nils-herrmann/clean_scirepeval huggingface dataset:
Serial block-face scanning electron microscopy for three-dimensional analysis of morphological changes in mitochondria regulated by Cdc48p/p97 ATPase.
Venue: Journal of structural biology

Cdc48p is a highly conserved cytosolic AAA chaperone that is involved in a wide range of cellular processes. It consists of two ATPase domains (D1 and D2), with regulatory regions at the N- and C-terminals. We have recently shown that Cdc48p regulates mitochondrial morphology, in that a loss of the ATPase activity or positive cooperativity in the D2 domain leads to severe fragmentations and aggregations of mitochondria in the cytoplasm. We have now used serial block-face scanning electron microscopy (SBF-SEM), an advanced three-dimensional (3D) electron microscopic technique to examine the structures and morphological changes of mitochondria in the yeast Saccharomyces cerevisiae. We found that mutants lacking ATPase activity of Cdc48p showed mitochondrial fragmentations and aggregations, without fusion of the outer membrane. This suggests that the ATPase activity of Cdc48p is necessary for fusion of the outer membranes of mitochondria. Our results also show that SBF-SEM has considerable advantages in morphological and quantitative studies on organelles and intracellular structures in entire cells.
Structural investigation of the interaction between the tandem SH3 domains of c-Cbl-associated protein and vinculin.
Venue: Journal of structural biology

c-Cbl-associated protein (CAP) is an important cytoskeletal adaptor protein involved in the regulation of adhesion turnover. The interaction between CAP and vinculin is critical for the recruitment of CAP to focal adhesions. The tandem SH3 domains (herein termed SH3a and SH3b) of CAP are responsible for its interaction with vinculin. However, the structural mechanism underlying the interaction between CAP and vinculin is poorly understood. In this manuscript, we report the solution structure of the tandem SH3 domains of CAP. Our NMR and ITC data indicate that the SH3a and SH3b domains of CAP simultaneously bind to a long proline-rich region of vinculin with different binding specificities. Furthermore, the crystal structures of the individual SH3a and SH3b domains complexed with their substrate peptides indicate that Q807(SH3a) and D881(SH3b) are the critical residues determining the different binding specificities of the SH3 domains. Based on the obtained structural information, a model of the SH3ab-vinculin complex was generated using MD simulation and SAXS data.
Alpbach special issue.
Venue: Journal of structural biology

1047-8477/ 2014 Coils, Collagen and Fibrous Proteins was held at Alpbach, Austria in September 2013. As usual, it attracted delegates with structural interests in those protein systems where the coiled-coil motif plays an important role. These days, of course, this is not confined to the world of fibrous proteins but is found equally commonly in the globular ones too. Developments in both biophysical and biochemical aspects of the field proved a fertile ground for discussion, as did the interactions between those scientists whose interests lay primarily either in the extracellular proteins or the intracellular proteins (but, prior to Alpbach, rarely in both). The Special Issue presented here reveals a selection of the contributions made at the Workshop, and thereby provides a snapshot of where the field is now and where it is going over the next few years. Increasing numbers of crystal structures of the coiled-coil conformations observed in globular and fibrous proteins have revealed many more details of the relationship between amino acid sequence and the conformation observed. There is, of course, much more to the coiled coil than its structure alone, and the role of specific sequences that bestow a distinct function on these motifs has become more evident. Another point of considerable interest to emerge was that the conformations of the short regions between stretches of continuous heptad substructure are now describable at the atomic level. This refers not only to the wellestablished stutters, stammers and skips (deletions of three, four and six residues, respectively) where previous crystal data and modeling studies have been presented, but also now to the three other less common possibilities (deletions of one, two and five residues). An analogous study of breaks in the triplet phasing in collagen sequences was also presented. It has also became clear that the silk structures, and not those with the short sequence repeats that dominated the field in its early years, are increasingly being studied in detail, and these too are proving extremely informative. Some aspects of the 6th Workshop were different from those that preceded it. Importantly, Andrei Lupas and Dek Woolfson have taken over from John Squire and me as the new organizers. In so doing they have highlighted a variety of globular coiled-coil protein systems as well as biomaterials and silks. Core areas of research in the collagens and other a-fibrous proteins have been retained, of course, as has the compilation of a Special Issue of the Journal of Structural Biology. Our thanks go to the Editor, Dr. Alasdair Steven, for his support and encouragement in this particular venture. His support of the specialized field of coiled coils and fibrous proteins remains of great importance to us. In summary, protein researchers of all types will find much of interest in this Special Issue and the reader is strongly encouraged to investigate some of the treasures included.
Reprint of: keratin intermediate filaments: differences in the sequences of the Type I and Type II chains explain the origin of the stability of an enzyme-resistant four-chain fragment.
Venue: Journal of structural biology

