Acta Crystallographica. Section D, Structural Biology最新文献

Membrane-protein quality control in Escherichia coli involves coordinated actions of the AAA+ protease FtsH, the insertase YidC and the regulatory complex HflKC. These systems maintain proteostasis by facilitating membrane-protein insertion, folding and degradation. To gain structural insights into a putative complex formed by FtsH and YidC, we performed single-particle cryogenic electron microscopy on detergent-solubilized membrane samples, from which FtsH and YidC were purified using Ni-NTA affinity and size-exclusion chromatography. Although SDS-PAGE analysis indicated high purity of these proteins, cryo-EM data sets unexpectedly yielded high-resolution structures of ArnA and AcrB at 4.0 and 2.9 Å resolution, respectively. ArnA is a bifunctional enzyme involved in lipid A modification and polymyxin resistance, while AcrB is a multidrug efflux transporter of the AcrAB-TolC system. ArnA and AcrB, known Ni-NTA purification contaminants, were also consistently detected by mass spectrometry in Strep-Tactin affinity-purified samples, validating their presence independently of affinity-tag selection. ArnA, which is typically cytoplasmic, was consistently found in membrane-isolated samples, indicating an association with membrane components. Only 2D class averages corresponding to the cytoplasmic AAA+ domain of FtsH were observed; neither side views of full-length FtsH nor densities corresponding to an intact FtsH-YidC complex could be identified, due to the conformational flexibility of the FtsH complex and its transient interaction with YidC, which limited particle alignment and stable classification in cryo-EM data sets. Two-dimensional class averages revealed additional particles resembling GroEL and cytochrome bo₃ oxidase. These results underscore the utility of cryo-EM in uncovering off-target yet structurally well defined complexes, which may reflect physiologically relevant interactions or purification biases during membrane-protein overexpression.

Helical symmetry is a structural feature of many biological assemblies, including cytoskeletons, viruses and pathological amyloid fibrils. The helical parameters twist and rise are unique metadata for helical structures. With the increasing number of helical structures being resolved through cryo-EM and deposited in the EMDB, there is a growing possibility of errors in the metadata associated with these entries. During our cryo-EM analysis of protein amyloids and the development of helical analysis tools, we realized that many deposited helical parameters appear to be inconsistent with the associated density maps. Here, we have developed a comprehensive validation process that examines the consistency of these parameters by combining high-throughput computational evaluation with manual verification. Multiple errors were identified and corrected for ∼14% of the total entries, including missing parameters, swapped twist and rise values, incorrect sign of twist angles, partial symmetries and bona fide errors. Our validation code, workflow and the validated parameters are publicly available.

The c-Src SH3 domain is one of the best-characterized modular domains from a biophysical and structural point of view. This SH3 domain displays noncanonical alternative folding, forming 3D domain-swapped oligomers and amyloid fibrils. These features make this small protein an ideal model for studying these phenomena. Residues in the regions that favour unfolding of the monomer and those in the hinge loop have been deeply studied in proteins undergoing 3D domain swapping. To study the role of these residues in the unfolding of the c-Src SH3 domain, we have constructed several chimeric proteins by interchanging residues in the RT and n-Src loops between the c-Src SH3 and Abl SH3 domains. The RT (the region between β1 and β2) and n-Src (the region between β2 and β3) loops create two sides of the shallow hydrophobic groove where proline-rich motif sequences bind to the SH3 domain. In addition to the structural information, we have performed a biophysical characterization of these chimeric constructs. The c-Src SH3 domain bearing the loops of the Abl SH3 shows minor changes in stability. Interestingly, these replacements do not prevent the formation of domain-swapped dimers. However, the interchange of one or two loops within the Abl SH3 domain produces a noticeable reduction in its stability but does not promote the formation of 3D domain-swapped oligomers. Thus, our results indicate that although the composition of the hinge loop is likely to play a role in the interchange of structural elements to form the intertwined dimers, it is not the sole driving force in their formation.

