nils-herrmann/clean_scirepeval huggingface dataset:
ESPRIT: an automated, library-based method for mapping and soluble expression of protein domains from challenging targets.
Venue: Journal of structural biology

Expression of sufficient quantities of soluble protein for structural biology and other applications is often a very difficult task, especially when multimilligram quantities are required. In order to improve yield, solubility or crystallisability of a protein, it is common to subclone shorter genetic constructs corresponding to single- or multi-domain fragments. However, it is not always clear where domain boundaries are located, especially when working on novel targets with little or no sequence similarity to other proteins. Several methods have been described employing aspects of directed evolution to the recombinant expression of challenging proteins. These combine the construction of a random library of genetic constructs of a target with a screening or selection process to identify solubly expressing protein fragments. Here we review several datasets from the ESPRIT (Expression of Soluble Proteins by Random Incremental Truncation) technology to provide a view on its capabilities. Firstly, we demonstrate how it functions using the well-characterised NF-kappaB p50 transcription factor as a model system. Secondly, application of ESPRIT to the challenging PB2 subunit of influenza polymerase has led to several novel atomic resolution structures; here we present an overview of the screening phase of that project. Thirdly, analysis of the human kinase TBK1 is presented to show how the ESPRIT technology rapidly addresses the compatibility of challenging targets with the Escherichia coli expression system.
Second harmonic generation imaging - a new method for unraveling molecular information of starch.
Venue: Journal of structural biology

We present a new method, second harmonic generation (SHG) imaging for the study of starch structure. SHG imaging can provide the structural organization and molecular orientation information of bio-tissues without centrosymmetry. In recent years, SHG has proven its capability in the study of crystallized bio-molecules such as collagen and myosin. Starch, the most important food source and a promising future energy candidate, has, for a decade, been shown to exhibit strong SHG response. By comparing SHG intensity from different starch species, we first identified that the SHG-active molecule is amylopectin, which accounts for the crystallinity in starch granules. With the aid of SHG polarization anisotropy, we extracted the complete χ((2)) tensor of amylopectin, which reflects the underlying molecular details. Through χ((2)) tensor analysis, three-dimensional orientation and packing symmetry of amylopectin are determined. The helical angle of the double-helix in amylopectin is also deduced from the tensor, and the value corresponds well to previous X-ray studies, further verifying amylopectin as SHG source. It is noteworthy that the nm-sized structure of amylopectin inside a starch granule can be determined by this far-field optical method with 1-μm excitation wavelength. Since SHG is a relatively new tool for plant research, a detailed understanding of SHG in starch structure will be useful for future high-resolution imaging and quantitative analyses for food/energy applications.
Automated electron microscopy for evaluating two-dimensional crystallization of membrane proteins.
Venue: Journal of structural biology

Membrane proteins fulfill many important roles in the cell and represent the target for a large number of therapeutic drugs. Although structure determination of membrane proteins has become a major priority, it has proven to be technically challenging. Electron microscopy of two-dimensional (2D) crystals has the advantage of visualizing membrane proteins in their natural lipidic environment, but has been underutilized in recent structural genomics efforts. To improve the general applicability of electron crystallography, high-throughput methods are needed for screening large numbers of conditions for 2D crystallization, thereby increasing the chances of obtaining well ordered crystals and thus achieving atomic resolution. Previous reports describe devices for growing 2D crystals on a 96-well format. The current report describes a system for automated imaging of these screens with an electron microscope. Samples are inserted with a two-part robot: a SCARA robot for loading samples into the microscope holder, and a Cartesian robot for placing the holder into the electron microscope. A standard JEOL 1230 electron microscope was used, though a new tip was designed for the holder and a toggle switch controlling the airlock was rewired to allow robot control. A computer program for controlling the robots was integrated with the Leginon program, which provides a module for automated imaging of individual samples. The resulting images are uploaded into the Sesame laboratory information management system database where they are associated with other data relevant to the crystallization screen.
Density functional theory analysis and spectral studies on amyloid peptide Abeta(28-35) and its mutants A30G and A30I.
Venue: Journal of structural biology

