ABSTRACT
Developments in direct electron detector technology have played a pivotal role in enabling high-resolution structural studies by cryo-EM at 200 and 300 keV. Yet, theory and recent experiments indicate advantages to imaging at 100 keV, energies for which the current detectors have not been optimized. In this study, we evaluated the Gatan Alpine detector, designed for operation at 100 and 200 keV. Compared to the Gatan K3, Alpine demonstrated a significant DQE improvement at these energies, specifically a â¼ 4-fold improvement at Nyquist at 100 keV. In single-particle cryo-EM experiments, Alpine datasets yielded better than 2 Å resolution reconstructions of apoferritin at 120 and 200 keV on a ThermoFisher Scientific (TFS) Glacios microscope fitted with a non-standard SP-Twin lens. We also achieved a â¼ 3.2 Å resolution reconstruction of a 115 kDa asymmetric protein complex, proving Alpine's effectiveness with complex biological samples. In-depth analysis revealed that Alpine reconstructions are comparable to K3 reconstructions at 200 keV, and remarkably, reconstruction from Alpine at 120 keV on a TFS Glacios surpassed all but the 300 keV data from a TFS Titan Krios with GIF/K3. Additionally, we show Alpine's capability for high-resolution data acquisition and screening on lower-end systems by obtaining â¼ 3 Å resolution reconstructions of apoferritin and aldolase at 100 keV and detailed 2D averages of a 55 kDa sample using a side-entry cryo holder. Overall, we show that Gatan Alpine performs well with the standard 200 keV imaging systems and may potentially capture the benefits of lower accelerating voltages, bringing smaller sized particles within the scope of cryo-EM.
ABSTRACT
The Rosetta software for macromolecular modeling, docking and design is extensively used in laboratories worldwide. During two decades of development by a community of laboratories at more than 60 institutions, Rosetta has been continuously refactored and extended. Its advantages are its performance and interoperability between broad modeling capabilities. Here we review tools developed in the last 5 years, including over 80 methods. We discuss improvements to the score function, user interfaces and usability. Rosetta is available at http://www.rosettacommons.org.
Subject(s)
Macromolecular Substances/chemistry , Models, Molecular , Proteins/chemistry , Software , Molecular Docking Simulation , Peptidomimetics/chemistry , Protein ConformationABSTRACT
Critical molecular events that control conformational transitions in most allosteric proteins are ill-defined. The mannose-specific FimH protein of Escherichia coli is a prototypic bacterial adhesin that switches from an 'inactive' low-affinity state (LAS) to an 'active' high-affinity state (HAS) conformation allosterically upon mannose binding and mediates shear-dependent catch bond adhesion. Here we identify a novel type of antibody that acts as a kinetic trap and prevents the transition between conformations in both directions. Disruption of the allosteric transitions significantly slows FimH's ability to associate with mannose and blocks bacterial adhesion under dynamic conditions. FimH residues critical for antibody binding form a compact epitope that is located away from the mannose-binding pocket and is structurally conserved in both states. A larger antibody-FimH contact area is identified by NMR and contains residues Leu-34 and Val-35 that move between core-buried and surface-exposed orientations in opposing directions during the transition. Replacement of Leu-34 with a charged glutamic acid stabilizes FimH in the LAS conformation and replacement of Val-35 with glutamic acid traps FimH in the HAS conformation. The antibody is unable to trap the conformations if Leu-34 and Val-35 are replaced with a less bulky alanine. We propose that these residues act as molecular toggle switches and that the bound antibody imposes a steric block to their reorientation in either direction, thereby restricting concerted repacking of side chains that must occur to enable the conformational transition. Residues homologous to the FimH toggle switches are highly conserved across a diverse family of fimbrial adhesins. Replacement of predicted switch residues reveals that another E. coli adhesin, galactose-specific FmlH, is allosteric and can shift from an inactive to an active state. Our study shows that allosteric transitions in bacterial adhesins depend on toggle switch residues and that an antibody that blocks the switch effectively disables adhesive protein function.
