@article{NanguneriFlottmannHorstmannetal.2012, author = {Nanguneri, Siddharth and Flottmann, Benjamin and Horstmann, Heinz and Heilemann, Mike and Kuner, Thomas}, title = {Three-Dimensional, Tomographic Super-Resolution Fluorescence Imaging of Serially Sectioned Thick Samples}, series = {PLoS One}, volume = {7}, journal = {PLoS One}, number = {5}, doi = {10.1371/journal.pone.0038098}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-134434}, pages = {e38098}, year = {2012}, abstract = {Three-dimensional fluorescence imaging of thick tissue samples with near-molecular resolution remains a fundamental challenge in the life sciences. To tackle this, we developed tomoSTORM, an approach combining single-molecule localization-based super-resolution microscopy with array tomography of structurally intact brain tissue. Consecutive sections organized in a ribbon were serially imaged with a lateral resolution of 28 nm and an axial resolution of 40 nm in tissue volumes of up to 50 \(\mu\)mx50\(\mu\)mx2.5\(\mu\)m. Using targeted expression of membrane bound (m)GFP and immunohistochemistry at the calyx of Held, a model synapse for central glutamatergic neurotransmission, we delineated the course of the membrane and fine-structure of mitochondria. This method allows multiplexed super-resolution imaging in large tissue volumes with a resolution three orders of magnitude better than confocal microscopy.}, language = {en} } @article{WilleSchuemannWreeetal.2015, author = {Wille, Michael and Sch{\"u}mann, Antje and Wree, Andreas and Kreutzer, Michael and Glocker, Michael O. and Mutzbauer, Grit and Schmitt, Oliver}, title = {The Proteome Profiles of the Cerebellum of Juvenile, Adult and Aged Rats-An Ontogenetic Study}, series = {International Journal of Molecular Sciences}, volume = {16}, journal = {International Journal of Molecular Sciences}, doi = {10.3390/ijms160921454}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-151347}, pages = {21454 -- 21485}, year = {2015}, abstract = {In this study, we searched for proteins that change their expression in the cerebellum (Ce) of rats during ontogenesis. This study focuses on the question of whether specific proteins exist which are differentially expressed with regard to postnatal stages of development. A better characterization of the microenvironment and its development may result from these study findings. A differential two-dimensional polyacrylamide gel electrophoresis (2DE) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) analysis of the samples revealed that the number of proteins of the functional classes differed depending on the developmental stages. Especially members of the functional classes of biosynthesis, regulatory proteins, chaperones and structural proteins show the highest differential expression within the analyzed stages of development. Therefore, members of these functional protein groups seem to be involved in the development and differentiation of the Ce within the analyzed development stages. In this study, changes in the expression of proteins in the Ce at different postnatal developmental stages (postnatal days (P) 7, 90, and 637) could be observed. At the same time, an identification of proteins which are involved in cell migration and differentiation was possible. Especially proteins involved in processes of the biosynthesis and regulation, the dynamic organization of the cytoskeleton as well as chaperones showed a high amount of differentially expressed proteins between the analyzed dates.}, language = {en} } @article{PauliPaulProppertetal.2021, author = {Pauli, Martin and Paul, Mila M. and Proppert, Sven and Mrestani, Achmed and Sharifi, Marzieh and Repp, Felix and K{\"u}rzinger, Lydia and Kollmannsberger, Philip and Sauer, Markus and Heckmann, Manfred and Sir{\´e}n, Anna-Leena}, title = {Targeted volumetric single-molecule localization microscopy of defined presynaptic structures in brain sections}, series = {Communications Biology}, volume = {4}, journal = {Communications Biology}, doi = {10.1038/s42003-021-01939-z}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-259830}, pages = {407}, year = {2021}, abstract = {Revealing the molecular organization of anatomically precisely defined brain regions is necessary for refined understanding of synaptic plasticity. Although three-dimensional (3D) single-molecule localization microscopy can provide