nils-herrmann/clean_scirepeval huggingface dataset:
An N-terminal extension to the hepatitis B virus core protein forms a poorly ordered trimeric spike in assembled virus-like particles
Venue: Journal of structural biology

Virus-like particles composed of the core antigen of hepatitis B virus (HBcAg) have been shown to be an effective platform for the display of foreign epitopes in vaccine development. Heterologous sequences have been successfully inserted at both amino and carboxy termini as well as internally at the major immunodominant epitope. We used cryogenic electron microscopy (CryoEM) and three-dimensional image reconstruction to investigate the structure of VLPs assembled from an N-terminal extended HBcAg that contained a polyhistidine tag. The insert was seen to form a trimeric spike on the capsid surface that was poorly resolved, most likely owing to it being flexible. We hypothesise that the capacity of N-terminal inserts to form trimers may have application in the development of multivalent vaccines to trimeric antigens. Our analysis also highlights the value of tools for local resolution assessment in studies of partially disordered macromolecular assemblies by cryoEM.
Use of a "silver bullet" to resolve crystal lattice dislocation disorder: a cobalamin complex of Δ1-pyrroline-5-carboxylate dehydrogenase from Mycobacterium tuberculosis.
Venue: Journal of structural biology

The use of small molecules as "silver bullets" that can bind to generate crosslinks between protein molecules has been advanced as a powerful means of enhancing success in protein crystallization (McPherson and Cudney, 2006). We have explored this approach in attempts to overcome an order-disorder phenomenon that complicated the structural analysis of the enzyme Δ(1)-pyrroline-5-carboxylate dehydrogenase from Mycobacterium tuberculosis (P5CDH, Mtb-PruA). Using the Silver Bullets Bio screen, we obtained new crystal packing using cobalamin as a co-crystallization agent. This crystal form did not display the order-disorder phenomenon previously encountered. Solution of the crystal structure showed that cobalamin molecules are present in the crystal contacts. Although the cobalamin binding probably does not have physiological relevance, it reflects similarities in the nucleotide-binding region of Mtb-PruA, with the nucleotide loop of cobalamin sharing the binding site for the adenine moiety of NAD(+).
Seeing tobacco mosaic virus through direct electron detectors
Venue: Journal of structural biology

With the introduction of direct electron detectors (DED) to the field of electron cryo-microscopy, a wave of atomic-resolution structures has become available. As the new detectors still require comparative characterization, we have used tobacco mosaic virus (TMV) as a test specimen to study the quality of 3D image reconstructions from data recorded on the two direct electron detector cameras, K2 Summit and Falcon II. Using DED movie frames, we explored related image-processing aspects and compared the performance of micrograph-based and segment-based motion correction approaches. In addition, we investigated the effect of dose deposition on the atomic-resolution structure of TMV and show that radiation damage affects negative carboxyl chains first in a side-chain specific manner. Finally, using 450,000 asymmetric units and limiting the effects of radiation damage, we determined a high-resolution cryo-EM map at 3.35 Å resolution. Here, we provide a comparative case study of highly ordered TMV recorded on different direct electron detectors to establish recording and processing conditions that enable structure determination up to 3.2 Å in resolution using cryo-EM.
Tomo3D 2.0--exploitation of advanced vector extensions (AVX) for 3D reconstruction.
Venue: Journal of structural biology

Tomo3D is a program for fast tomographic reconstruction on multicore computers. Its high speed stems from code optimization, vectorization with Streaming SIMD Extensions (SSE), multithreading and optimization of disk access. Recently, Advanced Vector eXtensions (AVX) have been introduced in the x86 processor architecture. Compared to SSE, AVX double the number of simultaneous operations, thus pointing to a potential twofold gain in speed. However, in practice, achieving this potential is extremely difficult. Here, we provide a technical description and an assessment of the optimizations included in Tomo3D to take advantage of AVX instructions. Tomo3D 2.0 allows huge reconstructions to be calculated in standard computers in a matter of minutes. Thus, it will be a valuable tool for electron tomography studies with increasing resolution needs.
On the use of Legionella/Rickettsia chimeras to investigate the structure and regulation of Rickettsia effector RalF.
Venue: Journal of structural biology

