nils-herrmann/clean_scirepeval huggingface dataset:
Algorithm for selection of optimized EPR distance restraints for de novo protein structure determination.
Venue: Journal of structural biology

A hybrid protein structure determination approach combining sparse Electron Paramagnetic Resonance (EPR) distance restraints and Rosetta de novo protein folding has been previously demonstrated to yield high quality models (Alexander et al. (2008)). However, widespread application of this methodology to proteins of unknown structures is hindered by the lack of a general strategy to place spin label pairs in the primary sequence. In this work, we report the development of an algorithm that optimally selects spin labeling positions for the purpose of distance measurements by EPR. For the α-helical subdomain of T4 lysozyme (T4L), simulated restraints that maximize sequence separation between the two spin labels while simultaneously ensuring pairwise connectivity of secondary structure elements yielded vastly improved models by Rosetta folding. 54% of all these models have the correct fold compared to only 21% and 8% correctly folded models when randomly placed restraints or no restraints are used, respectively. Moreover, the improvements in model quality require a limited number of optimized restraints, which is determined by the pairwise connectivities of T4L α-helices. The predicted improvement in Rosetta model quality was verified by experimental determination of distances between spin labels pairs selected by the algorithm. Overall, our results reinforce the rationale for the combined use of sparse EPR distance restraints and de novo folding. By alleviating the experimental bottleneck associated with restraint selection, this algorithm sets the stage for extending computational structure determination to larger, traditionally elusive protein topologies of critical structural and biochemical importance.
Hinge-loop mutation can be used to control 3D domain swapping and amyloidogenesis of human cystatin C.
Venue: Journal of structural biology

Cystatins are natural inhibitors of cysteine proteases, enzymes that are widely distributed in animals, plants, and microorganisms. Human cystatin C (hCC) has been also recognized as an aggregating protein directly involved in the formation of pathological amyloid fibrils, and these amyloidogenic properties greatly increase in a naturally occurring L68Q hCC variant. For a long time only dimeric structure of wild-type hCC has been known. The dimer is created through 3D domain swapping process, in which two parts of the cystatin structure become separated from each other and next exchanged between two molecules. Important role in the domain swapping plays the L1 loop, which connects the exchanging segments and, upon dimerization, transforms from a β-turn into a part of a long β-strand. In the very recently published first monomeric structure of human cystatin C (hCC-stab1), dimerization was abrogated due to clasping of the β-strands from the swapping domains by an engineered disulfide bridge. We have designed and constructed another mutated cystatin C with the smallest possible structural intervention, that is a single-point mutation replacing hydrophobic V57 from the L1 loop by polar asparagine, known as a stabilizer of a β-turn motif. V57N hCC mutant occurred to be stable in its monomeric form and crystallized as a monomer, revealing typical cystatin fold with a five-stranded antiparallel β-sheet wrapped around an α-helix. Here we report a 2.04 Å resolution crystal structure of V57N hCC and discuss the architecture of the protein in comparison to chicken cystatin, hCC-stab1 and dimeric hCC.
Structural NMR of protein oligomers using hybrid methods.
Venue: Journal of structural biology

Solving structures of native oligomeric protein complexes using traditional high-resolution NMR techniques remains challenging. However, increased utilization of computational platforms, and integration of information from less traditional NMR techniques with data from other complementary biophysical methods, promises to extend the boundary of NMR-applicable targets. This article reviews several of the techniques capable of providing less traditional and complementary structural information. In particular, the use of orientational constraints coming from residual dipolar couplings and residual chemical shift anisotropy offsets are shown to simplify the construction of models for oligomeric complexes, especially in cases of weak homo-dimers. Combining this orientational information with interaction site information supplied by computation, chemical shift perturbation, paramagnetic surface perturbation, cross-saturation and mass spectrometry allows high resolution models of the complexes to be constructed with relative ease. Non-NMR techniques, such as mass spectrometry, EPR and small angle X-ray scattering, are also expected to play increasingly important roles by offering alternative methods of probing the overall shape of the complex. Computational platforms capable of integrating information from multiple sources in the modeling process are also discussed in the article. And finally a new, detailed example on the determination of a chemokine tetramer structure will be used to illustrate how a non-traditional approach to oligomeric structure determination works in practice.
Role of DNA-DNA interactions on the structure and thermodynamics of bacteriophages Lambda and P4.
Venue: Journal of structural biology

