nils-herrmann/clean_scirepeval huggingface dataset:
Inducing phase changes in crystals of macromolecules: status and perspectives for controlled crystal dehydration.
Venue: Journal of structural biology

The increase in the number of large multi-component complexes and membrane protein crystal structures determined over the last few years can be ascribed to a number of factors such as better protein expression and purification systems, the emergence of high-throughput crystallization techniques and the advent of 3rd generation synchrotron sources. However, many systems tend to produce crystals that can be extremely heterogeneous in their diffraction properties. This prevents, in many cases, the collection of diffraction data of sufficient quality to yield useful biological or phase information. Techniques that can increase the diffraction quality of macromolecular crystals can therefore be essential in the successful conclusion of these challenging projects. No technique is universal but encouraging results have been recently achieved by carrying out the controlled dehydration of crystals of biological macromolecules. A new device that delivers a stream of air with a precisely controlled relative humidity to the complicated sample environment found at modern synchrotron beamlines has been conceived at the EMBL Grenoble and developed by the EMBL and the ESRF as part of the SPINE2 complexes project, a European Commission funded protein structure initiative. The device, the HC1b, has been available for three years at the ESRF macromolecular crystallography beamlines and many systems have benefitted from on-line controlled dehydration. Here we describe a standard dehydration experiment, highlight some successful cases and discuss the different possible uses of the device.
Exploiting powder X-ray diffraction for direct structure determination in structural biology: The P2X4 receptor trafficking motif YEQGL
Venue: Journal of structural biology

We report the crystal structure of the 5-residue peptide acetyl-YEQGL-amide, determined directly from powder X-ray diffraction data recorded on a conventional laboratory X-ray powder diffractometer. The YEQGL motif has a known biological role, as a trafficking motif in the C-terminus of mammalian P2X4 receptors. Comparison of the crystal structure of acetyl-YEQGL-amide determined here and that of a complex formed with the μ2 subunit of the clathrin adaptor protein complex AP2 reported previously, reveals differences in conformational properties, although there are nevertheless similarities concerning aspects of the hydrogen-bonding arrangement and the hydrophobic environment of the leucine sidechain. Our results demonstrate the potential for exploiting modern powder X-ray diffraction methodology to achieve complete structure determination of materials of biological interest that do not crystallize as single crystals of suitable size and quality for single-crystal X-ray diffraction.
Expression of protein complexes using multiple Escherichia coli protein co-expression systems: a benchmarking study.
Venue: Journal of structural biology

Escherichia coli (E. coli) remains the most commonly used host for recombinant protein expression. It is well known that a variety of experimental factors influence the protein production level as well as the solubility profile of over-expressed proteins. This becomes increasingly important for optimizing production of protein complexes using co-expression strategies. In this study, we focus on the effect of the choice of the expression vector system: by standardizing experimental factors including bacterial strain, cultivation temperature and growth medium composition, we compare the effectiveness of expression technologies used by the partners of the Structural Proteomics in Europe 2 (SPINE2-complexes) consortium. Four different protein complexes, including three binary and one ternary complex, all known to be produced in the soluble form in E. coli, are used as the benchmark targets. The respective genes were cloned by each partner into their preferred set of vectors. The resulting constructs were then used for comparative co-expression analysis done in parallel and under identical conditions at a single site. Our data show that multiple strategies can be applied for the expression of protein complexes in high yield. While there is no 'silver bullet' approach that was infallible even for this small test set, our observations are useful as a guideline to delineate co-expression strategies for particular protein complexes.
Classification of electron sub-tomograms with neural networks and its application to template-matching.
Venue: Journal of structural biology