Previous studies have shown that a strong interaction exists between oppositely directed 1B molecular segments in the intermediate filaments of trichocyte keratins. A similar interaction has been identified as having a significant role in the formation of unit-length filaments, a precursor to intermediate filament formation. The present study is concerned with the spatial relationship of these interacting segments and its dependence on differences in the amino acid sequences of the two-chain regions that constitute the 1B molecular segment. It is shown that along a particular line of contact both chain segments possess an elevated concentration of residues with a high propensity for dimer formation. The transition from the reduced to the oxidized state involves a simple axial displacement of one molecular segment relative to the other, with no attendant rotation of either segment. This changes the inter-relationship of the two 1B molecular segments from a loosely packed form to a more compact one. After the slippage eight of the cysteine residues in the dimer are precisely aligned to link up and form the disulfide linkages as observed. The two remaining cysteine residues are located on the outside of the dimer and are presumably involved in inter-dimer bonding. The existence of a unique line of contact requires that two chains in the molecule have different amino acid compositions with the clustering of dimer-favoring residues phased by half the pitch length of the coiled coil.
M-free: scoring the reference bias in sub-tomogram averaging and template matching.
Venue: Journal of structural biology

Cryo-electron tomography provides a snapshot of the cellular proteome. With template matching, the spatial positions of various macromolecular complexes within their native cellular context can be detected. However, the growing awareness of the reference bias introduced by the cross-correlation based approaches, and more importantly the lack of a reliable confidence measurement in the selection of these macromolecular complexes, has restricted the use of these applications. Here we propose a heuristic, in which the reference bias is measured in real space in an analogous way to the R-free value in X-ray crystallography. We measure the reference bias within the mask used to outline the area of the template, and do not modify the template itself. The heuristic works by splitting the mask into a working and a testing area in a volume ratio of 9:1. While the working area is used during the calculation of the cross-correlation function, the information from both areas is explored to calculate the M-free score. We show using artificial data, that the M-free score gives a reliable measure for the reference bias. The heuristic can be applied in template matching and in sub-tomogram averaging. We further test the applicability of the heuristic in tomograms of purified macromolecules, and tomograms of whole Mycoplasma cells.
Heparin induced dimerization of APP is primarily mediated by E1 and regulated by its acidic domain.
Venue: Journal of structural biology

The amyloid precursor protein (APP) and its cellular processing are believed to be centrally involved in the etiology of Alzheimer's disease (AD). In addition, many physiological functions have been described for APP, including a role in cell-cell- and cell-ECM-adhesion as well as in axonal outgrowth. We show here the molecular determinants of the oligomerization/dimerization of APP, which is central for its cellular (mis)function. Using size exclusion chromatography (SEC), dynamic light scattering and SEC-coupled static light scattering we demonstrate that the dimerization of APP is energetically induced by a heparin mediated dimerization of the E1 domain, which results in a dimeric interaction of E2. We also show that the acidic domain (AcD) interferes with the dimerization of E1 and propose a model where both, cis- and trans-dimerization occur dependent on cellular localization and function.
Crystal structure of the transport unit of the autotransporter adhesin involved in diffuse adherence from Escherichia coli.
Venue: Journal of structural biology

Several serious gastrointestinal diseases, which are widespread all over the world, are caused by enteropathogenic Escherichia coli. The monomeric autotransporter AIDA-I (adhesin involved in diffuse adherence) represents an important virulence factor of these strains and is involved in adhesion, biofilm formation, aggregation and invasion into host cells. Here, we present the crystal structure of the transport unit of AIDA-I at 3.0Å resolution, which forms a 12-stranded β-barrel harboring the linker domain in its pore. Mutagenesis studies of the C-terminal amino acid demonstrated the great impact of this terminal residue on membrane integration of AIDA-I and passenger translocation.
Spherical deconvolution improves quality of single particle reconstruction.
Venue: Journal of structural biology

One single-particle reconstruction technique is the reconstruction of macromolecules from projection images of randomly oriented particles (SPRR). In SPRR the reliability and consequent interpretation of the final reconstruction is affected by errors arising from incorrect assignment of projection angles to individual particles. In order to improve the resolution of SPRR we studied the influence of imperfect assignment on 3D blurring. We find that this blurring can be described as a Point Spread Function (PSF) that depends on the distance from geometrical center of the reconstructed volume and that blurring is higher at the periphery. This particular PSF can be described by an almost pure tangential angular function with a negligible radial component. We have developed a reliable algorithm for spherical deconvolution of the 3D reconstruction. This spherical deconvolution operation was tested on reconstructions of GroEL and mitochondrial ribosomes. We show that spherical deconvolution improves the quality of SPRR by reducing blurring and enhancing high frequency components, particularly near the periphery of the reconstruction.
In silico analysis and experimental verification of OSR1 kinase - Peptide interaction.
Venue: Journal of structural biology