A convenient strategy to interrogate the biology of regulatory proteins is to replace individual domains by an equivalent domain from a related protein of the same species or from an ortholog of another species. It is generally assumed that the overall properties of the native protein are retained in the chimera, and that functional differences reflect only the specific determinants contained in the swapped domains. Here we used this strategy to circumvent the difficulty in obtaining crystals of Rickettsia prowazekii RalF, a bacterial protein that functions as a guanine nucleotide exchange factor for eukaryotic Arf GTPases. A RalF homolog is encoded by Legionella pneumophila, in which a C-terminal capping domain auto-inhibits the catalytic Sec7 domain and localizes the protein to the Legionella-containing vacuole. The crystal structures of domain-swapped chimeras were determined and used to construct a model of Legionella RalF with a RMSD of less than 1Å with the crystal structure, which validated the use of this approach to build a model of Rickettsia RalF. In the Rickettsia RalF model, sequence differences in the capping domain that target it to specific membranes are accommodated by a shift of the entire domain with respect to the Sec7 domain. However, local sequence changes also give rise to an artifactual salt bridge in one of the chimeras, which likely explains why this chimera is recalcitrant to activation. These findings highlight the structural plasticity whereby chimeras can be engineered, but also underline that unpredictable differences can modify their biochemical responses.
The scrunchworm hypothesis: transitions between A-DNA and B-DNA provide the driving force for genome packaging in double-stranded DNA bacteriophages.
Venue: Journal of structural biology

Double-stranded DNA bacteriophages have motors that drive the genome into preformed capsids, using the energy released by hydrolysis of ATP to overcome the forces opposing DNA packaging. Viral packaging motors are the strongest of all biological motors, but it is not known how they generate these forces. Several models for the process of mechanochemical force generation have been put forward, but there is no consensus on which, if any, of these is correct. All the existing models assume that protein-generated forces drive the DNA forward. The scrunchworm hypothesis proposes that the DNA molecule is the active force-generating core of the motor, not simply a substrate on which the motor operates. The protein components of the motor dehydrate a section of the DNA, converting it from the B form to the A form and shortening it by about 23%. The proteins then rehydrate the DNA, which converts back to the B form. Other regions of the motor grip and release the DNA to capture the shortening-lengthening motions of the B→A→B cycle ("scrunching"), so that DNA is pulled into the motor and pushed forward into the capsid. This DNA-centric mechanism provides a quantitative physical explanation for the magnitude of the forces generated by viral packaging motors. It also provides a simple explanation for the fact that each of the steps in the burst cycle advances the DNA by 2.5 base pairs. The scrunchworm hypothesis is consistent with a large body of published data, and it makes four experimentally testable predictions.
Semi-automated selection of cryo-EM particles in RELION-1.3
Venue: Journal of structural biology

The selection of particles suitable for high-resolution cryo-EM structure determination from noisy micrographs may represent a tedious and time-consuming step. Here, a semi-automated particle selection procedure is presented that has been implemented within the open-source software RELION. At the heart of the procedure lies a fully CTF-corrected template-based picking algorithm, which is supplemented by a fast sorting algorithm and reference-free 2D class averaging to remove false positives. With only limited user-interaction, the proposed procedure yields results that are comparable to manual particle selection. Together with an improved graphical user interface, these developments further contribute to turning RELION from a stand-alone refinement program into a convenient image processing pipeline for the entire single-particle approach.
Third Harmonic Generation microscopy as a reliable diagnostic tool for evaluating lipid body modification during cell activation: the example of BV-2 microglia cells.
Venue: Journal of structural biology