Hydroxynitrile lyase from Hevea brasiliensis (HbHNL) and the esterase SABP2 from Nicotiana tabacum share the α/β-hydrolase fold, a Ser-His-Asp catalytic triad and 44% sequence identity, yet catalyze different reactions. Prior studies showed that three active-site substitutions in HbHNL conferred weak esterase activity. To investigate how regions beyond the active site influence catalytic efficiency and active-site geometry, we engineered HbHNL variants with increasing numbers of substitutions to match SABP2. Variant HNL16 has all amino acids within 6.5 Å of the active site identical to SABP2, HNL40 those within 10 Å and HNL71 those within 14 Å. HNL16 exhibited poor esterase activity, whereas both HNL40 and HNL71 showed efficient esterase catalysis, demonstrating that residues beyond the immediate active site are critical for functional conversion. X-ray structures of HNL40 and HNL71 reveal a progressive shift in backbone positions toward those of SABP2, with r.m.s.d. values of 0.51 Å (HNL40) and 0.41 Å (HNL71) over the C^α atoms, and even smaller r.m.s.d.s within the active-site region. Both HNL40 and HNL71 show a restored oxyanion hole and an additional tunnel connecting the active site to the protein surface. This work demonstrates the essential role of distant, indirectly acting residues to catalysis in α/β-hydrolase enzymes.

Rolf Hilgenfeld lived a life in the fast lane, devoted entirely to science, in which he absorbed a wealth of knowledge, conscientiously created new knowledge and passionately passed it on to his students.

Atomic coordinate models are important for the interpretation of 3D maps produced with cryoEM and cryoET (3D electron microscopy; 3DEM). In addition to visual inspection of such maps and models, quantitative metrics can inform about the reliability of the atomic coordinates, in particular how well the model is supported by the experimentally determined 3DEM map. A recently introduced metric, Q-score, was shown to correlate well with the reported resolution of the map for well fitted models. Here, we present new statistical analyses of Q-score based on its application to ∼10 000 maps and models archived in the EMDB (Electron Microscopy Data Bank) and PDB (Protein Data Bank). Further, we introduce two new metrics based on Q-score to represent each map and model relative to all entries in the EMDB and those with similar resolution. We explore through illustrative examples of proteins, nucleic acids and small molecules how Q-scores can indicate whether the atomic coordinates are well fitted to 3DEM maps and also whether some parts of a map may be poorly resolved due to factors such as molecular flexibility, radiation damage and/or conformational heterogeneity. These examples and statistical analyses provide a basis for how Q-scores can be interpreted effectively in order to evaluate 3DEM maps and atomic coordinate models prior to publication and archiving.

Electron cryo-microscopy (cryo-EM) is an imaging technique that is widely used in structural biology to determine the three-dimensional structure of biological molecules from noisy two-dimensional projections with unknown orientations. As the typical pipeline involves processing large amounts of data, efficient algorithms are crucial for fast and reliable results. The stochastic gradient descent (SGD) algorithm has been used to improve the speed of ab initio reconstruction, which results in an initial, low-resolution estimation of the volume representing the molecule of interest, but has yet to be applied successfully in the high-resolution regime, where expectation-maximization algorithms achieve state-of-the-art results, at a high computational cost. In this article, we investigate the conditioning of the optimization problem and show that the large condition number prevents the successful application of gradient descent-based methods at high resolution. Our results include a theoretical analysis of the condition number of the optimization problem in a simplified setting where the individual projection directions are known, an algorithm based on computing a diagonal preconditioner using Hutchinson's diagonal estimator and numerical experiments showing the improvement in the convergence speed when using the estimated preconditioner with SGD. The preconditioned SGD approach can potentially enable a simple and unified approach to ab initio reconstruction and high-resolution refinement with faster convergence speed and higher flexibility, and our results are a promising step in this direction.

