo design and structural analysis of such filaments. Here, we describe a rational approach to design self-assembling peptide nanotubes based on controlling lateral interactions between protofilaments having an unusual cross-α supramolecular architecture. Near-atomic resolution cryo-EM structural analysis of seven designed nanotubes provides insight into the designability of interfaces within these synthetic peptide assemblies and identifies a non-native structural interaction based on a pair of arginine residues. This arginine clasp motif can robustly mediate cohesive interactions between protofilaments within the cross-α nanotubes. The structure of the resultant assemblies can be controlled through the sequence and length of the peptide subunits, which generates synthetic peptide filaments of similar dimensions to flagella and pili., Peptide-based filamentous assemblies are successfully used for generation of structurally ordered materials, but their de novo design and structural characterization is challenging. Here, the authors provide a strategy for the design of self-assembling peptide nanotubes based on modifications of an arginine clasp interaction motif, and report the cryo-EM structures of seven designed nanotubes.

Lipases are enzymes necessary for the proper distribution and utilization of lipids in the human body. Lipoprotein lipase (LPL) is active in capillaries, where it plays a crucial role in preventing dyslipidemia by hydrolyzing triglycerides from packaged lipoproteins. Thirty years ago, the existence of a condensed and inactive LPL oligomer was proposed. Although recent work has shed light on the structure of the LPL monomer, the inactive oligomer remained opaque. Here we present a cryo-EM reconstruction of a helical LPL oligomer at 3.8-Å resolution. Helix formation is concentration-dependent, and helices are composed of inactive dihedral LPL dimers. Heparin binding stabilizes LPL helices, and the presence of substrate triggers helix disassembly. Superresolution fluorescent microscopy of endogenous LPL revealed that LPL adopts a filament-like distribution in vesicles. Mutation of one of the helical LPL interaction interfaces causes loss of the filament-like distribution. Taken together, this suggests that LPL is condensed into its inactive helical form for storage in intracellular vesicles.

Spinocerebellar ataxia type 5 (SCA5) is a neurodegenerative disease caused by mutations in the cytoskeletal protein β-III-spectrin. Previously, a SCA5 mutation resulting in a leucine-to-proline substitution (L253P) in the actin-binding domain (ABD) was shown to cause a 1000-fold increase in actin-binding affinity. However, the structural basis for this increase is unknown. Here, we report a 6.9 Å cryo-EM structure of F-actin complexed with the L253P ABD. This structure, along with co-sedimentation and pulsed-EPR measurements, demonstrates that high-affinity binding caused by the CH2-localized mutation is due to opening of the two CH domains. This enables CH1 to bind actin aided by an unstructured N-terminal region that becomes α-helical upon binding. This helix is required for association with actin as truncation eliminates binding. Collectively, these results shed light on the mechanism by which β-III-spectrin, and likely similar actin-binding proteins, interact with actin, and how this mechanism can be perturbed to cause disease., The disease causing L253P mutation in the actin-binding domain (ABD) of β-III-spectrin drastically increases actin-binding affinity. Here, the authors present the cryo-EM structure of F-actin complexed with the ABD mutant and double electron–electron resonance measurements show how the mutation affects the ABD conformational state.

Pili on the surface of Sulfolobus islandicus are used for many functions, and serve as receptors for certain archaeal viruses. The cells grow optimally at pH 3 and ~80 °C, exposing these extracellular appendages to a very harsh environment. The pili, when removed from cells, resist digestion by trypsin or pepsin, and survive boiling in sodium dodecyl sulfate or 5 M guanidine hydrochloride. We used electron cryo-microscopy to determine the structure of these filaments at 4.1 A resolution. An atomic model was built by combining the electron density map with bioinformatics without previous knowledge of the pilin sequence—an approach that should prove useful for assemblies where all of the components are not known. The atomic structure of the pilus was unusual, with almost one-third of the residues being either threonine or serine, and with many hydrophobic surface residues. While the map showed extra density consistent with glycosylation for only three residues, mass measurements suggested extensive glycosylation. We propose that this extensive glycosylation renders these filaments soluble and provides the remarkable structural stability. We also show that the overall fold of the archaeal pilin is remarkably similar to that of archaeal flagellin, establishing common evolutionary origins. The electron cryo-microscopy structure of Sulfolobus islandicus pili enabled the identification of SiL_2606 as the main pilin in these filaments and revealed that the pili are glycosylated, which probably explains how these structures remain soluble and stable even when cells grow at pH 3 and 80 °C.