Nonlinear optical processes have found widespread applications in fields ranging from fundamental physics to biomedicine. In this study, we attempted to evaluate cell activation by using the Third Harmonic Generation (THG) imaging microscopy as a new diagnostic tool. The BV-2 microglia cell line with or without activation by lipopolysaccharide was chosen as a representative biological model. The results showed that THG imaging could discriminate between the control versus activated state of BV-2 cells not only as to THG signal intensity but also as to THG signal area, while verifying once more that the majority of the intracellular detected signal corresponds to lipid bodies. Since THG imaging is a real time, non-destructive modality and does not require any prior cell processing and staining, the results presented here provide an important tool for normal versus activated cell discrimination, which could be proved very useful in the study of inflammation.
Sparse and incomplete factorial matrices to screen membrane protein 2D crystallization.
Venue: Journal of structural biology

Electron crystallography is well suited for studying the structure of membrane proteins in their native lipid bilayer environment. This technique relies on electron cryomicroscopy of two-dimensional (2D) crystals, grown generally by reconstitution of purified membrane proteins into proteoliposomes under conditions favoring the formation of well-ordered lattices. Growing these crystals presents one of the major hurdles in the application of this technique. To identify conditions favoring crystallization a wide range of factors that can lead to a vast matrix of possible reagent combinations must be screened. However, in 2D crystallization these factors have traditionally been surveyed in a relatively limited fashion. To address this problem we carried out a detailed analysis of published 2D crystallization conditions for 12 β-barrel and 138 α-helical membrane proteins. From this analysis we identified the most successful conditions and applied them in the design of new sparse and incomplete factorial matrices to screen membrane protein 2D crystallization. Using these matrices we have run 19 crystallization screens for 16 different membrane proteins totaling over 1300 individual crystallization conditions. Six membrane proteins have yielded diffracting 2D crystals suitable for structure determination, indicating that these new matrices show promise to accelerate the success rate of membrane protein 2D crystallization.
Stability of the βB2B3 crystallin heterodimer to increased oxidation by radical probe and ion mobility mass spectrometry.
Venue: Journal of structural biology

Ion mobility mass spectrometry was employed to study the structure of the βB2B3-crystallin heterodimer following oxidation through its increased exposure to hydroxyl radicals. The results demonstrate that the heterodimer can withstand limited oxidation through the incorporation of up to some 10 oxygen atoms per subunit protein without any appreciable change to its average collision cross section and thus conformation. These results are in accord with the oxidation levels and timescales applicable to radical probe mass spectrometry (RP-MS) based protein footprinting experiments. Following prolonged exposure, the heterodimer is increasingly degraded through cleavage of the backbone of the subunit crystallins rather than denaturation such that heterodimeric structures with altered conformations and ion mobilities were not detected. However, evidence from measurements of oxidation levels within peptide segments, suggest the presence of some aggregated structure involving C-terminal domain segments of βB3 crystallin across residues 115-126 and 152-166. The results demonstrate, for the first time, the ability of ion mobility in conjunction with RP-MS to investigate the stability of protein complexes to, and the onset of, free radical based oxidative damage that has important implications in cataractogenesis.
Improvement of protein binding sites prediction by selecting amino acid residues' features.
Venue: Journal of structural biology

One of the main focuses of bioinformatics community is the study of the relationship between the structure of the protein molecules and their functions. In the literature, there are various methods that consider different protein-derived information for predicting protein functions. In our research, we focus on predicting the protein binding sites, which could be used to functionally annotate the protein structures. In this paper we consider a set of sixteen amino acid residues' features, and by applying various feature selection techniques we estimate their significance. Although the number of features in our case is not high, we perform feature selection in order to improve the prediction power and time complexity of the prediction models. The results show that by applying proper feature selection technique, the predictive performance of the classification algorithms is improved, i.e., by considering the most relevant features we induce more accurate models than if we consider the entire set of features. Furthermore, the model complexity, as well as the training and testing times are decreased by performing feature selection. We also compare our approach with several existing methods for protein binding sites prediction. The results demonstrate that the existing methods considered in this research are specific and applicable to the group of proteins for which the model was developed, while our approach is more generic and can be applied to a wider class of proteins.
Distinct structural features of Rex-family repressors to sense redox levels in anaerobes and aerobes.
Venue: Journal of structural biology