The oxidative-stress-responsive kinase 1 (OSR1) and the STE20/SPS1-related proline/alanine-rich kinase (SPAK) are key enzymes in a signaling cascade regulating the activity of Na(+)-K(+)-2Cl(-) cotransporters (NKCC1-2) and Na(+)-Cl(-) cotransporter (NCC). Both kinases have a conserved carboxyl-terminal (CCT) domain, which recognizes a unique peptide motif present in OSR1- and SPAK-activating kinases (with-no-lysine kinase 1 (WNK1) and WNK4) as well as their substrates (NKCC1, NKCC2, and NCC). Utilizing various modalities of the Rosetta Molecular Modeling Software Suite including flexible peptide docking and protein design, we comprehensively explored the sequence space recognized by the CCT domain. Specifically, we studied single residue mutations as well as complete unbiased designs of a hexapeptide substrate. The computational study started from a crystal structure of the CCT domain of OSR1 in complex with a hexapeptide derived from WNK4. Point mutations predicted to be favorable include Arg to His or Trp substitutions at position 2 and a Phe to Tyr substitution at position 3 of the hexapeptide. In addition, de novo design yielded two peptides predicted to bind to the CCT domain: FRFQVT and TRFDVT. These results, which indicate a little bit more freedom in the composition of the peptide, were confirmed through the use of yeast two-hybrid screening.
Oxygen-aromatic contacts in intra-strand base pairs: analysis of high-resolution DNA crystal structures and quantum chemical calculations.
Venue: Journal of structural biology

Three-dimensional structures of biomolecules are stabilized by a large number of non-covalent interactions and some of them such as van der Waals, electrostatic and hydrogen bond interactions are well characterized. Delocalized π-electron clouds of aromatic residues are known to be involved in cation-π, CH-π, OH-π and π-π interactions. In proteins, many examples have been found in which the backbone carbonyl oxygen of one residue makes close contact with the aromatic center of aromatic residues. Quantum chemical calculations suggest that such contacts may provide stability to the protein secondary structures. In this study, we have systematically analyzed the experimentally determined high-resolution DNA crystal structures and identified 91 examples in which the aromatic center of one base is in close contact (<3.5Ǻ) with the oxygen atom of preceding (Group-I) or succeeding base (Group-II). Examples from Group-I are overwhelmingly observed and cytosine or thymine is the preferred base contributing oxygen atom in Group-I base pairs. A similar analysis of high-resolution RNA structures surprisingly did not yield many examples of oxygen-aromatic contact of similar type between bases. Ab initio quantum chemical calculations on compounds based on DNA crystal structures and model compounds show that interactions between the bases in base pairs with oxygen-aromatic contacts are energetically favorable. Decomposition of interaction energies indicates that dispersion forces are the major cause for energetically stable interaction in these base pairs. We speculate that oxygen-aromatic contacts in intra-strand base pairs in a DNA structure may have biological significance.
Molecular dynamics investigation of the active site dynamics of mycobacterial cyclopropane synthase during various stages of the cyclopropanation process.
Venue: Journal of structural biology

Mycobacterial cyclopropane synthase 1 (CmaA1) is one of the most important drug targets in anti tuberculosis drug discovery as it is responsible for cis-cyclopropanation at the distal position of unsaturated mycolates, which is an essential step for the pathogenicity, persistence and drug resistance. Five representative models of CmaA1 which correspond to different stages in the cyclopropanation process have been studied using molecular dynamics (MD) simulations. The MD simulations and structural analyses provide a detailed account of the structural changes in the active sites of CmaA1. CmaA1 has two distinct binding sites, i.e., cofactor binding site (CBS) and acyl substrate binding site (ASBS). The apo state of CmaA1 corresponds to a closed conformation where the CBS is inaccessible due to the existence of H-bond between Pro202 of loop10 (L10) and Asn11 of N-terminal α1 helix. However, cofactor binding leads to the breaking of this H-bond and thus the H-bond is absent in the holo form. The hydrophobic side chains orient towards the inner side of the ASBS upon cofactor binding to create a hydrophobic environment for the substrate. The cofactor and substrate tend to come close to each other facilitated by opening of L10 to exchange the methyl group from the cofactor to the substrate. The MD study also revealed that the system tends to regain the apo conformation within 40ns after releasing the product.
Single-step antibody-based affinity cryo-electron microscopy for imaging and structural analysis of macromolecular assemblies.
Venue: Journal of structural biology