Summary Intracellular protein filaments are ubiquitous for cellular functions, but forming bona fide biomimetic intracellular filaments of small molecules in living cells remains elusive. Here, we report the in situ formation of self-limiting intracellular filaments of a small peptide via enzymatic morphological transition of a phosphorylated and trimethylated heterochiral tetrapeptide. Enzymatic dephosphorylation reduces repulsive intermolecular electrostatic interactions and converts the peptidic nanoparticles into filaments, which exhibit distinct types of cross-β structures with either C7 or C2 symmetries, with the hydrophilic C-terminal residues at the periphery of the helix. Macromolecular crowding promotes the peptide filaments to form bundles, which extend from the plasma membrane to nuclear membrane and hardly interact with endogenous components, including cytoskeletons. Stereochemistry and post-translational modification (PTM) of peptides are critical for generating the intracellular bundles. This work may offer a way to gain lost functions or to provide molecular insights for understanding normal and aberrant intracellular filaments.

Uropathogenic E. coli (UPEC), which cause urinary tract infections (UTI), utilize type 1 pili, a chaperone usher pathway (CUP) pilus, to cause UTI and colonize the gut. The pilus rod, comprised of repeating FimA subunits, provides a structural scaffold for displaying the tip adhesin, FimH. We solved the 4.2 Å resolution structure of the type 1 pilus rod using cryo-electron microscopy. Residues forming the interactive surfaces that determine the mechanical properties of the rod were maintained by selection based on a global alignment of fimA sequences. We identified mutations that did not alter pilus production in vitro but reduced the force required to unwind the rod. UPEC expressing these mutant pili were significantly attenuated in bladder infection and intestinal colonization in mice. This study elucidates an unappreciated functional role for the molecular spring-like property of type 1 pilus rods in host-pathogen interactions and carries important implications for other pilus-mediated diseases., eLife digest Escherichia coli, or E. coli for short, is a type of bacteria commonly found in the guts of people and animals. Certain types of E. coli can cause urinary tract infections (UTIs): they travel from the digestive tract up to the bladder (and sometimes to the kidneys) where they provoke painful symptoms. To cause the infection, the bacteria must become solidly attached to the lining of the bladder; otherwise they will get flushed out whenever urine is expelled. Pili are hair-like structures that cover a bacterium and allow it to attach to surfaces. E. coli has many different types of pili, but one seems particularly important in UTIs: type 1 pili. These pili are formed of subunits that assemble into a long coil-shaped rod, which is tipped by adhesive molecules that can stick to body surfaces. The current hypothesis is that the pili act as shock absorbers: when the bladder empties, the pili’s coil-like structure can unwind into a flexible straight fiber. This would take some of the forces off the adhesive molecules that are attached to the bladder, and help the bacteria to remain in place when urine flows out. However, the exact structure of type 1 pili is still unclear, and the essential role of their coil-like shape unconfirmed. Here, Spaulding, Schreiber, Zheng et al. use a microscopy method called cryo-EM to reveal the structure of the type 1 pili at near atomic-level, and identify the key units necessary for their coiling properties. The experiments show that pili with certain mutations in these units unwind much more easily when the bacteria carrying them are ‘tugged on’ with molecular tweezers. The bacteria with mutant pili are also less able to cause UTIs in mice. The coiling ability of the type 1 pili is therefore essential for E. coli to invade and colonize the bladder. Every year, over 150 million people worldwide experience a UTI; for 25% of women, the infection regularly returns. Antibiotics usually treat the problem but bacteria are becoming resistant to these drugs. New treatments could be designed if scientists understand what roles pili play in the infection mechanisms.