Electrostatic interactions play an important role in both packaging of DNA inside bacteriophages and its release into bacterial cells. While at physiological conditions DNA strands repel each other, the presence of polyvalent cations such as spermine and spermidine in solutions leads to the formation of DNA condensates. In this study, we discuss packaging of DNA into bacteriophages P4 and Lambda under repulsive and attractive conditions using a coarse-grained model of DNA and capsids. Packaging under repulsive conditions leads to the appearance of the coaxial spooling conformations; DNA occupies all available space inside the capsid. Under the attractive potential both packed systems reveal toroidal conformations, leaving the central part of the capsids empty. We also present a detailed thermodynamic analysis of packaging and show that the forces required to pack the genomes in the presence of polyamines are significantly lower than those observed under repulsive conditions. The analysis reveals that in both the repulsive and attractive regimes the entropic penalty of DNA confinement has a significant non-negligible contribution into the total energy of packaging. Additionally we report the results of simulations of DNA condensation inside partially packed Lambda. We found that at low densities DNA behaves as free unconfined polymer and condenses into the toroidal structures; at higher densities rearrangement of the genome into toroids becomes hindered, and condensation results in the formation of non-equilibrium structures. In all cases packaging in a specific conformation occurs as a result of interplay between bending stresses experienced by the confined polymer and interactions between the strands.
NMR and small-angle scattering-based structural analysis of protein complexes in solution.
Venue: Journal of structural biology

Structural analysis of multi-domain protein complexes is a key challenge in current biology and a prerequisite for understanding the molecular basis of essential cellular processes. The use of solution techniques is important for characterizing the quaternary arrangements and dynamics of domains and subunits of these complexes. In this respect solution NMR is the only technique that allows atomic- or residue-resolution structure determination and investigation of dynamic properties of multi-domain proteins and their complexes. As experimental NMR data for large protein complexes are sparse, it is advantageous to combine these data with additional information from other solution techniques. Here, the utility and computational approaches of combining solution state NMR with small-angle X-ray and Neutron scattering (SAXS/SANS) experiments for structural analysis of large protein complexes is reviewed. Recent progress in experimental and computational approaches of combining NMR and SAS are discussed and illustrated with recent examples from the literature. The complementary aspects of combining NMR and SAS data for studying multi-domain proteins, i.e. where weakly interacting domains are connected by flexible linkers, are illustrated with the structural analysis of the tandem RNA recognition motif (RRM) domains (RRM1-RRM2) of the human splicing factor U2AF65 bound to a nine-uridine (U9) RNA oligonucleotide.
Effects of phosphorylation on the self-assembly of native full-length porcine amelogenin and its regulation of calcium phosphate formation in vitro.
Venue: Journal of structural biology