Folding and self-assembly of amyloid beta (Abeta) peptide are linked to Alzheimer's disease. To understand the initial stage of amyloid-beta peptide aggregation, conformational characteristics of monomers of wild-type (WT) Abeta(28-35) and its mutant peptides A30G and A30I were investigated using density functional theory calculations and experimental studies. Monomeric structures and their relative stabilities were obtained on the basis of systematic structural optimization in the gas-phase and in the aqueous medium. Computations were performed by hybrid Hartree-Fock-Density Functional Theory (HF-DFT) at B3LYP/6-31G * level. Experimentally, the conformational transitions in the early stages of the octapeptide Abeta(28-35) and its mutants A30G and A30I in solution were characterized by CD, Thioflavin assay and FRET spectroscopy. Examination of the secondary structures revealed that Abeta(28-35) and its mutant monomers exist in random coil conformation in the aqueous medium in agreement with the theoretical predictions, which upon aging is transformed to sheet with different kinetics. This study deals with the structurally important intermediates and it may help to understand the mechanism of amyloid fibril aggregation leading to the onset of Alzheimer's disease.
Monoclonal antibody MN423 as a stable mold facilitates structure determination of disordered tau protein.
Venue: Journal of structural biology

Flexibility of intrinsically disordered tau protein is important for performing its functions. It is believed that alteration of the flexibility is instrumental to the assembly of tau protein into paired helical filaments (PHF) in tauopathies. Tau flexibility represents the main obstacle for structure determination of its conformation in physiology and/or pathology. We have alleviated this inherited difficulty by using specific monoclonal antibodies as tau protein surrogate binding partners. In this work we compare two "antibody mold structures": (1) X-ray structure of the free form of the Alzheimer's disease PHF core-specific antibody MN423 and (2) previously solved structure of the complex of MN423 with the PHF core C-terminal tau peptide. We found that MN423 combining site is in both structures identical. As a consequence, recombinant tau assumes in the complex a fold determined by the antibody combining site. Obtained results show that MN423 functions as a molecular mold for the PHF core segment, and opens the way for structure determination of other PHF core segments providing that other conformation-specific antibodies are available. Data from in silico docking of tau peptide into antibody mold, obtained in this study, show that biochemical data and computational approaches provide results comparable to X-ray crystallography.
New baculovirus expression tools for recombinant protein complex production.
Venue: Journal of structural biology

Most eukaryotic proteins exist as large multicomponent assemblies with many subunits, which act in concert to catalyze specific cellular activities. Many of these molecular machines are only present in low amounts in their native hosts, which impede purification from source material. Unraveling their structure and function at high resolution will often depend on heterologous overproduction. Recombinant expression of multiprotein complexes for structural studies can entail considerable, sometimes inhibitory, investment in both labor and materials, in particular if altering and diversifying of the individual subunits are necessary for successful structure determination. Our laboratory has addressed this challenge by developing technologies that streamline the complex production and diversification process. Here, we review several of these developments for recombinant multiprotein complex production using the MultiBac baculovirus/insect cell expression system which we created. We also addressed parallelization and automation of gene assembly for multiprotein complex expression by developing robotic routines for multigene vector generation. In this contribution, we focus on several improvements of baculovirus expression system performance which we introduced: the modifications of the transfer plasmids, the methods for generation of composite multigene baculoviral DNA, and the simplified and standardized expression procedures which we delineated using our MultiBac system.
Micromachining tools and correlative approaches for cellular cryo-electron tomography.
Venue: Journal of structural biology

