Protein Science最新文献

{"title":"An intrinsically disordered region of Ubp10 regulates its binding and activity on ubiquitinated histone substrates.","authors":"Anneliese M Faustino, Melesse Nune, Raquel Merino-Urteaga, Edgar Manriquez-Sandoval, Matthew Poyton, Taekjip Ha, Cynthia Wolberger, Stephen D Fried","doi":"10.1002/pro.70237","DOIUrl":"10.1002/pro.70237","url":null,"abstract":"<p><p>Monoubiquitinated histone H2B at K123 in yeast (K120 in humans) is a transient modification that is both attached and removed during transcription. H2B is ubiquitinated in yeast by the E2/E3 pair, Rad6/Bre1, and deubiquitinated by two enzymes, Ubp8 and Ubp10. Previous studies had shown that Ubp10 has higher activity on ubiquitinated H2A/H2B dimers than on intact nucleosomes, but that activity on nucleosomes is higher in the presence of the histone chaperone, FACT. By contrast, the Ubp8 complex has equal activity on both histone substrates and is unaffected by FACT. We report here the results of single-molecule FRET experiments showing that FACT unwraps DNA and evicts ubiquitinated H2A/H2B dimers, the preferred substrate of Ubp10. To explore the basis for the differing activity of Ubp10 on ubiquitinated H2A/H2B dimers and nucleosomes, we employed crosslinking mass spectrometry combined with structural modeling. These studies revealed that Ubp10 forms a different set of interactions with H2A/H2B in free versus nucleosomal states. Acidic stretches within the N-terminal intrinsically disordered region (IDR) of Ubp10 interact extensively with H2A/H2B heterodimers, whereas this portion of Ubp10 interacts more with the tails of histones H3 and H4 in the nucleosome. The importance of these interactions for affinity is consistent with binding studies showing the IDR is necessary for substrate interactions. Structural modeling using the crosslinks as constraints suggests that the complex formed by Ubp10 with free H2A/H2B dimers could not be formed within a nucleosome due to steric clash with the DNA, H3, and H4, thereby explaining its low activity on ubiquitinated nucleosomes.</p>","PeriodicalId":20761,"journal":{"name":"Protein Science","volume":"34 8","pages":"e70237"},"PeriodicalIF":5.2,"publicationDate":"2025-08-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144732923","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Structural investigations connect the disordered N-terminal extension of HypB to the activities of HypB and SlyD in E. coli.","authors":"Wayne W H Law, Dmitry Pichugin, Ranjith Muhandiram, Deborah B Zamble, Voula Kanelis","doi":"10.1002/pro.70231","DOIUrl":"10.1002/pro.70231","url":null,"abstract":"<p><p>The activities of [NiFe]-hydrogenase enzymes, which are critical to many microbes, require insertion of a Ni(II) ion into the bimetallic catalytic center. Delivery of Ni(II) to [NiFe]-hydrogenases depends, in part, on the metallochaperone HypB, which lies at the center of a Ni(II) transfer pathway that includes the metal storage protein SlyD and the metallochaperone HypA. SlyD is a source of Ni(II) ions for HypB, whereas Ni(II) from HypB is transferred to HypA. In this work, we examine how the intrinsically disordered N-terminal extension (NTE) of HypB modulates the action of the HypB GTPase domain (G-domain). The NTE contains a high-affinity Ni(II) binding site, while the G-domain contains a lower affinity Ni(II) binding site that is affected by binding of guanine nucleotides. The HypB G-domain is also affected by SlyD and provides Ni(II) to HypA. Our NMR data show that, although disordered, the HypB NTE possesses residual structure and makes transient interactions with the HypB G-domain and with SlyD. A set of common residues in the center of the NTE are affected by SlyD and G-domain binding, and also by binding of Ni(II) to the high-affinity site located at the N terminus of the protein. The NTE interacts with residues in or near the Ni(II)- and GDP-binding sites in the G-domain, which are also affected when SlyD binds the NTE. Thus, the data showcase a complex interaction network between HypB and SlyD, and provide molecular details regarding how the HypB NTE links the activities of the HypB G-domain and SlyD.