The self-assembly of the predominant extracellular enamel matrix protein amelogenin plays an essential role in regulating the growth and organization of enamel mineral during early stages of dental enamel formation. The present study describes the effect of the phosphorylation of a single site on the full-length native porcine amelogenin P173 on self-assembly and on the regulation of spontaneous calcium phosphate formation in vitro. Studies were also conducted using recombinant non-phosphorylated (rP172) porcine amelogenin, along with the most abundant amelogenin cleavage product (P148) and its recombinant form (rP147). Amelogenin self-assembly was assessed using dynamic light scattering (DLS) and transmission electron microscopy (TEM). Using these approaches, we have shown that self-assembly of each amelogenin is very sensitive to pH and appears to be affected by both hydrophilic and hydrophobic interactions. Furthermore, our results suggest that the phosphorylation of the full-length porcine amelogenin P173 has a small but potentially important effect on its higher-order self-assembly into chain-like structures under physiological conditions of pH, temperature, and ionic strength. Although phosphorylation has a subtle effect on the higher-order assembly of full-length amelogenin, native phosphorylated P173 was found to stabilize amorphous calcium phosphate for extended periods of time, in sharp contrast to previous findings using non-phosphorylated rP172. The biological relevance of these findings is discussed.
Multiple dense particle tracking in fluorescence microscopy images based on multidimensional assignment.
Venue: Journal of structural biology

Multiple particle tracking (MPT) has seen numerous applications in live-cell imaging studies of subcellular dynamics. Establishing correspondence between particles in a sequence of frames with high particle density, particles merging and splitting, particles entering and exiting the frame, temporary particle disappearance, and an ill-performing detection algorithm is the most challenging part of MPT. Here we propose a tracking method based on multidimensional assignment to address these problems. We combine an Interacting Multiple Model (IMM) filter, multidimensional assignment, particle occlusion handling, and merge-split event detection in a single software analysis package. The main advantage of a multidimensional assignment is that both spatial and temporal information can be used by using several later frames as reference. The IMM filter, which is used to maintain and predict the state of each track, contains several models which correspond to different types of biologically realistic movements. It works especially well with multidimensional assignment, because there tends to be a higher probability of correct particle association over time. First the method generates many particle-correspondence hypotheses, merge-split hypotheses and misdetection hypotheses within the framework of a sliding window over the frames of the image sequence. Then it builds a multidimensional assignment problem (MAP) accordingly. The particle is tracked with gap-filling, and merging and splitting events are then detected using the MAP solution. The tracking method is validated on both simulated tracks and microscopy image sequences. The results of these experiments show that the method is more accurate and robust than other "tracking from detected features" methods in dense particle situations.
Crystal structure of the mucin-binding domain of Spr1345 from Streptococcus pneumoniae.
Venue: Journal of structural biology

The surface protein Spr1345 from Streptococcus pneumoniae R6 is a 22-kDa mucin-binding protein (MucBP) involved in adherence and colonization of the human lung and respiratory tract. It is composed of a mucin-binding domain (MucBD) and a proline-rich domain (PRD) followed by an LPxTG motif, which is recognized and cleaved by sortase, resulting in a mature form of 171 residues (MF171) that is anchored to the cell wall. We found that the MucBD alone possesses comparable in vitro mucin-binding affinity to the mature form, and can be specifically enriched at the surface of human lung carcinoma A549 cells. Using single-wavelength anomalous dispersion (SAD) phasing method with the iodine signals, we solved the crystal structure of the MucBD at 2.0Å resolution, the first structure of MucBDs from pathogenic bacteria. The overall structure adopts an immunoglobulin-like fold with an elongated rod-like shape, composed of six anti-parallel β-strands and a long loop. Structural comparison suggested that the conserved C-terminal moiety may participate in the recognition of mucins. These findings provided structural insights into host-pathogen interaction mediated by mucins, which might be useful for designing novel vaccines and antibiotic drugs against human diseases caused by pneumococci.
Dual-axis electron tomography of biological specimens: Extending the limits of specimen thickness with bright-field STEM imaging.
Venue: Journal of structural biology