A principal limitation of cryo-transmission electron microscopy performed on cells or tissues is the accessible specimen thickness. This is exacerbated in tomography applications, where the aspect ratio (and thus the apparent specimen thickness) changes considerably during specimen tilting. Cryo-ultramicrotomy is the most obvious way of dealing with this problem; however, frozen-hydrated sections suffer from potentially inconsistent compression that cannot be corrected with certainty, and furthermore, yields of sections that satisfy all of the conditions necessary for tomographic imaging are poor. An alternative approach that avoids mechanical deformations is the use of focused ion beam (FIB) instrumentation, where thinning of the frozen-hydrated specimen occurs through the process of sputtering with heavy ions, typically gallium. Here, we use correlative cryo-fluorescence microscopy to navigate large cellular volumes and to localize specific cellular targets. We show that the selected targets in frozen-hydrated specimens can be accessed directly by focused ion beam milling. We also introduce a novel cryo-planing procedure as a method that could facilitate thinning of large areas of vitreous ice prior to cryo-fluorescence, FIB thinning, and cryo-electron tomography.
A novel coiled-coil repeat variant in a class of bacterial cytoskeletal proteins.
Venue: Journal of structural biology

In recent years, a number of bacterial coiled-coil proteins have been characterised which have roles in cell growth and morphology. Several have been shown to have a cytoskeletal function and some have been proposed to have an IF-like character in particular. We recently demonstrated in Streptomyces coelicolor a cytoskeletal role of Scy, a large protein implicated in filamentous growth, whose sequence is dominated by an unusual coiled-coil repeat. We present a detailed analysis of this 51-residue repeat and conclude that it is likely to form a parallel dimeric non-canonical coiled coil based on hendecads but with regions of local underwinding reflecting highly periodic modifications in the sequence. We also demonstrate that traditional sequence similarity searching is insufficient to identify all but the close orthologues of such repeat-dominated proteins, but that by an analysis of repeat periodicity and composition, remote homologues can be found. One clear candidate, despite a great size discrepancy and unremarkable sequence identity, is the known filament-former FilP in the same species. Both proteins appear distinct from the archetypal bacterial IF-like protein; they therefore may constitute a new class of bacterial filamentous protein. The similar sequence characteristics of both suggest their likely oligomer state and a possible mechanism for higher-order assembly into filaments. Another remote homologue in Actinomyces was highlighted by this method. Further, a known coiled-coil protein, DivIVA, appears to share some of these sequence characteristics.
A transition from strong right-handed to canonical left-handed supercoiling in a conserved coiled-coil segment of trimeric autotransporter adhesins.
Venue: Journal of structural biology

Trimeric autotransporter adhesins (TAAs) represent an important class of pathogenicity factors in proteobacteria. Their defining feature is a conserved membrane anchor, which forms a 12-stranded beta-barrel through the outer membrane. The proteins are translocated through the pore of this barrel and, once export is complete, the pore is occluded by a three-stranded coiled coil with canonical heptad (7/2) sequence periodicity. In many TAAs this coiled coil is extended by a segment of varying length, which has pentadecad (15/4) periodicity. We used X-ray crystallography and biochemical methods to analyze the transition between these two periodicities in the coiled-coil stalk of the Yersinia adhesin YadA. Our results show how the strong right-handed supercoil of the 15/4-periodic part locally undergoes further over-winding to 19/5, before switching at a fairly constant rate over 14 residues to the canonical left-handed supercoil of the 7/2-periodic part. The transition region contains two YxD motifs, which are characteristic for right-handed coiled-coil segments of TAAs. This novel coiled-coil motif forms a defined network of inter- and intrahelical hydrogen bonds, thus serving as a structural determinant. Supercoil fluctuations have hitherto been described in coiled coils whose main sequence periodicity is disrupted locally by discontinuities. Here we present the first detailed analysis of two fundamentally different coiled-coil periodicities being accommodated in the same structure.
Coiled-coils and fibrous proteins.
Venue: Journal of structural biology