The bacterial flagellar filament has long been studied to understand how a polymer composed of a single protein can switch between different supercoiled states with high cooperativity. Here we present near-atomic resolution cryo-EM structures for flagellar filaments from both Gram-positive Bacillus subtilis and Gram-negative Pseudomonas aeruginosa. Seven mutant flagellar filaments in B. subtilis and two in P. aeruginosa capture two different states of the filament. These reliable atomic models of both states reveal conserved molecular interactions in the interior of the filament among B. subtilis, P. aeruginosa and Salmonella enterica. Using the detailed information about the molecular interactions in two filament states, we successfully predict point mutations that shift the equilibrium between those two states. Further, we observe the dimerization of P. aeruginosa outer domains without any perturbation of the conserved interior of the filament. Our results give new insights into how the flagellin sequence has been “tuned” over evolution., Bacterial flagellar filaments are composed almost entirely of a single protein—flagellin—which can switch between different supercoiled states in a highly cooperative manner. Here the authors present near-atomic resolution cryo-EM structures of nine flagellar filaments, and begin to shed light on the molecular basis of filament switching.

Urinary tract infections (UTIs) are caused by a wide range of pathogens, but the most common causative agent of UTIs is uropathogenic Escherichia coli (UPEC). Virtually all uropathogenic strains of ...

The bacterial flagellar filament has been an important model system in structural biology. Current models are based upon the notion that subunits can switch between two stable states, one forming left-handed 11-start protofilaments and the other forming right-handed ones (Calladine, 1975; Kamiya et al., 1980). Pioneering work on the structure of two flagellin mutants came from the Namba lab, studying the Salmonella filament (Maki-Yonekura et al., 2010; Samatey et al., 2001). We have been using cryo-EM to study the structure of a gram-positive bacterium, Bacillus subtilis. We have now reconstructed five mutants, all at a better resolution than obtained for Salmonella. For one mutant, we have reached ∼ 3.6 A, which allows for a full atomic model to be built with a high-degree of confidence. These results are being combined into a new model for how structural polymorphism is dictated by the switching between two states, only possible with the recent advances allowing for near-atomic resolution reconstructions of these filaments.Calladine, C.R. (1975). Construction of bacterial flagella. Nature 255, 121-124.Kamiya, R., Asakura, S., and Yamaguchi, S. (1980). Formation of helical filaments by copolymerization of two types of ’straight‘ flagellins. Nature 286, 628-630.Maki-Yonekura, S., Yonekura, K., and Namba, K. (2010). Conformational change of flagellin for polymorphic supercoiling of the flagellar filament. NatStructMolBiol 17, 417-422.Samatey, F.A., Imada, K., Nagashima, S., Vonderviszt, F., Kumasaka, T., Yamamoto, M., and Namba, K. (2001). Structure of the bacterial flagellar protofilament and implications for a switch for supercoiling. Nature 410, 331-337.

Yeast tRNA-thiouridine modification protein 1 (Tum1p), a crucial component of the Urm1 system, is believed to play important roles in protein urmylation and tRNA-thiouridine modification. Previous studies have demonstrated that the conserved residue Cys259 in the C-terminal rhodanese-like domain of Tum1p is essential for these sulfur-transfer activities. Here, recombinant Tum1p protein has been cloned and overexpressed in Escherichia coli strain BL21 (DE3). After purification, crystals of Tum1p were obtained by the hanging-drop vapour-diffusion method and diffracted to 1.9 Å resolution. The preliminary X-ray data showed that the tetragonal Tum1p crystal belonged to space group I4(1), with unit-cell parameters a = b = 120.94, c = 48.35 Å. The asymmetric unit of the crystal was assumed to contain one protein molecule, giving a Matthews coefficient of 2.41 Å(3) Da(-1) and a solvent content of 49.0%.