Subject(s)
Adhesins, Bacterial/metabolism , Bacterial Adhesion/physiology , Fimbriae Proteins/metabolism , Fimbriae, Bacterial/metabolism , Adhesins, Escherichia coli/metabolism , Escherichia coli/metabolism , Models, Molecular , Protein BindingABSTRACT
To create new enzymes and biosensors from scratch, precise control over the structure of small-molecule binding sites is of paramount importance, but systematically designing arbitrary protein pocket shapes and sizes remains an outstanding challenge. Using the NTF2-like structural superfamily as a model system, we developed an enumerative algorithm for creating a virtually unlimited number of de novo proteins supporting diverse pocket structures. The enumerative algorithm was tested and refined through feedback from two rounds of large-scale experimental testing, involving in total the assembly of synthetic genes encoding 7,896 designs and assessment of their stability on yeast cell surface, detailed biophysical characterization of 64 designs, and crystal structures of 5 designs. The refined algorithm generates proteins that remain folded at high temperatures and exhibit more pocket diversity than naturally occurring NTF2-like proteins. We expect this approach to transform the design of small-molecule sensors and enzymes by enabling the creation of binding and active site geometries much more optimal for specific design challenges than is accessible by repurposing the limited number of naturally occurring NTF2-like proteins.
Subject(s)
Nucleocytoplasmic Transport Proteins/chemistry , Algorithms , Binding Sites , Computer Simulation , High-Throughput Screening Assays , Models, Molecular , Protein Conformation , Protein Engineering , Protein StabilityABSTRACT
Developments in direct electron detector technology have played a pivotal role in enabling high-resolution structural studies by cryo-EM at 200 and 300 keV. Yet, theory and recent experiments indicate advantages to imaging at 100 keV, energies for which the current detectors have not been optimized. In this study, we evaluated the Gatan Alpine detector, designed for operation at 100 and 200 keV. Compared to the Gatan K3, Alpine demonstrated a significant DQE improvement at these voltages, specifically a ~4-fold improvement at Nyquist at 100 keV. In single-particle cryo-EM experiments, Alpine datasets yielded better than 2 Å resolution reconstructions of apoferritin at 120 and 200 keV on a ThermoFisher Scientific (TFS) Glacios microscope. We also achieved a ~3.2 Å resolution reconstruction for a 115 kDa asymmetric protein complex, proving its effectiveness with complex biological samples. In-depth analysis revealed that Alpine reconstructions are comparable to K3 reconstructions at 200 keV, and remarkably, reconstruction from Alpine at 120 keV on a TFS Glacios surpassed all but the 300 keV data from a TFS Titan Krios with GIF/K3. Additionally, we show Alpine's capability for high-resolution data acquisition and screening on lower-end systems by obtaining ~3 Å resolution reconstructions of apoferritin and aldolase at 100 keV and detailed 2D averages of a 55 kDa sample using a side-entry cryo holder. Overall, we show that Gatan Alpine performs well with the standard 200 keV imaging systems and may potentially capture the benefits of lower accelerating voltages, possibly bringing smaller sized particles within the scope of cryo-EM.
ABSTRACT
Transthyretin (TTR) is a natively tetrameric thyroxine transporter found in blood and cerebrospinal fluid whose misfolding and aggregation causes transthyretin amyloidosis. A rational drug design campaign identified the small molecule tafamidis (Vyndaqel/Vyndamax) as an effective stabilizer of the native TTR fold, and this aggregation inhibitor is regulatory agency-approved for the treatment of TTR amyloidosis. Despite 50 years of structural studies on TTR and this triumph of structure-based drug design, there remains a notable dearth of structural information available to understand ligand binding allostery and amyloidogenic TTR unfolding intermediates. We used single-particle cryo-electron microscopy (cryo-EM) to investigate the conformational landscape of this 55 kiloDalton tetramer in the absence and presence of one or two ligands, revealing inherent asymmetries in the tetrameric architecture and previously unobserved conformational states. These findings provide critical mechanistic insights into negatively cooperative ligand binding and the structural pathways responsible for TTR amyloidogenesis. This study underscores the capacity of cryo-EM to provide new insights into protein structures that have been historically considered too small to visualize and to identify pharmacological targets suppressed by the confines of the crystal lattice, opening uncharted territory in structure-based drug design.
