Journal of Structural Biology: X最新文献

NMR spectroscopy provides structural and functional information about biomolecules and their complexes. The complexity of these systems can make the NMR data difficult to interpret, particularly for newer users of NMR technology, who may have limited understanding of the tools available and how they are used. To alleviate this problem, we have created software based on standardized workflows for both solution and solid-state NMR spectroscopy of proteins. These tools assist with manual and automated peak picking and with chemical shift assignment and validation. They provide users with an optimized path through spectral analysis that can help them perform the necessary tasks more efficiently.

Solid-state NMR (ssNMR) has become a well-established technique to study large and insoluble protein assemblies. However, its application to nucleic acid–protein complexes has remained scarce, mainly due to the challenges presented by overlapping nucleic acid signals. In the past decade, several efforts have led to the first structure determination of an RNA molecule by ssNMR. With the establishment of these tools, it has become possible to address the problem of structure determination of nucleic acid–protein complexes by ssNMR. Here we review first and more recent ssNMR methodologies that study nucleic acid–protein interfaces by means of chemical shift and peak intensity perturbations, direct distance measurements and paramagnetic effects. At the end, we review the first structure of an RNA–protein complex that has been determined from ssNMR-derived intermolecular restraints.

Cell surface receptors such as the T-cell receptor (TCR) and B-cell receptor (BCR) engage with external stimuli to transmit information into the cell and initiate a cascade of signaling events that lead to gene expression that drives the immune response. At the heart of controlling T- and B-cell cell signaling, phospholipase Cγ hydrolyzes membrane associated PIP₂, leading to generation of the second messengers IP₃ and DAG. These small molecules trigger mobilization of intracellular Ca²⁺ and promote transcription factor transport into the nucleus launching the adaptive immune response. The TEC family kinases are responsible for phosphorylating and activating PLCγ, and our group aims to understand mechanisms that regulate immune cell signal transduction by focusing on this kinase/phospholipase axis in T-cells and B-cells. Here, we review the current molecular level understanding of how the TEC kinases (ITK and BTK) and PLCγ1/2 are autoinhibited prior to activation of cell surface receptors, how TEC kinases are activated to specifically recognize the PLCγ substrate, and how conformational changes induced by phosphorylation trigger PLCγ activation.

Structural hierarchy of bone – observed across multiple scales and in three dimensions (3D) – is essential to its mechanical performance. While the mineralized extracellular matrix of bone consists predominantly of carbonate-substituted hydroxyapatite, type I collagen fibrils, water, and noncollagenous organic constituents (mainly proteins and small proteoglycans), it is largely the 3D arrangement of these inorganic and organic constituents at each length scale that endow bone with its exceptional mechanical properties. Focusing on recent volumetric imaging studies of bone at each of these scales – from the level of individual mineralized collagen fibrils to that of whole bones – this graphical review builds upon and re-emphasizes the original work of James Bell Pettigrew and D’Arcy Thompson who first described the ubiquity of spiral structure in Nature. Here we illustrate and discuss the omnipresence of twisted, curved, sinusoidal, coiled, spiraling, and braided motifs in bone in at least nine of its twelve hierarchical levels – a visualization undertaking that has not been possible until recently with advances in 3D imaging technologies (previous 2D imaging does not provide this information). From this perspective, we hypothesize that the twisting motif occurring across each hierarchical level of bone is directly linked to enhancement of function, rather than being simply an energetically favorable way to assemble mineralized matrix components. We propose that attentive consideration of twists in bone and the skeleton at different scales will likely develop, and will enhance our understanding of structure–function relationships in bone.

Advancements in the field of cryo-electron tomography have greatly contributed to our current understanding of prokaryotic cell organization and revealed intracellular structures with remarkable architecture. In this review, we present some of the prominent advancements in cryo-electron tomography, illustrated by a subset of structural examples to demonstrate the power of the technique. More specifically, we focus on technical advances in automation of data collection and processing, sample thinning approaches, correlative cryo-light and electron microscopy, and sub-tomogram averaging methods. In turn, each of these advances enabled new insights into bacterial cell architecture, cell cycle progression, and the structure and function of molecular machines. Taken together, these significant advances within the cryo-electron tomography workflow have led to a greater understanding of prokaryotic biology. The advances made the technique available to a wider audience and more biological questions and provide the basis for continued advances in the near future.