</p>","PeriodicalId":20761,"journal":{"name":"Protein Science","volume":"34 8","pages":"e70231"},"PeriodicalIF":5.2,"publicationDate":"2025-08-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12284835/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144691296","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Resolving the structure and assembly of the honeybee silk heterotetrameric coiled coil.","authors":"Caitlin L Johnston, Chacko Jobichen, Lyndall J Briggs, Michelle Michie, Jian-Wei Liu, Craig J Morton, Andrew C Warden, Tara D Sutherland","doi":"10.1002/pro.70230","DOIUrl":"10.1002/pro.70230","url":null,"abstract":"<p><p>Coiled coil structures, first proposed by Crick in the 1950s, are protein structural motifs found across diverse biological systems. Honeybee silk was among the earliest identified coiled coils, with X-ray diffraction studies in the 1960s revealing its characteristic helical packing. Decades of research have provided insights into silk composition and formation, yet the molecular details of its coiled coil assembly and final structure remained unresolved. In this study, we generated a structural model of the tetrameric coiled coil using AlphaFold and validated it with crosslinking mass spectrometry and medium-resolution cryo-electron microscopy. The model reveals that the four proteins (F1-F4) adopt an antiparallel configuration in a defined clockwise arrangement (F1-F3-F2-F4). Furthermore, we experimentally investigated the formation of this coiled coil complex using biochemical techniques, including blue-native PAGE and circular dichroism spectroscopy. The sum of these experimental results and the structural predictions has allowed for the elucidation of key transitional steps in the assembly pathway, suggesting molecular interactions that may drive tetramer formation. These findings support a stepwise assembly model in which F2 and F4 form a stable core, F3 binds to the complex, and F1 initiates formation of the final, highly ordered structure. These structural insights establish a framework for understanding and directing coiled coil assembly, the fundamental building block of honeybee silk. By resolving this structure and its assembly process, this work lays the foundation for future rational design of functional sequences and materials with tailored properties.</p>","PeriodicalId":20761,"journal":{"name":"Protein Science","volume":"34 8","pages":"e70230"},"PeriodicalIF":5.2,"publicationDate":"2025-08-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12284834/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144691295","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Recognizing amino acid sidechains in a medium-resolution cryo-electron density map.","authors":"Dibyendu Mondal, Vipul Kumar, Tadej Satler, Rakesh Ramachandran, Daniel Saltzberg, Ilan Chemmama, Kala Bharath Pilla, Ignacia Echeverria, Benjamin M Webb, Meghna Gupta, Klim Verba, Andrej Sali","doi":"10.1002/pro.70217","DOIUrl":"10.1002/pro.70217","url":null,"abstract":"<p><p>Building an accurate atomic structure model of a protein into a cryo-electron microscopy (cryo-EM) map at worse than 3 Å resolution is difficult. To facilitate this task, we devised a method for assigning the amino acid residue sequence to the backbone fragments traced in an input cryo-EM map (EMSequenceFinder). EMSequenceFinder relies on a Bayesian scoring function for ranking 20 standard amino acid residue types at a given backbone position, based on the fit to a density map, map resolution, and secondary structure propensity. The fit to a density is quantified by a convolutional neural network that was trained on ~5.56 million amino acid residue densities extracted from cryo-EM maps at 3-10 Å resolution and corresponding atomic structure models deposited in the Electron Microscopy Data Bank (EMDB). We benchmarked EMSequenceFinder by predicting the sequences of 58,044 distinct ɑ-helix and β-strand fragments, given the fragment backbone coordinates fitted in their density maps. EMSequenceFinder identifies the correct sequence as the best-scoring sequence in 77.8% of these cases. We also assessed EMSequenceFinder on separate datasets of cryo-EM maps at resolutions from 4 to 6 Å. The accuracy of EMSequenceFinder (58%) was better than that of three tested state-of-the-art methods, including findMysequence (45%), ModelAngelo (27%), and sequence_from_map in Phenix (12.9%). We further illustrate EMSequenceFinder by threading the Severe Acute Respiratory Syndrome Coronavirus 2 Non-Structural Protein 2 sequence into eight cryo-EM maps at resolutions from 3.7 to 7.0 Å. EMSequenceFinder is implemented in our open-source Integrative Modeling Platform (IMP) program. Thus, it is expected to be helpful for integrative structure modeling based on a cryo-EM map and other information, such as models of protein complex components and chemical crosslinks between them. EMSequenceFinder is available as part of our open-source IMP distribution at https://integrativemodeling.org/.