ABSTRACT
Cryogenic electron microscopy (cryoEM) has emerged as a powerful technique for probing the atomic structure of macromolecular complexes. Sample preparation for cryoEM requires preserving specimens in a thin layer of vitreous ice, typically suspended within the holes of a fenestrated support film. However, all commonly used sample preparation approaches for cryoEM studies expose the specimen to the air-water interface, introducing a strong hydrophobic effect on the specimen that often results in denaturation, aggregation, and complex dissociation. Further, preferred hydrophobic interactions between regions of the specimen and the air-water interface impact the orientations adopted by the macromolecules, resulting in 3D reconstructions with anisotropic directional resolution. Adsorption of cryoEM specimens to a monolayer of graphene has been shown to help mitigate interactions with the air-water interface while minimizing the introduction of background noise. Graphene supports also offer the benefit of substantially lowering the required concentration of proteins required for cryoEM imaging. Despite the advantages of these supports, graphene-coated grids are not widely used by the cryoEM community due to the prohibitive expense of commercial options and the challenges associated with large-scale in-house production. This paper describes an efficient method for preparing batches of cryoEM grids that have nearly full coverage of monolayer graphene.
Subject(s)
Graphite , Cryoelectron Microscopy , Adsorption , Anisotropy , WaterABSTRACT
Buffer-composition and sample-preparation guidelines for cryo-electron microscopy are geared towards maximizing imaging contrast and reducing electron-beam-induced motion. These pursuits often involve the minimization or the complete removal of additives that are commonly used to facilitate proper protein folding and minimize aggregation. Among these admonished additives is glycerol, a widely used osmolyte that aids protein stability. In this work, it is shown that the inclusion of glycerol does not preclude high-resolution structure determination by cryoEM, as demonstrated by an â¼2.3â Å resolution reconstruction of mouse apoferritin (â¼500â kDa) and an â¼3.3â Å resolution reconstruction of rabbit muscle aldolase (â¼160â kDa) in the presence of 20%(v/v) glycerol. While it was found that generating thin ice that is amenable to high-resolution imaging requires long blot times, the addition of glycerol did not result in increased beam-induced motion or an inability to pick particles. Overall, these findings indicate that glycerol should not be discounted as a cryoEM sample-buffer additive, particularly for large, fragile complexes that are prone to disassembly or aggregation upon its removal.
Subject(s)
Cryoelectron Microscopy/methods , Glycerol/chemistry , Animals , Apoferritins/chemistry , Buffers , Fructose-Bisphosphate Aldolase/chemistry , Mice , Models, Molecular , Protein Folding , RabbitsABSTRACT
While native scaffolds offer a large diversity of shapes and topologies for enzyme engineering, their often unpredictable behavior in response to sequence modification makes de novo generated scaffolds an exciting alternative. Here we explore the customization of the backbone and sequence of a de novo designed eight stranded ß-barrel protein to create catalysts for a retro-aldolase model reaction. We show that active and specific catalysts can be designed in this fold and use directed evolution to further optimize activity and stereoselectivity. Our results support previous suggestions that different folds have different inherent amenability to evolution and this property could account, in part, for the distribution of natural enzymes among different folds.