the required resolution, imaging more than a few micrometers deep into tissue remains challenging. To quantify presynaptic active zones (AZ) of entire, large, conditional detonator hippocampal mossy fiber (MF) boutons with diameters as large as 10 mu m, we developed a method for targeted volumetric direct stochastic optical reconstruction microscopy (dSTORM). An optimized protocol for fast repeated axial scanning and efficient sequential labeling of the AZ scaffold Bassoon and membrane bound GFP with Alexa Fluor 647 enabled 3D-dSTORM imaging of 25 mu m thick mouse brain sections and assignment of AZs to specific neuronal substructures. Quantitative data analysis revealed large differences in Bassoon cluster size and density for distinct hippocampal regions with largest clusters in MF boutons. Pauli et al. develop targeted volumetric dSTORM in order to image large hippocampal mossy fiber boutons (MFBs) in brain slices. They can identify synaptic targets of individual MFBs and measured size and density of Bassoon clusters within individual untruncated MFBs at nanoscopic resolution.}, language = {en} } @article{VolceanovHerbstBiniosseketal.2014, author = {Volceanov, Larisa and Herbst, Katharina and Biniossek, Martin and Schilling, Oliver and Haller, Dirk and N{\"o}lke, Thilo and Subbarayal, Prema and Rudel, Thomas and Zieger, Barbara and H{\"a}cker, Georg}, title = {Septins Arrange F-Actin-Containing Fibers on the Chlamydia trachomatis Inclusion and Are Required for Normal Release of the Inclusion by Extrusion}, series = {MBIO}, volume = {5}, journal = {MBIO}, number = {5}, issn = {2150-7511}, doi = {10.1128/mBio.01802-14}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-115421}, pages = {e01802-14}, year = {2014}, abstract = {Chlamydia trachomatis is an obligate intracellular human pathogen that grows inside a membranous, cytosolic vacuole termed an inclusion. Septins are a group of 13 GTP-binding proteins that assemble into oligomeric complexes and that can form higher-order filaments. We report here that the septins SEPT2, -9, -11, and probably -7 form fibrillar structures around the chlamydial inclusion. Colocalization studies suggest that these septins combine with F actin into fibers that encase the inclusion. Targeting the expression of individual septins by RNA interference (RNAi) prevented the formation of septin fibers as well as the recruitment of actin to the inclusion. At the end of the developmental cycle of C. trachomatis, newly formed, infectious elementary bodies are released, and this release occurs at least in part through the organized extrusion of intact inclusions. RNAi against SEPT9 or against the combination of SEPT2/7/9 substantially reduced the number of extrusions from a culture of infected HeLa cells. The data suggest that a higher-order structure of four septins is involved in the recruitment or stabilization of the actin coat around the chlamydial inclusion and that this actin recruitment by septins is instrumental for the coordinated egress of C. trachomatis from human cells. The organization of F actin around parasite-containing vacuoles may be a broader response mechanism of mammalian cells to the infection by intracellular, vacuole-dwelling pathogens. IMPORTANCE Chlamydia trachomatis is a frequent bacterial pathogen throughout the world, causing mostly eye and genital infections. C. trachomatis can develop only inside host cells; it multiplies inside a membranous vacuole in the cytosol, termed an inclusion. The inclusion is covered by cytoskeletal "coats" or "cages," whose organization and function are poorly understood. We here report that a relatively little-characterized group of proteins, septins, is required to organize actin fibers on the inclusion and probably through actin the release of the inclusion. Septins are a group of GTP-binding proteins that can organize into heteromeric complexes and then into large filaments. Septins have previously been found to be involved in the interaction of the cell with bacteria in the cytosol. Our observation that they also organize a reaction to bacteria living in vacuoles suggests that they have a function in the recognition of foreign compartments by a parasitized human cell.