Classification of electron sub-tomograms is a challenging task, due the missing-wedge and the low signal-to-noise ratio of the data. Classification algorithms tend to classify data according to their orientation to the missing-wedge, rather than to the underlying signal. Here we use a neural network approach, called the Kernel Density Estimator Self-Organizing Map (KerDenSOM3D), which we have implemented in three-dimensions (3D), also having compensated for the missing-wedge, and we comprehensively compare it to other classification methods. For this purpose, we use various simulated macromolecules, as well as tomographically reconstructed in vitro GroEL and GroEL/GroES molecules. We show that the performance of this classification method is superior to previously used algorithms. Furthermore, we show how this algorithm can be used to provide an initial cross-validation of template-matching approaches. For the example of sub-tomogram classification extracted from cellular tomograms of Mycoplasma pneumonia and Spiroplasma melliferum cells, we show the bias of template-matching, and by using differing search and classification areas, we demonstrate how the bias can be significantly reduced.
Simulation and correction of electron images of tilted planar weak-phase samples.
Venue: Journal of structural biology

The phase contrast theory describes the transfer of information from a weak-phase object to the image plane of a transmission electron microscope. For a tilted sample where the distance from the focal plane varies continuously across the field of view, the recently introduced Tilted Contrast Imaging Function (TCIF) model provides the mathematical description of this information transfer. Here we expand the TCIF model to account for astigmatism, and present several methods to generate simulated images of tilted samples and compare them to experimental results. We analyze in depth the differences between TCIF and the classical Contrast Transfer Function (CTF) model, which assumes invariant defocus, and discuss how they can affect the interpretation of experimental data. In addition, we apply the TCIF model to simulated test objects in order to explore the performance of techniques that aim to correct the artifacts introduced by the imaging function, and evaluate how well they recover the original information after optimizing the parameters.
Strategies for the crystallization of viruses: using phase diagrams and gels to produce 3D crystals of Grapevine fanleaf virus.
Venue: Journal of structural biology

The small icosahedral plant RNA nepovirus Grapevine fanleaf virus (GFLV) is specifically transmitted by a nematode and causes major damage to vineyards worldwide. To elucidate the molecular mechanisms underlying the recognition between the surface of its protein capsid and cellular components of its vector, host and viral proteins synthesized upon infection, the wild type GFLV strain F13 and a natural mutant (GFLV-TD) carrying a Gly₂₉₇Asp mutation were purified, characterized and crystallized. Subsequently, the geometry and volume of their crystals was optimized by establishing phase diagrams. GFLV-TD was twice as soluble as the parent virus in the crystallization solution and its crystals diffracted X-rays to a resolution of 2.7 Å. The diffraction limit of GFLV-F13 crystals was extended from 5.5 to 3 Å by growth in agarose gel. Preliminary crystallographic analyses indicate that both types of crystals are suitable for structure determination. Keys for the successful production of GFLV crystals include the rigorous quality control of virus preparations, crystal quality improvement using phase diagrams, and crystal lattice reinforcement by growth in agarose gel. These strategies are applicable to the production of well-diffracting crystals of other viruses and macromolecular assemblies.
A novel heterotetrameric structure of the crenarchaeal PCNA2-PCNA3 complex.
Venue: Journal of structural biology

Proliferating cell nuclear antigen (PCNA) is a key protein that orchestrates the arrangement of DNA-processing proteins on DNA during DNA metabolism. In crenarchaea, PCNA forms a heterotrimer (PCNA123) consisting of PCNA1, PCNA2, and PCNA3, while in most eukaryotes and many archaea PCNAs form a homotrimer. Interestingly, unique oligomeric PCNAs from Sulfolobus tokodaii were reported in which PCNA2 and PCNA3 form a heterotrimer without PCNA1. In this paper, we describe the crystal structure of the stoPCNA2-stoPCNA3 complex. While most DNA sliding clamps form ring-shaped structures, our crystal structure showed an elliptic ring-like heterotetrameric complex, differing from a previous reports. Furthermore, we investigated the composition and the dimension of the stoPCNA2-stoPCNA3 complex in the solution using gel-filtration column chromatography and small-angle X-ray scattering analyses, respectively. These results indicate that stoPCNA2 and stoPCNA3 form the heterotetramer in solution. Based on our heterotetrameric structure, we propose a possible biological role for the heterotetrameric complex as a Holliday junction clamp.
The surface of evaporated carbon films is an insulating, high-bandgap material.
Venue: Journal of structural biology