</p>","PeriodicalId":20761,"journal":{"name":"Protein Science","volume":"34 8","pages":"e70217"},"PeriodicalIF":5.2,"publicationDate":"2025-08-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12302279/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144732858","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Two cysteines control Tse1 secretion by H1-T6SS in Pseudomonas aeruginosa.","authors":"Marie M Grandjean, Jean-Pierre Duneau, Edwige B Garcin, Laetitia Houot, Olivier Bornet, Christophe Bordi, Latifa Elantak, Corinne Sebban-Kreuzer","doi":"10.1002/pro.70226","DOIUrl":"10.1002/pro.70226","url":null,"abstract":"<p><p>Type Six Secretion Systems (T6SS) are molecular machines that export toxic effector proteins into bacterial competitors or eukaryotic cells. Pseudomonas aeruginosa's H1-T6SS secretes Tse1, which contains a disulfide bond between cysteines at positions 7 and 148, linking its N- and C-terminal regions. The role of this disulfide bond in Tse1 activity and mechanism of action during bacterial competition is unknown. In this study, we investigated the role of the C7-C148 disulfide bond within Tse1. First, NMR spectroscopy experiments suggest a redox-active instead of a structural disulfide bond. Moreover, while the presence of this bond did not alter Tse1's amidase activity or toxicity in Escherichia coli, substituting cysteines C7 or C148 in P. aeruginosa strains affected the bacterium's capacity to lyse prey cells. Secretome analysis showed that the Tse1C148S variant was not secreted via the H1-T6SS, whereas the Tse1C7S variant was secreted. These findings suggest that cysteine 148 is likely important for Tse1's assembly with the T6SS machinery, while cysteine 7 appears to be involved in its disassembly, potentially through the formation of the disulfide bond. This study points to a potential redox regulation mechanism during the assembly and disassembly of Tse1 with Hcp1, consistent with a \"bridge of delivery\" model.</p>","PeriodicalId":20761,"journal":{"name":"Protein Science","volume":"34 8","pages":"e70226"},"PeriodicalIF":5.2,"publicationDate":"2025-08-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12302283/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144732859","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Deep sequencing combined with high-throughput screening enables efficient development of a pH-dependent high-affinity binding domain targeting HER3.","authors":"Marit Möller, Malin Jönsson, Magnus Lundqvist, Johan Rockberg, John Löfblom, Hanna Tegel, Sophia Hober","doi":"10.1002/pro.70247","DOIUrl":"10.1002/pro.70247","url":null,"abstract":"<p><p>In vitro methods for developing binding domains have been well-established for many years, owing to the cost-efficient synthesis of DNA and high-throughput selection and screening technologies. However, generating high-affinity binding domains often requires the development of focused maturation libraries for a second selection, which typically demands a detailed understanding of the binding surfaces from the initial selection, a process that can be time-consuming. In this study, we accelerated this process by using deep sequencing data from the first selection to guide the design of the maturation library. Additionally, we employed a high-throughput screening system using flow cytometry based on Escherichia coli display to identify conditional binding domains from the selection output. This approach enabled the development of a high-affinity binder targeting the cancer biomarker HER3, with a binding affinity of 3.3 nM at extracellular pH 7.4, 100 times higher than the first-generation binding domain. Notably, the binding domain features a pH-dependent release mechanism, enabling rapid release in slightly acidic environments (pH ≈6), which resemble endosomal conditions. When conjugated to the cytotoxin mertansine (DM1), the binding domain demonstrated specific cytotoxic activity against HER3-expressing cell lines, with an IC50 of 2-5 nM. The presented approach enables the efficient development of conditional binding domains which hold promise for therapeutic applications.