}, language = {en} } @article{HurdGruebelWojciechowskietal.2021, author = {Hurd, Paul J. and Gr{\"u}bel, Kornelia and Wojciechowski, Marek and Maleszka, Ryszard and R{\"o}ssler, Wolfgang}, title = {Novel structure in the nuclei of honey bee brain neurons revealed by immunostaining}, series = {Scientific Reports}, volume = {11}, journal = {Scientific Reports}, doi = {10.1038/s41598-021-86078-5}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-260059}, pages = {6852}, year = {2021}, abstract = {In the course of a screen designed to produce antibodies (ABs) with affinity to proteins in the honey bee brain we found an interesting AB that detects a highly specific epitope predominantly in the nuclei of Kenyon cells (KCs). The observed staining pattern is unique, and its unfamiliarity indicates a novel previously unseen nuclear structure that does not colocalize with the cytoskeletal protein f-actin. A single rod-like assembly, 3.7-4.1 mu m long, is present in each nucleus of KCs in adult brains of worker bees and drones with the strongest immuno-labelling found in foraging bees. In brains of young queens, the labelling is more sporadic, and the rod-like structure appears to be shorter (similar to 2.1 mu m). No immunostaining is detectable in worker larvae. In pupal stage 5 during a peak of brain development only some occasional staining was identified. Although the cellular function of this unexpected structure has not been determined, the unusual distinctiveness of the revealed pattern suggests an unknown and potentially important protein assembly. One possibility is that this nuclear assembly is part of the KCs plasticity underlying the brain maturation in adult honey bees. Because no labelling with this AB is detectable in brains of the fly Drosophila melanogaster and the ant Camponotus floridanus, we tentatively named this antibody AmBNSab (Apis mellifera Brain Neurons Specific antibody). Here we report our results to make them accessible to a broader community and invite further research to unravel the biological role of this curious nuclear structure in the honey bee central brain.}, language = {en} } @article{WagnerVolkmerSharanetal.2014, author = {Wagner, Ines and Volkmer, Michael and Sharan, Malvika and Villaveces, Jose M. and Oswald, Felix and Surendranath, Vineeth and Habermann, Bianca H.}, title = {morFeus: a web-based program to detect remotely conserved orthologs using symmetrical best hits and orthology network scoring}, series = {BMC Bioinformatics}, volume = {15}, journal = {BMC Bioinformatics}, number = {263}, doi = {10.1186/1471-2105-15-263}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-115590}, year = {2014}, abstract = {Background: Searching the orthologs of a given protein or DNA sequence is one of the most important and most commonly used Bioinformatics methods in Biology. Programs like BLAST or the orthology search engine Inparanoid can be used to find orthologs when the similarity between two sequences is sufficiently high. They however fail when the level of conservation is low. The detection of remotely conserved proteins oftentimes involves sophisticated manual intervention that is difficult to automate. Results: Here, we introduce morFeus, a search program to find remotely conserved orthologs. Based on relaxed sequence similarity searches, morFeus selects sequences based on the similarity of their alignments to the query, tests for orthology by iterative reciprocal BLAST searches and calculates a network score for the resulting network of orthologs that is a measure of orthology independent of the E-value. Detecting remotely conserved orthologs of a protein using morFeus thus requires no manual intervention. We demonstrate the performance of morFeus by comparing it to state-of-the-art orthology resources and methods. We provide an example of remotely conserved orthologs, which were experimentally shown to be functionally equivalent in the respective organisms and therefore meet the criteria of the orthology-function conjecture. Conclusions: Based on our results, we conclude that morFeus is a powerful and specific search method for detecting remotely conserved orthologs.