Acta Crystallographica. Section D, Structural Biology最新文献

Polo-like kinase 1 (PLK1) is a major regulator of cell division and has been pursued as a drug target for cancer therapy for a long time. Crystallization of the kinase domain has proven to be exceptionally challenging. Previously, we published a crystallization approach using a PLK1-specific designed ankyrin-repeat protein (DARPin) as a crystallization facilitator. Here, we report an alternative route: crystallization was successful after the introduction of a double mutation which reduced surface entropy and enabled the formation of a new crystal contact. This new PLK1 crystallization system was used to determine the first co-complex crystal structure of the Bayer thiazolidinone lead series, as well as crystal structures with representatives of two competitor inhibitor series. The molecular binding modes of these three inhibitors are analysed and discussed, and the surface-entropy reduction approach is compared with the surface modifications employed by us and others to enable the crystallization of PLK1.

Protein structure determination has long been one of the primary challenges of structural biology, to which deep machine learning (ML)-based approaches have increasingly been applied. However, these ML models generally do not directly incorporate the experimental measurements, such as X-ray crystallographic diffraction data. To this end, we explore an approach that more tightly couples these traditional crystallographic and recent ML-based methods by training a hybrid 3D vision transformer and convolutional network on inputs from both domains. We make use of two distinct input constructs: Patterson maps, which are directly obtainable from crystallographic data, and `partial structure' template maps derived from predicted structures deposited in the AlphaFold Protein Structure Database with subsequently omitted residues. With these, we predict electron-density maps that are then post-processed into atomic models through standard crystallographic refinement processes. Introducing an initial data set of small protein fragments taken from Protein Data Bank entries and placing them in hypothetical crystal settings, we demonstrate that our method is effective at both improving the phases of the crystallographic structure factors and completing the regions missing from partial structure templates, as well as improving the agreement of the electron-density maps with the ground-truth atomic structures.

Similarity between two periodic functions is commonly assessed by comparing their Fourier coefficients within resolution shells. In particular, this approach is widely used in both crystallography and cryo-electron microscopy (cryoEM). The definition of these shells, that is the choice of resolution scale for their boundaries, can be guided by the specific goals of the analysis, by the expected features of the studied functions or simply by convention. In cryoEM, shell boundaries are traditionally defined uniformly in inverse resolution. This convention results in a vast imbalance in the number of Fourier coefficients per shell, which may bias statistical comparisons and can make function plots misleading. Constructing resolution shells with approximately equal numbers of Fourier coefficients can be achieved automatically by defining shell boundaries uniformly on the inverse cubic resolution scale. This transformation effectively zooms into the high-resolution region, which is typically the primary focus of analysis. For Fourier shell correlation (FSC) calculations between half-maps, the characteristic sigmoidal curves were observed to transform into profiles that permit piecewise linear interpolation, which may make FSC analysis more robust.

Foldit is a citizen science video game in which players tackle a variety of complex biochemistry puzzles. Here, we describe a new series of puzzles in which Foldit players improve the accuracy of models in the public repository of experimental protein structure models, the Protein Data Bank (PDB). Analyzing the results of these puzzles showed that the Foldit players were able to considerably improve the deposited structures. We describe a mechanism by which the efforts of the Foldit players can be fed back into the structural biology scientific record by using Foldit results as improved input for the PDB-REDO databank. These efforts highlight the continued need for the engagement of the lay population in science.

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.