The Rex-family repressors sense redox levels by alternative binding to NADH or NAD(+). Unlike other Rex proteins that regulate aerobic respiration, RSP controls ethanol fermentation in the obligate anaerobe Thermoanaerobacter ethanolicus JW200(T). It is also found in other anaerobic microorganisms. Here we present the crystal structures of apo-RSP, RSP/NADH and RSP/NAD(+)/DNA, which are the first structures of Rex-family members from an obligate anaerobe. RSP functions as a homodimer. It assumes an open conformation when bound to the operator DNA and a closed conformation when not DNA-bound. The DNA binds to the N-terminal winged-helix domain and the dinucleotide, either reduced or oxidized, binds to the C-terminal Rossmann-fold domain. The two distinct orientations of nicotinamide ring, anti in NADH and syn in NAD(+), give rise to two sets of protein-ligand interactions. Consequently, NADH binding makes RSP into a closed conformation, which does not bind to DNA. Both the conserved residues and the DNA specificity of RSP show a number of variations from those of the aerobic Rex, reflecting different structural bases for redox-sensing by the anaerobic and aerobic Rex-family members.
The structure of ibuprofen bound to cyclooxygenase-2.
Venue: Journal of structural biology

The cyclooxygenases (COX-1 and COX-2) catalyze the rate-limiting step in the biosynthesis of prostaglandins, and are the pharmacological targets of non-steroidal anti-inflammatory drugs (NSAIDs) and COX-2 selective inhibitors (coxibs). Ibuprofen (IBP) is one of the most commonly available over-the-counter pharmaceuticals in the world. The anti-inflammatory and analgesic properties of IBP are thought to arise from inhibition of COX-2 rather than COX-1. While an X-ray crystal structure of IBP bound to COX-1 has been solved, no such structure exists for the cognate isoform COX-2. We have determined the crystal structure of muCOX-2 with a racemic mixture of (R/S)-IBP. Our structure reveals that only the S-isomer of IBP was bound, indicating that the S-isomer possesses higher affinity for COX-2 than the R-isomer. Mutational analysis of Arg-120 and Tyr-355 at the entrance of the cyclooxygenase channel confirmed their role in binding and inhibition of COX-2 by IBP. Our results provide the first atomic level detail of the interaction between IBP and COX-2.
Internal skeletal analysis of the colonial azooxanthellate scleractinian Dendrophyllia cribrosa using microfocus X-ray CT images: underlying basis for its rigid and highly adaptive colony structure.
Venue: Journal of structural biology

Dendrophyllid Scleractinia exhibit a variety of colonial morphologies, formed under the strict constraints on (1) budding sites, (2) orientations of the directive septa of offsets, (3) inclination of budding direction, and (4) those constraints in every generation. Dendrophyllia cribrosa exhibits a sympodial dendroid form, characteristically large coralla, and occasional fusions of adjacent branches within the same colony. Adjacent corallites are bound and supported by coenosteum skeleton. This study examined the inner skeletal structures at the junctions of fused branches using a non-destructive microfocus X-ray computed tomography (CT) imaging approach, and considered the reasons for the large colonial sizes and their adaptive significance. Three-dimensional reconstructions of two-dimensional X-ray CT images reveal that individual corallites are not directly connected in fused parts. Additionally, no completely buried individuals were found within fused skeleton. When adjacent branches approach one another, constituent corallites change their growth directions to avoid collisions between the branches. The adjacent branches fuse without a reduction in the number of constituent corallites, leading to the establishment of reticular and rigid colonial structures. In addition, a nearly even distribution of individuals on the colony surface facilitates efficient intake of nutrients. Thus, the growth of large D. cribrosa colonies involves avoidance of collision between constituent individuals, the reinforcement of colonial structure, and efficient uptake of nutrients. These observations provide insights on the dynamics of interrelationships between colony-making mechanisms and the adaptive strategies required under habitat conditions such as specific current activities.
Crystal structure of the essential Mycobacterium tuberculosis phosphopantetheinyl transferase PptT, solved as a fusion protein with maltose binding protein.
Venue: Journal of structural biology