</p>","PeriodicalId":20761,"journal":{"name":"Protein Science","volume":"34 8","pages":"e70247"},"PeriodicalIF":5.2,"publicationDate":"2025-08-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144732856","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Extracellular catalysis of environmental substrates by Shewanella oneidensis MR-1 occurs via active sites on the C-terminal domains of MtrC.","authors":"Alejandro Morales-Florez, Colin W J Lockwood, Benjamin W Nash, Marcus J Edwards, Jessica H van Wonderen, Amit Sachdeva, Julea N Butt, Thomas A Clarke","doi":"10.1002/pro.70243","DOIUrl":"10.1002/pro.70243","url":null,"abstract":"<p><p>The Gram-negative Shewanellaceae family is well known for its ability to transfer catabolically derived electrons to extracellular terminal electron acceptors through electron conduits that permeate the outer membrane. The primary conduit is MtrCAB, a trimeric porin-cytochrome complex that contains the cell surface exposed decaheme cytochrome MtrC. This donates electrons to extracellular substrates, including OmcA, soluble metals, organic electron shuttles, and insoluble metal oxides. However, it is not clear whether this broad substrate specificity requires specific sites for binding and reduction, or whether reduction occurs through non-specific interactions near exposed hemes on the cytochrome surface. Shewanella oneidensis MtrC is composed of four domains, with the hemes closely packed and distributed evenly between domains II and IV. The domains are arranged to allow electron transport across the cytochrome via interdomain electron transfer, but the significance of this conserved feature is not understood. Here we use site-directed mutagenesis to generate an MtrC variant that is comprised only of domains I and II (MtrC_DI,II). The properties of this MtrC_DI,II are effectively identical to domains I and II of full-length MtrC. Whole-cell assays revealed that S. oneidensis cells replacing full-length MtrC with MtrC_DI,II had significantly lower rates of OmcA, flavin mononucleotide, and Fe(III) citrate reduction. Our results demonstrate that MtrC domains III and IV contain sites for association of specific substrates, enabling the reduction of extracellular electron acceptors in S. oneidensis.</p>","PeriodicalId":20761,"journal":{"name":"Protein Science","volume":"34 8","pages":"e70243"},"PeriodicalIF":5.2,"publicationDate":"2025-08-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12304082/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144732857","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Navigating infection by pathogenic spirochetes: The host-bacteria interface at the atomic level.","authors":"Libor Hejduk, Norbert Müller, Adriana Rathner, Ján Štěrba, Shang-Cheng Hung, Chia-Lin Chyan, Ryan O M Rego, Martin Strnad","doi":"10.1002/pro.70185","DOIUrl":"10.1002/pro.70185","url":null,"abstract":"<p><p>Pathogenic spirochetes bind and interact with various host structures and molecules throughout the course of infection. By utilizing their outer surface molecules, spirochetes can effectively modulate their dissemination, interact with immune system regulators, and select specific destination niches within the host. The three-dimensional structures of multiple spirochetal surface proteins have been elucidated, providing insight into their modus operandi. This review focuses on the structural characteristics of these sticky molecules and their functional implications, highlighting how these features contribute to the pathogenicity of spirochetes and their ability to persist in the host and vector environments. Recognizing the structural motifs and ligands to which these important virulence determinants bind could open new avenues for developing strategies to block colonization by spirochetal pathogens.