}, language = {en} } @article{SarukhanyanShityakovDandekar2018, author = {Sarukhanyan, Edita and Shityakov, Sergey and Dandekar, Thomas}, title = {In silico designed Axl receptor blocking drug candidates against Zika virus infection}, series = {ACS Omega}, volume = {3}, journal = {ACS Omega}, number = {5}, doi = {10.1021/acsomega.8b00223}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-176739}, pages = {5281-5290}, year = {2018}, abstract = {After a large outbreak in Brazil, novel drugs against Zika virus became extremely necessary. Evaluation of virus-based pharmacological strategies concerning essential host factors brought us to the idea that targeting the Axl receptor by blocking its dimerization function could be critical for virus entry. Starting from experimentally validated compounds, such as RU-301, RU-302, warfarin, and R428, we identified a novel compound 2′ (R428 derivative) to be the most potent for this task amongst a number of alternative compounds and leads. The improved affinity of compound 2′ was confirmed by molecular docking as well as molecular dynamics simulation techniques using implicit solvation models. The current study summarizes a new possibility for inhibition of the Axl function as a potential target for future antiviral therapies.}, language = {en} } @article{DiestelReschMeinhardtetal.2013, author = {Diestel, Uschi and Resch, Marcus and Meinhardt, Kathrin and Weiler, Sigrid and Hellmann, Tina V. and Mueller, Thomas D. and Nickel, Joachim and Eichler, Jutta and Muller, Yves A.}, title = {Identification of a Novel TGF-beta-Binding Site in the Zona Pellucida C-terminal (ZP-C) Domain of TGF-\(\beta\)-Receptor-3 (TGFR-3)}, series = {PLoS ONE}, volume = {8}, journal = {PLoS ONE}, number = {6}, doi = {10.1371/journal.pone.0067214}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-130904}, pages = {e67214}, year = {2013}, abstract = {The zona pellucida (ZP) domain is present in extracellular proteins such as the zona pellucida proteins and tectorins and participates in the formation of polymeric protein networks. However, the ZP domain also occurs in the cytokine signaling co-receptor transforming growth factor beta (TGF-\(\beta\)) receptor type 3 (TGFR-3, also known as betaglycan) where it contributes to cytokine ligand recognition. Currently it is unclear how the ZP domain architecture enables this dual functionality. Here, we identify a novel major TGF-beta-binding site in the FG loop of the C-terminal subdomain of the murine TGFR-3 ZP domain (ZP-C) using protein crystallography, limited proteolysis experiments, surface plasmon resonance measurements and synthetic peptides. In the murine 2.7 angstrom crystal structure that we are presenting here, the FG-loop is disordered, however, well-ordered in a recently reported homologous rat ZP-C structure. Surprisingly, the adjacent external hydrophobic patch (EHP) segment is registered differently in the rat and murine structures suggesting that this segment only loosely associates with the remaining ZP-C fold. Such a flexible and temporarily-modulated association of the EHP segment with the ZP domain has been proposed to control the polymerization of ZP domain-containing proteins. Our findings suggest that this flexibility also extends to the ZP domain of TGFR-3 and might facilitate co-receptor ligand interaction and presentation via the adjacent FG-loop. This hints that a similar C-terminal region of the ZP domain architecture possibly regulates both the polymerization of extracellular matrix proteins and cytokine ligand recognition of TGFR-3.}, language = {en} } @article{HaarmannNehenDeissetal.2015, author = {Haarmann, Axel and Nehen, Mathias and Deiß, Annika and Buttmann, Mathias}, title = {Fumaric acid esters do not reduce inflammatory NF-\(\kappa\)B/p65 nuclear translocation, ICAM-1 expression and T-cell adhesiveness of human brain microvascular endothelial cells}, series = {International Journal of Molecular Sciences}, volume = {16}, journal = {International Journal of Molecular Sciences}, doi = {10.3390/ijms160819086}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-148295}, pages = {19086-19095}, year = {2015}, abstract = {Dimethyl fumarate (DMF) is approved for disease-modifying treatment of patients with relapsing-remitting multiple sclerosis. Animal experiments suggested that part of its therapeutic effect is due to a reduction of T-cell infiltration of the central nervous system (CNS) by uncertain mechanisms. Here we evaluated whether DMF and its primary metabolite monomethyl fumarate (MMF) modulate pro-inflammatory intracellular signaling and T-cell adhesiveness of nonimmortalized single donor human brain microvascular endothelial cells at low passages. Neither DMF nor MMF at concentrations