The proteins SFPQ (splicing factor proline- and glutamine-rich) and NONO (non-POU domain-containing octamer-binding protein) are members of the Drosophila behaviour/human splicing (DBHS) protein family, sharing 76% sequence identity in their conserved DBHS domain. These proteins are critical for elements of pre- and post-transcriptional regulation in mammals and are primarily located in paraspeckles: ribonucleoprotein bodies templated by NEAT1 long noncoding RNA. Regions that are structured and predicted to be disordered (IDRs) in DBHS proteins facilitate various interactions, including dimerization, polymerization, nucleic acid binding and liquid-liquid phase separation, all of which have consequences for cell health, the pathology of some neurological diseases and cancer. To date, very limited structural work has been carried out on characterizing the IDRs of the DBHS proteins, largely due to their predicted disordered nature and the fact that this is often a bottleneck for conventional structural techniques. This is a problem worth addressing, as the IDRs have been shown to be critical to the material state of the protein as well as its function. In this study, we used small-angle X-ray scattering (SAXS) and small-angle neutron scattering (SANS), together with lysine cross-linking mass spectrometry (XL-MS), to investigate the regions of SFPQ flanking the structured DBHS domain and the possibility of dimer partner exchange of full-length proteins. Our results demonstrate experimentally that the N- and C-terminal regions on either side of the folded DBHS domain are long, disordered and flexible in solution. Realistic modelling of disordered chains to fit the scattering data and the compaction of the different protein variants suggests that it is physically possible for the IDRs to be close enough to interact. The mass-spectrometry data additionally indicate that the C-terminal IDR can potentially interact with the folded DBHS domain and also shares some conformational space with the N-terminal IDR. Our small-angle neutron scattering (SANS) experiments reveal that full-length SFPQ is capable of swapping dimer partners with itself, which has implications for our understanding of the combinatorial dimerization of DBHS proteins within cells. Our study provides insight into possible interactions between different IDRs either in cis or in trans and how these may relate to protein function, and the possible impact of mutations in these regions. The dynamic dimer partner exchange of a full-length protein inferred from this study is a phenomenon that is integral to the function of DBHS proteins, allowing changes in gene-regulatory activity by altering levels of the various heterodimers or homodimers.

Factor XIIa (FXIIa) is generated from its zymogen factor XII (FXII) by contact with polyanions such as inorganic polyphosphates. FXIIa cleaves the substrates prekallikrein and factor XI, triggering inflammatory cascades and plasma coagulation. From the N-terminus, FXII has fibronectin type II (FnII), epidermal growth factor-1 (EGF1), fibronectin type I (FnI), EGF2 and kringle domains. The N-terminal domains of FXII mediate polyanion and Zn²⁺ binding. To understand how ligand binding to polyanions and Zn²⁺ is coordinated across multiple domains, we determined the crystal structure of recombinant FXII domains 1-5 (FXII^HC5) to 3.4 Å resolution. A separate crystal structure of the isolated FXII FnII domain at 1.2 Å resolution revealed two bound Zn²⁺ ions. In FXII^HC5 a head-to-tail interaction is formed between the FnII and kringle domains, co-localizing the lysine-binding sites of the kringle domain and the cation-binding site of the FnII domain. Two FXII^HC5 monomers interlock, burying a large surface area of 2067 Ų, such that two kringle domains point outwards separated by a distance of 20 Å. The polyanion-binding site in the EGF1 domain is localized onto a plane together with the FnII and FnI domains. Using native mass spectrometry, we detected a major FXII^HC5 monomer peak and a minor dimer peak. Small-angle X-ray scattering and gel-filtration chromatography revealed the presence of monomers and dimers in solution. These FXII N-terminal domain structures provide a holistic framework to understand how the mosaic domain structure of FXII assembles diverse ligand-binding sites in three dimensions.

Multi-crystal processing of X-ray diffraction data has become highly automated to keep pace with the current high-throughput capabilities afforded by beamlines. A significant challenge, however, is the automated clustering of such data based on subtle differences such as ligand binding or conformational shifts. Intensity-based hierarchical clustering has been shown to be a viable method of identifying such subtle structural differences, but the interpretation of the resulting dendrograms is difficult to automate. Using isomorphous crystals of bovine, porcine and human insulin, the existing clustering methods in the multi-crystal processing software xia2.multiplex were validated and their limits were tested. It was determined that weighting the pairwise correlation coefficient calculations with the intensity uncertainties was required for accurate calculation of the pairwise correlation coefficient matrix (correlation clustering) and dimension optimization was required when expressing this matrix as a set of coordinates representing data sets (cosine-angle clustering). Finally, the introduction of the OPTICS spatial density-based clustering algorithm into DIALS allowed the automatic output of species-pure clusters of bovine, porcine and human insulin data sets.