The absence of imaging lenses after the specimen in the scanning transmission electron microscope (STEM) enables electron tomography to be performed in the STEM mode on micrometer-thick plastic-embedded specimens without the deleterious effect of chromatic aberration, which limits spatial resolution and signal-to-noise ratio in conventional TEM. Using Monte Carlo calculations to simulate electron scattering from gold nanoparticles situated at the top and bottom surfaces of a plastic section, we assess the optimal acquisition strategy for axial bright-field STEM electron tomography at a beam-energy of 300keV. Dual tilt-axis STEM tomography with optimized axial bight-field detector geometry is demonstrated by application to micrometer-thick sections of beta cells from mouse pancreatic islet. The quality of the resulting three-dimensional reconstructions is comparable to that obtained from much thinner (0.3-micrometer) sections using conventional TEM tomography. The increased range of specimen thickness accessible to axial STEM tomography without the need for serial sectioning enables the 3-D visualization of more complex and larger subcellular structures.
The Kaposi's sarcoma-associated herpesvirus ORF6 DNA binding protein forms long DNA-free helical protein filaments.
Venue: Journal of structural biology

The Kaposi's sarcoma-associated herpesvirus ORF6 has a 41% sequence identity with Balf2 protein of Epstein-Barr virus and 23% with ICP8 protein of Herpes Simplex type I. Balf2 and ICP8 are multi-functional DNA binding proteins with roles central to viral DNA replication and recombination. In this study, we cloned the KSHV ORF6 gene, expressed the full length ORF6 protein in insect cells and purified it to homogeneity. Gel filtration revealed the protein to be present in a broad spectrum of sizes ranging from monomers to high molecular weight oligomers. Transmission electron microscopy (TEM) using negative staining under conditions favoring monomers and small oligomers revealed fields of globular particles measuring 11nm in diameter consistent with the size of a protein monomer. Incubation of ORF6 protein at room temperature for extended periods of time resulted in the bulk of the protein forming very long helical filaments. Measurements from negative staining revealed that the filaments were up to 2600nm in length, with a width of 13.7nm and a long gentle helical periodicity of 42.9nm along the filament axis. Using rapid freezing and freeze-drying, it was possible to show that the filaments consist of two protein chains wrapped around each other. The possibility that these protein filaments generate a scaffold upon which viral DNA replication, recombination, and encapsidation occur in the infected cell nucleus is discussed.
Toward understanding the mechanism of action of the yeast multidrug resistance transporter Pdr5p: a molecular modeling study.
Venue: Journal of structural biology

Pleotropic drug resistant protein 5 (Pdr5p) is a plasma membrane ATP-binding cassette (ABC) transporter and the major drug efflux pump in Saccharomyces cerevisiae. The Pdr5p family of fungal transporters possesses a number of structural features significantly different from other modeled or crystallized ABC transporters, which include a reverse topology, an atypical ATP-binding site, a very low sequence similarity in the transmembrane section and long linkers between domains. These features present a considerable hurdle in molecular modeling studies of these important transporters. Here, we report the creation of an atomic model of Pdr5p based on a combination of homology modeling and ab initio methods, incorporating information from consensus transmembrane segment prediction, residue lipophilicity, and sequence entropy. Reported mutations in the transmembrane substrate-binding pocket that altered drug-resistance were used to validate the model, and one mutation that changed the communication pattern between transmembrane and nucleotide-binding domains was used in model improvement. The predictive power of the model was demonstrated experimentally by the increased sensitivity of yeast mutants to clotrimazole having alanine substitutions for Thr1213 and Gln1253, which are predicted to be in the substrate-binding pocket, without reducing the amount of Pdr5p in the plasma membrane. The quality and reliability of our model are discussed in the context of various approaches used for modeling different parts of the structure.
The beginning of a beautiful friendship: Cross-linking/mass spectrometry and modelling of proteins and multi-protein complexes
Venue: Journal of structural biology