The electrical conductance and the optical density of evaporated carbon films are measured as a function of the thickness of the films. The resulting data show that up to a thickness of approximately 4 nm, carbon films are optically transparent and electrically insulating. The same data also suggest that this insulating character persists near to the surface when the overall thickness is further increased. Since a support film with poor surface conductivity is undesirable for many applications in electron microscopy, we suggest that the usefulness of evaporated carbon films in electron microscopy might be further improved by doping or by other methods that improve the electrical conductivity near the surface.
Stabilizing role of glutamic acid 222 in the structure of Enhanced Green Fluorescent Protein.
Venue: Journal of structural biology

Enhanced Green Fluorescent Protein (EGFP) is a variant of wild-type Green Fluorescent Protein from the jellyfish Aequorea victoria, whose mutations S65T and F64L increase brightness and folding efficiency. EGFP is extensively used in cell biology and biochemistry as a colocalization or expression reporter. Surprisingly, the structure of this very popular protein has not been determined yet. We report here its crystallographic structure at 1.5Å resolution which shows significant differences in the vicinity of residue 64 and of the chromophore. In particular, two conformations are observed for the key residue glutamic acid 222, in apparent contradiction with the single fluorescence lifetime of the protein. We then show that X-ray induced decarboxylation of Glu222 during diffraction data collection results in the disruption of a hydrogen-bond network near the chromophore. Using single-crystal microspectrophotometry, we demonstrate that this correlates with a significant loss of the fluorescence properties. We thus propose a mechanism of bleaching of the protein at low temperature. Taken together, these two sets of results highlight the stabilizing role of Glu222 to the chromophore cavity of EGFP.
Nucleolar structure across evolution: the transition between bi- and tri-compartmentalized nucleoli lies within the class Reptilia.
Venue: Journal of structural biology

Two types of nucleolus can be distinguished among eukaryotic cells: a tri-compartmentalized nucleolus in amniotes and a bi-compartmentalized nucleolus in all the others. However, though the nucleolus' ultrastructure is well characterized in mammals and birds, it has been so far much less studied in reptiles. In this work, we examined the ultrastructural organization of the nucleolus in various tissues from different reptilian species (three turtles, three lizards, two crocodiles, and three snakes). Using cytochemical and immunocytological methods, we showed that in reptiles both types of nucleolus are present: a bi-compartmentalized nucleolus in turtles and a tri-compartmentalized nucleolus in the other species examined in this study. Furthermore, in a given species, the same type of nucleolus is present in all the tissues, however, the importance and the repartition of those nucleolar components could vary from one tissue to another. We also reveal that, contrary to the mammalian nucleolus, the reptilian fibrillar centers contain small clumps of condensed chromatin and that their surrounding dense fibrillar component is thicker. Finally, we also report that Cajal bodies are detected in reptiles. Altogether, we believe that these results have profound evolutionarily implications since they indicate that the point of transition between bipartite and tripartite nucleoli lies at the emergence of the amniotes within the class Reptilia.
Micro-structure and chemical composition of vateritic deformities occurring in the bivalve Corbicula fluminea (Müller, 1774).
Venue: Journal of structural biology