</p>","PeriodicalId":20761,"journal":{"name":"Protein Science","volume":"34 7","pages":"e70185"},"PeriodicalIF":4.5,"publicationDate":"2025-07-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12183334/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144369174","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"A high throughput assay for measuring secreted protein based on a de novo fluorescent reporter reveals regulatory and structural insights in Salmonella type three secretion system.","authors":"Samuel Alexander Leach, Jordan Scott Summers, Edward Wen, Danielle Tullman-Ercek","doi":"10.1002/pro.70183","DOIUrl":"10.1002/pro.70183","url":null,"abstract":"<p><p>Intracellular protein production in bacteria is limited by the need for lysis and costly purification. A promising alternative is to engineer the host organism for protein secretion. While the Salmonella enterica serovar Typhimurium (Salmonella typhimurium) Type 3 Secretion System (T3SS) has been utilized for protein secretion, its study and eventual applicability for recombinant protein production are constrained by the lack of high-throughput assays to quantitatively measure secretion titer. Developing such assays is challenging, as proteins must remain unfolded for secretion, limiting the use of several common reporter proteins. In this work, we develop a high-throughput secretion assay using mini-fluorescence activating protein (mFAP). mFAP forms a chromophore only upon addition of an exogenous substrate, allowing secretion and subsequent fluorescence detection. We demonstrate mFAP secretion via the T3SS with an N-terminal secretion tag and show that the fluorescent signal in the secreted fraction is rapid and linear over three orders of magnitude. Using this assay, we screen S. typhimurium strains with secretion-enhancing mutations, identifying a constitutively active strain and revealing temporally controlled secretion dynamics. We also show that this assay may be applicable to other secretion systems, providing a universal tool for tracking heterologous protein secretion.</p>","PeriodicalId":20761,"journal":{"name":"Protein Science","volume":"34 7","pages":"e70183"},"PeriodicalIF":4.5,"publicationDate":"2025-07-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12198052/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144497891","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Unraveling antibody-induced mechanical stability of antigen: Insights from single-molecule studies.","authors":"Soham Chakraborty, Shivam Pandit, Krishnendu Sinha, Madhu Bhatt, Debojyoti Chowdhury, Suman Chakrabarty, Shubhasis Haldar","doi":"10.1002/pro.70201","DOIUrl":"10.1002/pro.70201","url":null,"abstract":"<p><p>Antigen-antibody interaction, as a prominent ligand-receptor reaction, plays a crucial role in immunological responses. Notably, antigens can contain multiple ligand binding sites that define their intermolecular interactions more intricately and thereby make them context-dependent. Here, we have investigated the binding-induced effect of the largest antibody isotype, IgM, on protein L mechanical stability using single-molecule magnetic tweezers. Our results showed that IgM elevates the protein L mechanical stability by increasing its unfolding time. Interestingly, we were able to resolve distinct IgM-bound states of protein L by characterizing their unfolding dwell time: while the IgM-unbound state has the lowest dwell time, it increases with the IgM concentration via binding to either one or both of its binding sites, reconciling the IgM-induced protein L mechanical stability. To delve into the plausible mechanism of such intricate phenomena, we performed steered a molecular dynamic simulation of protein L and determined its unfolding rupture force at those multiple IgM-bound states, their corresponding molecular insights, and interaction gymnastics through binding interfaces. Additionally, we unraveled the mechanical response of these binding interfaces to be different; and during dimer IgM complex formation, these binding interfaces synergistically increase the mechanical stability of the complex. This provides the underlying principles of IgM-induced protein L stability under mechanical constraints. Overall, this study provides an in-depth understanding of a generic mechanism of antibody-induced mechanical stability of antigenic substrate under physiological sheer stress.</p>","PeriodicalId":20761,"journal":{"name":"Protein Science","volume":"34 7","pages":"e70201"},"PeriodicalIF":4.5,"publicationDate":"2025-07-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12168486/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144302721","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}