RNases are varied in the RNA structures and sequences they target for cleavage and are an important type of enzyme in cells. Despite the numerous examples of RNases known, and of those with determined three-dimensional structures, relatively few examples exist with the RNase bound to intact cognate RNA substrate prior to cleavage. To better understand RNase structure and sequence specificity for RNA targets, in vitro methods used to assemble these enzyme complexes trapped in a pre-cleaved state have been developed for a number of different RNases. We have surveyed the Protein Data Bank for such structures and in this review detail methodologies that have successfully been used and relate them to the corresponding structures. We also offer ideas and suggestions for future method development. Many strategies within this review can be used in combination with X-ray crystallography, as well as cryo-EM, and other structure-solving techniques. Our hope is that this review will be used as a guide to resolve future yet-to-be-determined RNase–substrate complex structures.

Many amyloid-forming proteins, which are normally intrinsically disordered, undergo a disorder-to-order transition to form fibrils with a rigid β-sheet core flanked by disordered domains. Solid-state NMR (ssNMR) and cryogenic electron microscopy (cryoEM) excel at resolving the rigid structures within amyloid cores but studying the dynamically disordered domains remains challenging. This challenge is exemplified by mutant huntingtin exon 1 (HttEx1), which self-assembles into pathogenic neuronal inclusions in Huntington disease (HD). The mutant protein’s expanded polyglutamine (polyQ) segment forms a fibril core that is rigid and sequestered from the solvent. Beyond the core, solvent-exposed surface residues mediate biological interactions and other properties of fibril polymorphs. Here we deploy magic angle spinning ssNMR experiments to probe for semi-rigid residues proximal to the fibril core and examine how solvent dynamics impact the fibrils’ segmental dynamics. Dynamic spectral editing (DYSE) 2D ssNMR based on a combination of cross-polarization (CP) ssNMR with selective dipolar dephasing reveals the weak signals of solvent-mobilized glutamine residues, while suppressing the normally strong background of rigid core signals. This type of ‘intermediate motion selection’ (IMS) experiment based on cross-polarization (CP) ssNMR, is complementary to INEPT- and CP-based measurements that highlight highly flexible or highly rigid protein segments, respectively. Integration of the IMS-DYSE element in standard CP-based ssNMR experiments permits the observation of semi-rigid residues in a variety of contexts, including in membrane proteins and protein complexes. We discuss the relevance of semi-rigid solvent-facing residues outside the fibril core to the latter’s detection with specific dyes and positron emission tomography tracers.

Anticalins are generated via combinatorial protein design on the basis of the lipocalin protein scaffold and constitute a novel class of small and robust engineered binding proteins that offer prospects for applications in medical therapy as well as in vivo diagnostics as an alternative to antibodies. The lipocalins are natural binding proteins with diverse ligand specificities which share a simple architecture with a central eight-stranded antiparallel β-barrel and an α-helix attached to its side. At the open end of the β-barrel, four structurally variable loops connect the β-strands in a pair-wise manner and, together, shape the ligand pocket. Using targeted random mutagenesis in combination with molecular selection techniques, this loop region can be reshaped to generate pockets for the tight binding of various ligands ranging from small molecules over peptides to proteins. While such Anticalin proteins can be derived from different natural lipocalins, the human lipocalin 2 (Lcn2) scaffold proved particularly successful for the design of binding proteins with novel specificities and, over the years, more than 20 crystal structures of Lcn2-based Anticalins have been elucidated. In this graphical structural biology review we illustrate the conformational variability that emerged in the loop region of these functionally diverse artificial binding proteins in comparison with the natural scaffold. Our present analysis provides picturesque evidence of the high structural plasticity around the binding site of the lipocalins which explains the proven tolerance toward excessive mutagenesis, thus demonstrating remarkable resemblance to the complementarity-determining region of antibodies (immunoglobulins).

Cryo-electron tomography is uniquely suited to provide insights into the molecular architecture of cells and tissue in the native state. While frozen hydrated specimens tolerate sufficient electron doses to distinguish different types of particles in a tomogram, the accumulating beam damage does not allow resolving their detailed molecular structure individually. Statistical methods for subtomogram averaging and classification that coherently enhance the signal of particles corresponding to copies of the same type of macromolecular allow obtaining much higher resolution insights into macromolecules. Here, I review the developments in subtomogram analysis at Wolfgang Baumeister’s laboratory that make the dream of structural biology in the native cell become reality.

Recent advances in hardware, software and computing power have led to increasingly ambitious applications of cryo-electron tomography and subtomogram averaging. It is now possible to reveal both structures and biophysical relationships like protein binding partners and small molecule occupancy in these experiments. However, some data processing choices require the user to prioritize structure or biophysical context. Here, we present a modified subtomogram averaging approach that preserves both capabilities. By increasing the accuracy of particle-picking, performing alignment and averaging on all subtomograms, and decreasing reliance on symmetry and tight masks, the usability of tomography and subtomogram averaging data for biophysical analyses is greatly increased without negatively impacting structural refinements.