Vateritic deformities occurring in the invasive heterodont bivalve Corbicula fluminea from several locations in the UK were characterised in detail for the first time using scanning electron microscopy, X-ray diffraction and different geochemical techniques (electron microprobe, ICP-AES, and mass spectrometry). Large volumes of vaterite are produced abnormally in the animals' shells in the form of yellow-green bulges. These are distinguished from the aragonitic parts of the shell by their characteristic micro-structures, content of organic material, trace elemental composition and carbon stable isotope signatures. The most commonly observed micro-structures include columnar vaterite, lamellar vaterite and different irregular structures occurring in all parts of the shell. There are indications that organic material is present largely as intracrystalline impurities or nano-scale phases and not as envelopes around microstructural units. These micro-structures are novel, nothing equivalent having yet been described for other vateritic systems. Euhedral vaterite crystals also occur occasionally. The vaterite has generally higher Mg/Ca and lower Na/Ca, K/Ca than the aragonite. In addition, δ¹³C is also always lower. Microstructural characteristics would suggest loss of biological control probably due to physiological stress(es) inducing the switch to vaterite production. The vaterite might be stabilised by its higher content of organic material and magnesium.
Deciphering correct strategies for multiprotein complex assembly by co-expression: application to complexes as large as the histone octamer.
Venue: Journal of structural biology

Macromolecular complexes are responsible for most of the essential mechanisms in cells, leading to a broad interest in their purification and characterization. Co-expression is now widely recognized as a major technique for assembling multiprotein complexes and many co-expression systems are currently available for performing co-expression experiments in different hosts. However, comparative knowledge on co-expression strategies is still crucially lacking. Using versatile co-expression systems for Escherichia coli, the pET-MCN and pET-MCP series, and ternary protein complexes as examples, we demonstrate how to successfully delineate correct co-expression strategies. Specifically, an appropriate, complex-dependent approach alleviates stoichiometry imbalance and yield problems, and even failure in producing complexes. Importantly, some of the parameters influencing co-expression strategies appear independent of the expression host, thus having implications for co-expression in eukaryotic hosts. By further using these strategies, we show that co-expression in E. coli enables reconstitution of protein complexes as large as the deubiquitination module of the SAGA transcription factor and the histone octamer.
Solution NMR structure and dynamics of human apo-S100A1 protein.
Venue: Journal of structural biology

S100A1 belongs to the EF-hand superfamily of calcium binding proteins. It is a representative of the S100 protein family based on amino acid sequence, three-dimensional structure, and biological function as a calcium signal transmitter. It is a homodimer of noncovalently bound subunits. S100A1, like most of other members of the S100 protein family, is a multifunctional, regulatory protein involved in a large variety of biological processes and closely associated with several human diseases. The three-dimensional structure of human apo-(i.e. calcium free)-S100A1 protein was determined by NMR spectroscopy (PDB 2L0P) and its backbone dynamics established by ¹⁵N magnetic relaxation. Comparison of these results with the structure and backbone dynamics previously determined for bovine apo-S100A1 protein modified by disulfide formation with β-mercaptoethanol at Cys 85 revealed that the secondary structure of both these proteins was almost identical, whereas the global structure of the latter was much more mobile than that of human apo-S100 protein. Differences between the structures of human and rat apo-S100A1 are also discussed.
Designed ankyrin repeat protein binders for the crystallization of AcrB: plasticity of the dominant interface.
Venue: Journal of structural biology

The formation of well-diffracting crystals is a major bottleneck in structural analysis of membrane proteins by X-ray crystallography. One approach to improve crystal quality is the use of DARPins as crystallization chaperones. Here, we present a detailed analysis of the interaction between DARPins and the integral membrane protein AcrB. We find that binders selected in vitro by ribosome display share a common epitope. The comparative analysis of three crystal structures of AcrB-DARPin complexes allowed us to study the plasticity of the interaction with this dominant binding site. Seemingly redundant AcrB-DARPin crystals show substantially different diffraction quality as a result of subtle differences in the binding geometry. This work exemplifies the importance to screen a number of crystallization chaperones to obtain optimal diffraction data. Crystallographic analysis is complemented by biophysical characterization of nine AcrB binders. We observe that small variations in the interface can lead to differing behavior of the DARPins with regards to affinity, stoichiometry of the complexes and specificity for their target.
Mapping elasticity moduli of atherosclerotic plaque in situ via atomic force microscopy.
Venue: Journal of structural biology