Phosphopantetheinyl transferases (PPTases) are key enzymes in the assembly-line production of complex molecules such as fatty acids, polyketides and polypeptides, where they activate acyl or peptidyl carrier proteins, transferring a 4'-phosphopantetheinyl moiety from coenzyme A (CoA) to a reactive serine residue on the carrier protein. The human pathogen Mycobacterium tuberculosis encodes two PPTases, both essential and therefore attractive drug targets. We report the structure of the type-II PPTase PptT, obtained from crystals of a fusion protein with maltose binding protein. The structure, at 1.75Å resolution (R=0.156, Rfree=0.191), reveals an α/β fold broadly similar to other type-II PPTases, but with differences in peripheral structural elements. A bound CoA is clearly defined with its pantetheinyl arm tucked into a hydrophobic pocket. Interactions involving the CoA diphosphate, bound Mg(2+) and three active site acidic side chains suggest a plausible pathway for proton transfer during catalysis.
Separation of replication and transcription domains in nucleoli.
Venue: Journal of structural biology

In mammalian cells, active ribosomal genes produce the 18S, 5.8S and 28S RNAs of ribosomal particles. Transcription levels of these genes are very high throughout interphase, and the cell needs a special strategy to avoid collision of the DNA polymerase and RNA polymerase machineries. To investigate this problem, we measured the correlation of various replication and transcription signals in the nucleoli of HeLa, HT-1080 and NIH 3T3 cells using a specially devised software for analysis of confocal images. Additionally, to follow the relationship between nucleolar replication and transcription in living cells, we produced a stable cell line expressing GFP-RPA43 (subunit of RNA polymerase I, pol I) and RFP-PCNA (the sliding clamp protein) based on human fibrosarcoma HT-1080 cells. We found that replication and transcription signals are more efficiently separated in nucleoli than in the nucleoplasm. In the course of S phase, separation of PCNA and pol I signals gradually increased. During the same period, separation of pol I and incorporated Cy5-dUTP signals decreased. Analysis of single molecule localization microscopy (SMLM) images indicated that transcriptionally active FC/DFC units (i.e. fibrillar centers with adjacent dense fibrillar components) did not incorporate DNA nucleotides. Taken together, our data show that replication of the ribosomal genes is spatially separated from their transcription, and FC/DFC units may provide a structural basis for that separation.
Crystal structure of the VapBC-15 complex from Mycobacterium tuberculosis reveals a two-metal ion dependent PIN-domain ribonuclease and a variable mode of toxin-antitoxin assembly.
Venue: Journal of structural biology

Although PIN (PilT N-terminal)-domain proteins are known to have ribonuclease activity, their specific mechanism of action remains unknown. VapCs form a family of ribonucleases that possess a PIN-domain assembly and are known as toxins. The activities of VapCs are impaired by VapB antitoxins. Here we present the crystal structure of the VapBC-15 toxin-antitoxin complex from Mycobacterium tuberculosis determined to 2.1Å resolution. The VapB-15 and VapC-15 components assemble into one heterotetramer (VapB2C2) and two heterotrimers (VapBC2) in each asymmetric unit of the crystal. The active site of VapC-15 toxin consists of a cluster of acidic amino acid residues and two divalent metal ions, forming a well organised ribonuclease active site. The distribution of the catalytic-site residues of the VapC-15 toxin is similar to that of T4 RNase H and of Methanococcus jannaschii FEN-1, providing strong evidence that these three proteins share a similar mechanism of activity. The presence of both VapB2C2 and VapBC2 emphasizes the fact that the same antitoxin can bind the toxin in 1:1 and 1:2 ratios. The crystal structure determination of the VapBC-15 complex reveals for the first time a PIN-domain ribonuclease protein that shows two metal ions at the active site and a variable mode of toxin-antitoxin assembly. The structure further shows that VapB-15 antitoxin binds to the same groove meant for the binding of putative substrate (RNA), resulting in the inhibition of VapC-15's toxicity.