After more than a decade of method development, cross-linking in combination with mass spectrometry and bioinformatics is finally coming of age. This technology now provides improved opportunities for modelling by mapping structural details of functional complexes in solution. The structure of proteins or protein complexes is ascertained by identifying amino acid pairs that are positioned in close proximity to each other. The validity of this technique has recently been benchmarked for large multi-protein complexes, by comparing cross-link data with that from a crystal structure of RNA polymerase II. Here, the specific nature of this cross-linking data will be discussed to assess the technical challenges and opportunities for model building. We believe that once remaining technological challenges of cross-linking/mass spectrometry have been addressed and cross-linking/mass spectrometry data has been incorporated into modelling algorithms it will quickly become an indispensable companion of protein and protein complex modelling and a corner-stone of integrated structural biology.
RosettaEPR: an integrated tool for protein structure determination from sparse EPR data.
Venue: Journal of structural biology

Site-directed spin labeling electron paramagnetic resonance (SDSL-EPR) is often used for the structural characterization of proteins that elude other techniques, such as X-ray crystallography and nuclear magnetic resonance (NMR). However, high-resolution structures are difficult to obtain due to uncertainty in the spin label location and sparseness of experimental data. Here, we introduce RosettaEPR, which has been designed to improve de novo high-resolution protein structure prediction using sparse SDSL-EPR distance data. The "motion-on-a-cone" spin label model is converted into a knowledge-based potential, which was implemented as a scoring term in Rosetta. RosettaEPR increased the fractions of correctly folded models (  see text] <7.5Å) and models accurate at medium resolution (  see text] <3.5Å) by 25%. The correlation of score and model quality increased from 0.42 when using no restraints to 0.51 when using bounded restraints and again to 0.62 when using RosettaEPR. This allowed for the selection of accurate models by score. After full-atom refinement, RosettaEPR yielded a 1.7Å model of T4-lysozyme, thus indicating that atomic detail models can be achieved by combining sparse EPR data with Rosetta. While these results indicate RosettaEPR's potential utility in high-resolution protein structure prediction, they are based on a single example. In order to affirm the method's general performance, it must be tested on a larger and more versatile dataset of proteins.
Chromosomal dynamics of cell cycle regulator gene p21 during transcriptional activation.
Venue: Journal of structural biology

The radial position of a gene within its chromosome territory (CT) in the interphase nucleus is thought to depend on the transcriptional activity of the gene and on transcriptional activity, gene density, and conformation of the chromosomal surrounding. In this study we analyzed the position of the cell cycle regulator gene p21 within the CT of human chromosome 6 (HSA6) upon transcriptional activation. Whereas the majority of active p21 genes is located in the interior of the CT of HSA6, induction of p21 transcription correlates with increased variation of gene localization within the CT and with a higher percentage of p21 genes located at the periphery of the CT. Additionally it demonstrates once more that transcription can take place throughout CTs. Comparison of the p21 locus with two non-coding regions on HSA6 showed that both non-coding sequences are located more frequently in the interior of the CT than p21 genes although they are situated in chromosomal neighborhoods with widely differing gene density and regional transcriptional activity. Thus our data support models describing an influence of the transcriptional activity of a gene on the localization within its CT. However, our data also indicate that additional factors such as chromatin remodeling are implicated in the positioning of genes within the respective chromosome territory.
NMR structure of the human Mediator MED25 ACID domain.
Venue: Journal of structural biology

MED25 (ARC92/ACID1) is a 747 residues subunit specific to higher eukaryote Mediator complex, an essential component of the RNA polymerase II general transcriptional machinery. MED25 is a target of the Herpes simplex virus transactivator protein VP16. MED25 interacts with VP16 through a central MED25 PTOV (Prostate tumour overexpressed)/ACID (Activator interacting domain) domain of unknown structure. As a first step towards understanding the mechanism of recruitment of transactivation domains by MED25, we report here the NMR structure of the MED25 ACID domain. The domain architecture consists of a closed β-barrel with seven strands (Β1-Β7) and three α-helices (H1-H3), an architecture showing similarities to that of the SPOC (Spen paralog and ortholog C-terminal domain) domain-like superfamily. Preliminary NMR chemical shift mapping showed that VP16 H2 (VP16C) interacts with MED25 ACID through one face of the β-barrel, defined by strands B4-B7-B6.