Subject(s)
Protein Engineering , Proteins , Proteins/genetics , Protein Engineering/methodsABSTRACT
Small heat shock proteins (sHSPs) are nature's 'first responders' to cellular stress, interacting with affected proteins to prevent their aggregation. Little is known about sHSP structure beyond its structured α-crystallin domain (ACD), which is flanked by disordered regions. In the human sHSP HSPB1, the disordered N-terminal region (NTR) represents nearly 50% of the sequence. Here, we present a hybrid approach involving NMR, hydrogen-deuterium exchange mass spectrometry, and modeling to provide the first residue-level characterization of the NTR. The results support a model in which multiple grooves on the ACD interact with specific NTR regions, creating an ensemble of 'quasi-ordered' NTR states that can give rise to the known heterogeneity and plasticity of HSPB1. Phosphorylation-dependent interactions inform a mechanism by which HSPB1 is activated under stress conditions. Additionally, we examine the effects of disease-associated NTR mutations on HSPB1 structure and dynamics, leveraging our emerging structural insights.
Subject(s)
Heat-Shock Proteins, Small/genetics , Heat-Shock Proteins/genetics , Molecular Chaperones/genetics , Protein Aggregates/genetics , Protein Interaction Domains and Motifs/genetics , Amino Acid Sequence/genetics , Humans , Mass Spectrometry , Models, Molecular , Protein Conformation , Protein Multimerization/genetics , Scattering, Small AngleABSTRACT
Active sites and ligand-binding cavities in native proteins are often formed by curved ß sheets, and the ability to control ß-sheet curvature would allow design of binding proteins with cavities customized to specific ligands. Toward this end, we investigated the mechanisms controlling ß-sheet curvature by studying the geometry of ß sheets in naturally occurring protein structures and folding simulations. The principles emerging from this analysis were used to design, de novo, a series of proteins with curved ß sheets topped with α helices. Nuclear magnetic resonance and crystal structures of the designs closely match the computational models, showing that ß-sheet curvature can be controlled with atomic-level accuracy. Our approach enables the design of proteins with cavities and provides a route to custom design ligand-binding and catalytic sites.
Subject(s)
Protein Conformation, beta-Strand , Protein Engineering/methods , Catalytic Domain , Crystallography, X-Ray , Ligands , Nuclear Magnetic Resonance, Biomolecular , Protein Binding , Protein FoldingABSTRACT
The diversity and flexibility of life offers a wide variety of molecules and systems useful for biosensing. A biosensor device should be robust, specific and reliable. Inorganic arsenic is a highly toxic water contaminant with worldwide distribution that poses a threat to public health. With the goal of developing an arsenic biosensor, we designed an incoherent feed-forward loop (I-FFL) genetic circuit to correlate its output pulse with the input signal in a relatively time-independent manner. The system was conceived exclusively based on the available BioBricks in the iGEM Registry of Standard Biological Parts. The expected behavior in silico was achieved; upon arsenic addition, the system generates a short-delayed reporter protein pulse that is dose dependent to the contaminant levels. This work is an example of the power and variety of the iGEM Registry of Standard Biological Parts, which can be reused in different sophisticated system designs like I-FFLs. Besides the scientific results, one of the main impacts of this synthetic biology project is the influence it had on team's members training and career choices which are summarized at the end of this article.
ABSTRACT
Design of polar interactions is a current challenge for protein design. The de novo designed protein Top7, like almost all designed proteins, has an entirely nonpolar core. Here we describe the replacing of a sizable fraction (5 residues) of this core with a designed polar hydrogen bond network. The polar core design is expressed at high levels in E. coli, has a folding free energy of 10 kcal/mol, and retains the multiphasic folding kinetics of the original Top7. The NMR structure of the design shows that conformations of three of the five residues, and the designed hydrogen bonds between them, are very close to those in the design model. The remaining two residues, which are more solvent exposed, sample a wide range of conformations in the NMR ensemble. These results show that hydrogen bond networks can be designed in protein cores, but also highlight challenges that need to be overcome when there is competition with solvent.