We describe a general approach to designing two-dimensional (2D) protein arrays mediated by noncovalent protein-protein interfaces. Protein homo-oligomers are placed into one of the seventeen 2D ...layer groups, the degrees of freedom of the lattice are sampled to identify configurations with shape-complementary interacting surfaces, and the interaction energy is minimized using sequence design calculations. We used the method to design proteins that self-assemble into layer groups P 3 2 1, P 4 21 2, and P 6. Projection maps of micrometer-scale arrays, assembled both in vitro and in vivo, are consistent with the design models and display the target layer group symmetry. Such programmable 2D protein lattices should enable new approaches to structure determination, sensing, and nanomaterial engineering.
Over the past decade, the Rosetta biomolecular modeling suite has informed diverse biological questions and engineering challenges ranging from interpretation of low-resolution structural data to ...design of nanomaterials, protein therapeutics, and vaccines. Central to Rosetta's success is the energy function: a model parametrized from small-molecule and X-ray crystal structure data used to approximate the energy associated with each biomolecule conformation. This paper describes the mathematical models and physical concepts that underlie the latest Rosetta energy function, called the Rosetta Energy Function 2015 (REF15). Applying these concepts, we explain how to use Rosetta energies to identify and analyze the features of biomolecular models. Finally, we discuss the latest advances in the energy function that extend its capabilities from soluble proteins to also include membrane proteins, peptides containing noncanonical amino acids, small molecules, carbohydrates, nucleic acids, and other macromolecules.
Accurate and rapid calculation of protein-small molecule interaction free energies is critical for computational drug discovery. Because of the large chemical space spanned by drug-like molecules, ...classical force fields contain thousands of parameters describing atom-pair distance and torsional preferences; each parameter is typically optimized independently on simple representative molecules. Here, we describe a new approach in which small molecule force field parameters are jointly optimized guided by the rich source of information contained within thousands of available small molecule crystal structures. We optimize parameters by requiring that the experimentally determined molecular lattice arrangements have lower energy than all alternative lattice arrangements. Thousands of independent crystal lattice-prediction simulations were run on each of 1386 small molecule crystal structures, and energy function parameters of an implicit solvent energy model were optimized, so native crystal lattice arrangements had the lowest energy. The resulting energy model was implemented in Rosetta, together with a rapid genetic algorithm docking method employing grid-based scoring and receptor flexibility. The success rate of bound structure recapitulation in cross-docking on 1112 complexes was improved by more than 10% over previously published methods, with solutions within <1 Å in over half of the cases. Our results demonstrate that small molecule crystal structures are a rich source of information for guiding molecular force field development, and the improved Rosetta energy function should increase accuracy in a wide range of small molecule structure prediction and design studies.
Advances in high-resolution cryo-electron microscopy (cryo-EM) require the development of validation metrics to independently assess map quality and model geometry. We report EMRinger, a tool that ...assesses the precise fitting of an atomic model into the map during refinement and shows how radiation damage alters scattering from negatively charged amino acids. EMRinger (https://github.com/fraser-lab/EMRinger) will be useful for monitoring progress in resolving and modeling high-resolution features in cryo-EM.
Near-atomic model of microtubule-tau interactions Kellogg, Elizabeth H; Hejab, Nisreen M A; Poepsel, Simon ...
Science (American Association for the Advancement of Science),
06/2018, Volume:
360, Issue:
6394
Journal Article
Peer reviewed
Open access
Tau is a developmentally regulated axonal protein that stabilizes and bundles microtubules (MTs). Its hyperphosphorylation is thought to cause detachment from MTs and subsequent aggregation into ...fibrils implicated in Alzheimer's disease. It is unclear which tau residues are crucial for tau-MT interactions, where tau binds on MTs, and how it stabilizes them. We used cryo-electron microscopy to visualize different tau constructs on MTs and computational approaches to generate atomic models of tau-tubulin interactions. The conserved tubulin-binding repeats within tau adopt similar extended structures along the crest of the protofilament, stabilizing the interface between tubulin dimers. Our structures explain the effect of phosphorylation on MT affinity and lead to a model of tau repeats binding in tandem along protofilaments, tethering together tubulin dimers and stabilizing polymerization interfaces.