Several studies have suggested that evolving mechanical stresses and strains drive atherosclerotic plaque development and vulnerability. Especially, stress distribution in the plaque fibrous capsule is an important determinant for the risk of vulnerable plaque rupture. Knowledge of the stiffness of atherosclerotic plaque components is therefore of critical importance. In this work, force mapping experiments using atomic force microscopy (AFM) were conducted in apolipoprotein E-deficient (ApoE(-/-)) mouse, which represents the most widely used experimental model for studying mechanisms underlying the development of atherosclerotic lesions. To obtain the elastic material properties of fibrous caps and lipidic cores of atherosclerotic plaques, serial cross-sections of aortic arch lesions were probed at different sites. Atherosclerotic plaque sub-structures were subdivided into cellular fibrotic, hypocellular fibrotic and lipidic rich areas according to histological staining. Hertz's contact mechanics were used to determine elasticity (Young's) moduli that were related to the underlying histological plaque structure. Cellular fibrotic regions exhibit a mean Young modulus of 10.4±5.7kPa. Hypocellular fibrous caps were almost six-times stiffer, with average modulus value of 59.4±47.4kPa, locally rising up to ∼250kPa. Lipid rich areas exhibit a rather large range of Young's moduli, with average value of 5.5±3.5kPa. Such precise quantification of plaque stiffness heterogeneity will allow investigators to have prospectively a better monitoring of atherosclerotic disease evolution, including arterial wall remodeling and plaque rupture, in response to mechanical constraints imposed by vascular shear stress and blood pressure.
Modeling protein structure at near atomic resolutions with Gorgon.
Venue: Journal of structural biology

Electron cryo-microscopy (cryo-EM) has played an increasingly important role in elucidating the structure and function of macromolecular assemblies in near native solution conditions. Typically, however, only non-atomic resolution reconstructions have been obtained for these large complexes, necessitating computational tools for integrating and extracting structural details. With recent advances in cryo-EM, maps at near-atomic resolutions have been achieved for several macromolecular assemblies from which models have been manually constructed. In this work, we describe a new interactive modeling toolkit called Gorgon targeted at intermediate to near-atomic resolution density maps (10-3.5 Å), particularly from cryo-EM. Gorgon's de novo modeling procedure couples sequence-based secondary structure prediction with feature detection and geometric modeling techniques to generate initial protein backbone models. Beyond model building, Gorgon is an extensible interactive visualization platform with a variety of computational tools for annotating a wide variety of 3D volumes. Examples from cryo-EM maps of Rotavirus and Rice Dwarf Virus are used to demonstrate its applicability to modeling protein structure.
Scoring functions for cryoEM density fitting.
Venue: Journal of structural biology

In fitting atomic structures into cryoEM density maps of macromolecular assemblies, the cross-correlation function (CCF) is the most prevalent method of scoring the goodness-of-fit. However, there are still many possible, less studied ways of scoring fits. In this paper, we introduce four scores new to cryoEM fitting and compare their performance to three known scores. Our benchmark consists of (a) 4 protein assemblies with simulated maps at 5-20 Å resolution, including the heptameric ring of GroEL; and (b) 4 experimental maps of GroEL at ∼6-23 Å resolution with corresponding fitted atomic models. We perturb each fit 1000 times and assess each new fit with each score. The correlation between a score and the Cα RMSD of each fit from the "correctly" fitted structure shows that the CCF is one of the best scores, but in certain situations could be augmented or even replaced by other scores. For instance, our implementation of a score based on mutual information outperforms or is comparable to the CCF in almost all test cases, and our new "envelope score" works as well as the CCF at sub-nanometer resolution but is an order of magnitude faster to calculate. The results also suggest that the width of the Gaussian function used to blur the atomic structure into a density map can significantly affect the fitting process. Finally, we show that our score-testing method, when combined with the Laplacian CCF or the mutual information scores, can be used as a statistical tool for improving cryoEM density fitting.