Single particle cryo-electron microscopy (cryo-EM) is an emerging powerful tool for structural studies of macromolecular assemblies (i.e., protein complexes and viruses). Although single particle cryo-EM requires less concentrated and smaller amounts of samples than X-ray crystallography, it remains challenging to study specimens that are low-abundance, low-yield, or short-lived. The recent development of affinity grid techniques can potentially further extend single particle cryo-EM to these challenging samples by combining sample purification and cryo-EM grid preparation into a single step. Here we report a new design of affinity cryo-EM approach, cryo-SPIEM, that applies a traditional pathogen diagnosis tool Solid Phase Immune Electron Microscopy (SPIEM) to the single particle cryo-EM method. This approach provides an alternative, largely simplified and easier to use affinity grid that directly works with most native macromolecular complexes with established antibodies, and enables cryo-EM studies of native samples directly from cell cultures. In the present work, we extensively tested the feasibility of cryo-SPIEM with multiple samples including those of high or low molecular weight, macromolecules with low or high symmetry, His-tagged or native particles, and high- or low-yield macromolecules. Results for all these samples (non-purified His-tagged bacteriophage T7, His-tagged Escherichiacoli ribosomes, native Sindbis virus, and purified but low-concentration native Tulane virus) demonstrated the capability of cryo-SPIEM approach in specifically trapping and concentrating target particles on TEM grids with minimal view constraints for cryo-EM imaging and determination of 3D structures.
Structural insights into regulatory mechanisms of MO25-mediated kinase activation.
Venue: Journal of structural biology

The tumor suppressor kinase LKB1 and germinal center kinases (GCKs) are key regulators of various cellular functions. The adaptor molecule MO25 not only recruits and activates LKB1 through the pseudokinase STRAD, but also may directly activate GCKs like MST3, MST4, STK25, OSR1 and SPAK. Targeting MO25 in a pathological setting has been recently studied in mouse. Yet the regulatory mechanism of MO25-mediated kinase activation is not fully understood. Here, our structural studies of MO25-related kinases reveal that MO25 binds to and activates GCK kinases or pseudokinase through a unified structural mechanism, featuring an active conformation of the αC helix and A-loop stabilized by MO25. Compared to GCKs that are directly activated by MO25-binding, activation of LKB1 has evolved additional layer of regulatory machinery, i.e., MO25 "activates" the pseudokinase STRAD, which in turn activates LKB1. Importantly, the structures of MO25α-STK25 and MO25α-MST3 determined in this work represent a transition/intermediate state and a fully activated state, respectively during the MO25-mediated kinase activating process.
Stretching desmin filaments with receding meniscus reveals large axial tensile strength.
Venue: Journal of structural biology

Desmin forms the intermediate filament system of muscle cells where it plays important role in maintaining mechanical integrity and elasticity. Although the importance of intermediate-filament elasticity in cellular mechanics is being increasingly recognized, the molecular basis of desmin's elasticity is not fully understood. We explored desmin elasticity by molecular combing with forces calculated to be as large as 4nN. Average filament contour length increased 1.55-fold axial on average. Molecular combing together with EGTA-treatment caused the fragmentation of the filament into short, 60 to 120-nm-long and 4-nm-wide structures. The fragments display a surface periodicity of 38nm, suggesting that they are composed of laterally attached desmin dimers. The axis of the fragments may deviate significantly from that of the overstretched filament, indicating that they have a large orientational freedom in spite of being axially interconnected. The emergence of protofibril fragments thus suggests that the interconnecting head or tail domains of coiled-coil desmin dimers are load-bearing elements during axial stretch.
Nano-thrombelastography of fibrin during blood plasma clotting.
Venue: Journal of structural biology

Hemostasis is a complex process that relies on the sensitive balance between the formation and breakdown of the thrombus, a three-dimensional polymer network of the fibrous protein fibrin. Neither the details of the fibrinogen-fibrin transition, nor the exact mechanisms of fibrin degradation are fully understood at the molecular level. In the present work we investigated the nanoscale-changes in the viscoelasticity of the 3D-fibrin network during fibrinogenesis and streptokinase (STK)-induced fibrinolysis by using a novel application of force spectroscopy, named nano-thrombelastography. In this method the changes in the bending of an oscillating atomic-force-microscope (AFM) cantilever in human blood-plasma droplet were followed as a function of time. Whereas the global features of the time-dependent change in cantilever deflection corresponded well to a macroscopic thrombelastogram, the underlying force spectra revealed large, sample-dependent oscillations in the range of 3-50nN and allowed the separation of elastic and viscous components of fibrin behavior. Upon STK treatment the nano-thrombelastogram signal decayed gradually. The decay was driven by a decrease in thrombus elasticity, whereas thrombus viscosity decayed with a time delay. In scanning AFM images mature fibrin appeared as 17-nm-high and 12-196-nm-wide filaments. STK-treatment resulted in the decrease of filament height and the appearance of a surface roughness with 23.7nm discrete steps that corresponds well to the length of a fibrinogen monomer. Thus, the initial decay of thrombus elasticity during fibrinolysis may be caused by the axial rupture of fibrin fibers.