The Fifth Workshop on ‘‘Coiled-Coils, Collagen and Co-proteins” was held at Alpbach in Austria from September 6 to 12, 2009. The Workshop this time was generously sponsored by Elsevier and by the M.E. Mueller Foundation/M.E. Mueller Institute for Structural Biology. The format, as usual, was to invite Chairs and a few keynote contributors for each session, but then at the Workshop itself to allow the Chairs to invite delegates to present something of their latest work. The Chairs arranged the programme accordingly and allotted time to each depending on the numbers involved. Of course particular sessions can (and did) prove especially popular and a degree of flexibility in extending some sessions and shortening others was necessary. This format, whilst a challenge on occasions to the Chairs and the Organisers, has proved remarkably successful over the years. Significant benefits of the Workshops include both the opportunity of meeting in an informal environment with colleagues in related fields with whom they would not normally collaborate, and also the chance to learn of new developments in techniques and software (for example) that are relevant across many facets of the field of fibrous proteins. Developments in structure and function in one protein system can often foster ideas and approaches in others. Considerable cross-fertilisation has occurred in past Workshops, with perhaps even more taking place in this latest one. The Guest of Honour was Professor Kenneth Holmes FRS, an eminent scientist who has made major contributions in the structural biology field, particularly on tobacco mosaic virus and on muscle and, in the latter case, more specifically, on thin filament structure and function. At Alpbach, Ken Holmes presented a fascinating and beautifully illustrated lecture on the research that he has undertaken over a span of more than 50 years. He mentioned in context the remarkable galaxy of other eminent scientists, a Who’s Who of 20th century structural biologists, with whom he has collaborated over the years. The first paper in this Special Issue is by Ken Holmes and in it he provides a delightful personal account of his life with fibres. Learning a little of the history of world class scientists such as Ken, their successes and their (rare) failures, provides an insight into the individuals concerned, and is something that we hear about all too infrequently.
Differential splicing of the large sarcomeric protein nebulin during skeletal muscle development.
Venue: Journal of structural biology

We studied differential splicing of nebulin, a giant filamentous F-actin binding protein (M(r) approximately 700-800kDa) that is found in skeletal muscle. Nebulin spans the thin filament length, its C-terminus is anchored in the Z-disc, and its N-terminal region is located toward the thin filament pointed end. Various lines of evidence indicate that nebulin plays important roles in thin filament and Z-disc structure in skeletal muscle. In the present work we studied nebulin in a range of muscle types during postnatal development and performed transcript studies with a mouse nebulin exon microarray, developed by us, whose results were confirmed by RT-PCR. We also performed protein studies with high-resolution SDS-agarose gels and Western blots, and structural studies with electron microscopy. We found during postnatal development of the soleus muscle major changes in splicing in both the super-repeat region and the Z-disc region of nebulin; interestingly, these changes were absent in other muscle types. Three novel Z-disc exons, previously described in the mouse gene, were upregulated during postnatal development of soleus muscle and this was correlated with a significant increase in Z-disc width. These findings support the view that nebulin plays an important role in Z-disc width regulation. In summary, we discovered changes in both the super-repeat region and the Z-disc region of nebulin, that these changes are muscle-type specific, and that they correlate with differences in sarcomere structure.
Atomic structure of vimentin coil 2.
Venue: Journal of structural biology

Intermediate filaments (IFs) are essential cytoskeletal components in metazoan cells. They assemble from elementary dimers that are built around the central alpha-helical coiled-coil rod domain representing the IF 'signature'. The rod consists of two similarly-sized parts, coil 1 and coil 2, connected by a non-alpha-helical linker L12. Coil 2 is absolutely conserved in length across all IF types and was initially predicted to consist of a short coiled-coil segment 2A based on a heptad pattern of hydrophobic residues, another linker L2 and a coiled-coil segment 2B. Here we present the crystal structure of human vimentin fragment including residues 261-335 i.e. approximately the first half of coil 2. The N-terminal part of this fragment reveals a parallel alpha-helical bundle characterized by 3.5 consecutive hendecad repeats. It is immediately followed by a regular left-handed coiled coil. The distinct non-helical linker L2 is therefore not observed. Together with the previously determined crystal structure of the major part of segment 2B (Strelkov et al., 2002), we can now build a complete atomic model of the 21nm long vimentin coil 2 dimer being a relatively rigid rod.
12 Arylstibonic acids that inhibit the DNA binding of five B-ZIP dimers.
Venue: Journal of structural biology