The threat of a major coronavirus pandemic urges the development of strategies to combat these pathogens. Human coronavirus NL63 (HCoV-NL63) is an α-coronavirus that can cause severe ...lower-respiratory-tract infections requiring hospitalization. We report here the 3.4-Å-resolution cryo-EM reconstruction of the HCoV-NL63 coronavirus spike glycoprotein trimer, which mediates entry into host cells and is the main target of neutralizing antibodies during infection. The map resolves the extensive glycan shield obstructing the protein surface and, in combination with mass spectrometry, provides a structural framework to understand the accessibility to antibodies. The structure reveals the complete architecture of the fusion machinery including the triggering loop and the C-terminal domains, which contribute to anchoring the trimer to the viral membrane. Our data further suggest that HCoV-NL63 and other coronaviruses use molecular trickery, based on epitope masking with glycans and activating conformational changes, to evade the immune system of infected hosts.
Protein structure prediction using Rosetta in CASP12 Ovchinnikov, Sergey; Park, Hahnbeom; Kim, David E. ...
Proteins, structure, function, and bioinformatics,
March 2018, Volume:
86, Issue:
S1
Journal Article
Peer reviewed
Open access
We describe several notable aspects of our structure predictions using Rosetta in CASP12 in the free modeling (FM) and refinement (TR) categories. First, we had previously generated (and published) ...models for most large protein families lacking experimentally determined structures using Rosetta guided by co‐evolution based contact predictions, and for several targets these models proved better starting points for comparative modeling than any known crystal structure—our model database thus starts to fulfill one of the goals of the original protein structure initiative. Second, while our “human” group simply submitted ROBETTA models for most targets, for six targets expert intervention improved predictions considerably; the largest improvement was for T0886 where we correctly parsed two discontinuous domains guided by predicted contact maps to accurately identify a structural homolog of the same fold. Third, Rosetta all atom refinement followed by MD simulations led to consistent but small improvements when starting models were close to the native structure, and larger but less consistent improvements when starting models were further away.
A key issue in macromolecular structure modeling is the granularity of the molecular representation. A fine‐grained representation can approximate the actual structure more accurately, but may ...require many more degrees of freedom than a coarse‐grained representation and hence make conformational search more challenging. We investigate this tradeoff between the accuracy and the size of protein conformational search space for two frequently used representations: one with fixed bond angles and lengths and one that has full flexibility. We performed large‐scale explorations of the energy landscapes of 82 protein domains under each model, and find that the introduction of bond angle flexibility significantly increases the average energy gap between native and non‐native structures. We also find that incorporating bonded geometry flexibility improves low resolution X‐ray crystallographic refinement. These results suggest that backbone bond angle relaxation makes an important contribution to native structure energetics, that current energy functions are sufficiently accurate to capture the energetic gain associated with subtle deformations from chain ideality, and more speculatively, that backbone geometry distortions occur late in protein folding to optimize packing in the native state.
Most biomolecular modeling energy functions for structure prediction, sequence design, and molecular docking have been parametrized using existing macromolecular structural data; this contrasts ...molecular mechanics force fields which are largely optimized using small-molecule data. In this study, we describe an integrated method that enables optimization of a biomolecular modeling energy function simultaneously against small-molecule thermodynamic data and high-resolution macromolecular structural data. We use this approach to develop a next-generation Rosetta energy function that utilizes a new anisotropic implicit solvation model, and an improved electrostatics and Lennard-Jones model, illustrating how energy functions can be considerably improved in their ability to describe large-scale energy landscapes by incorporating both small-molecule and macromolecule data. The energy function improves performance in a wide range of protein structure prediction challenges, including monomeric structure prediction, protein-protein and protein-ligand docking, protein sequence design, and prediction of the free energy changes by mutation, while reasonably recapitulating small-molecule thermodynamic properties.
Metabolic energy is stored in cells primarily as triacylglycerols in lipid droplets (LDs), and LD dysregulation leads to metabolic diseases. The formation of monolayer-bound LDs from the endoplasmic ...reticulum (ER) bilayer is poorly understood, but the ER protein seipin is essential to this process. In this study, we report a cryo-electron microscopy structure and functional characterization of
seipin. The structure reveals a ring-shaped dodecamer with the luminal domain of each monomer resolved at ∼4.0 Å. Each luminal domain monomer exhibits two distinctive features: a hydrophobic helix (HH) positioned toward the ER bilayer and a β-sandwich domain with structural similarity to lipid-binding proteins. This structure and our functional testing in cells suggest a model in which seipin oligomers initially detect forming LDs in the ER via HHs and subsequently act as membrane anchors to enable lipid transfer and LD growth.
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