Previously, we identified an arylstibonic acid, NSC13778 that specifically binds to the basic region of the C/EBPalpha B-ZIP domain and disrupts DNA binding. We now examine a panel of 14 additional arylstibonic acid derivatives of NSC13778 for their ability to inhibit the DNA binding of five B-ZIP dimers (c-Fos|JunD, VBP, C/EBPalpha, C/EBPbeta, and CREB). They show various specificities at inhibiting the DNA binding of five B-ZIP domains. NSC13746 inhibits the DNA binding of C/EBPbeta and CREB at 100nM and promiscuously inhibiting the DNA binding of all five proteins in the 1muM range. Dialysis experiments indicate that NSC 13746 binding to the B-ZIP domain is reversible. Thermal denaturation studies indicate that NSC13746 binds the B-ZIP domain. Some compounds specifically inhibit DNA binding, with VBP and c-Fos|JunD being most easily disrupted. These compounds inhibit, with similar specificities to the pure B-ZIP domains, the DNA binding of nuclear extract to the AP1 DNA sequence but no inhibition is observed to SP1 containing oligonucleotide. Transient transfection assays indicate that NSC13746 can inhibit the TPA induced activation of two B-ZIP dependent reporters. These experiments suggest that arylstibonic acids are promising leads for inhibiting the DNA binding of a group of B-ZIP proteins in cells.
Laminin chain assembly is regulated by specific coiled-coil interactions
Venue: Journal of structural biology

Laminins are large heterotrimeric, multidomain proteins that play a central role in organising and establishing all basement membranes. Despite a total of 45 potential heterotrimeric chain combinations formed through the coiled-coil domain of the 11 identified laminin chains (α1–5, β1–3, γ1–3), to date only 15 different laminin isoforms have been reported. This observation raises the question whether laminin assembly is regulated by differential gene expression or specific chain recognition. To address this issue, we here perform a complete analysis of laminin chain assembly and specificity. Using biochemical and biophysical techniques, all possible heterotrimeric combinations from recombinant C-terminal coiled-coil fragments of all chains were analysed. Apart from laminin 323 (α3, β2, γ3), for which no biochemical evidence of its existence in vivo is available, these experiments confirmed all other known laminin isoforms and identified two novel potential chain combinations, laminins 312 (α3, β1, γ2) and 422 (α4, β2, γ4). Our findings contribute to the understanding of basement membrane structure, function and diversity.
Recent advances in the production of proteins in insect and mammalian cells for structural biology.
Venue: Journal of structural biology

The production of proteins in sufficient quantity and of appropriate quality is an essential pre-requisite for structural studies. Escherichia coli remains the dominant expression system in structural biology with nearly 90% of the structures in the Protein Data Bank (PDB) derived from proteins produced in this bacterial host. However, many mammalian and eukaryotic viral proteins require post-translation modification for proper folding and/or are part of large multimeric complexes. Therefore expression in higher eukaryotic cell lines from both invertebrate and vertebrate is required to produce these proteins. Although these systems are generally more time-consuming and expensive to use than bacteria, there have been improvements in technology that have streamlined the processes involved. For example, the use of multi-host vectors, i.e., containing promoters for not only E. coli but also mammalian and baculovirus expression in insect cells, enables target genes to be evaluated in both bacterial and higher eukaryotic hosts from a single vector. Culturing cells in micro-plate format allows screening of large numbers of vectors in parallel and is amenable to automation. The development of large-scale transient expression in mammalian cells offers a way of rapidly producing proteins with relatively high throughput. Strategies for selenomethionine-labelling (important for obtaining phase information in crystallography) and controlling glycosylation (important for reducing the chemical heterogeneity of glycoproteins) have also been reported for higher eukaryotic cell expression systems.