of 10 or 50 \(\mu\)M blocked the IL-1\(\beta\)-induced nuclear translocation of NF-\(\kappa\)B/p65, whereas the higher concentration of DMF inhibited the nuclear entry of p65 in human umbilical vein endothelium cultured in parallel. DMF and MMF also did not alter the IL-1\(\beta\)-stimulated activation of p38 MAPK in brain endothelium. Furthermore, neither DMF nor MMF reduced the basal or IL-1\(\beta\)-inducible expression of ICAM-1. In accordance, both fumaric acid esters did not reduce the adhesion of activated Jurkat T cells to brain endothelium under basal or inflammatory conditions. Therefore, brain endothelial cells probably do not directly mediate a potential blocking effect of fumaric acid esters on the inflammatory infiltration of the CNS by T cells.}, language = {en} } @article{RickmanLachAbhyankaretal.2015, author = {Rickman, Kimberly A. and Lach, Francis P. and Abhyankar, Avinash and Donovan, Frank X. and Sanborn, Erica M. and Kennedy, Jennifer A. and Sougnez, Carrie and Gabriel, Stacey B. and Elemento, Olivier and Chandrasekharappa, Settara C. and Schindler, Detlev and Auerbach, Arleen D. and Smogorzewska, Agata}, title = {Deficiency of UBE2T, the E2 Ubiquitin Ligase Necessary for FANCD2 and FANCI Ubiquitination, Causes FA-T Subtype of Fanconi Anemia}, series = {Cell Reports}, volume = {12}, journal = {Cell Reports}, doi = {10.1016/j.celrep.2015.06.014}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-151525}, pages = {35 -- 41}, year = {2015}, abstract = {Fanconi anemia (FA) is a rare bone marrow failure and cancer predisposition syndrome resulting from pathogenic mutations in genes encoding proteins participating in the repair of DNA interstrand crosslinks (ICLs). Mutations in 17 genes (FANCA-FANCS) have been identified in FA patients, defining 17 complementation groups. Here, we describe an individual presenting with typical FA features who is deficient for the ubiquitin-conjugating enzyme (E2), UBE2T. UBE2T is known to interact with FANCL, the E3 ubiquitin-ligase component of the multiprotein FA core complex, and is necessary for the monoubiquitination of FANCD2 and FANCI. Proband fibroblasts do not display FANCD2 and FANCI monoubiquitination, do not form FANCD2 foci following treatment with mitomycin C, and are hypersensitive to crosslinking agents. These cellular defects are complemented by expression of wild-type UBE2T, demonstrating that deficiency of the protein UBE2T can lead to Fanconi anemia. UBE2T gene gains an alias of FANCT.}, language = {en} } @article{GeyerChalmersMacKintoshetal.2013, author = {Geyer, Kathrin K. and Chalmers, Iain W. and MacKintosh, Neil and Hirst, Julie E. and Geoghegan, Rory and Badets, Mathieu and Brophy, Peter M. and Brehm, Klaus and Hoffmann, Karl F.}, title = {Cytosine methylation is a conserved epigenetic feature found throughout the phylum Platyhelminthes}, series = {BMC Genomics}, volume = {14}, journal = {BMC Genomics}, number = {462}, issn = {1471-2164}, doi = {10.1186/1471-2164-14-462}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-121892}, year = {2013}, abstract = {Background: The phylum Platyhelminthes (flatworms) contains an important group of bilaterian organisms responsible for many debilitating and chronic infectious diseases of human and animal populations inhabiting the planet today. In addition to their biomedical and veterinary relevance, some platyhelminths are also frequently used models for understanding tissue regeneration and stem cell biology. Therefore, the molecular (genetic and epigenetic) characteristics that underlie trophic specialism, pathogenicity or developmental maturation are likely to be pivotal in our continued studies of this important metazoan group. Indeed, in contrast to earlier studies that failed to detect evidence of cytosine or adenine methylation in parasitic flatworm taxa, our laboratory has recently defined a critical role for cytosine methylation in Schistosoma mansoni oviposition, egg maturation and ovarian development. Thus, in order to identify whether this epigenetic modification features in other platyhelminth species or is a novelty of S. mansoni, we conducted a study simultaneously surveying for DNA methylation machinery components and DNA methylation marks throughout the phylum using both parasitic and non-parasitic representatives. Results: Firstly, using both S. mansoni DNA methyltransferase 2 (SmDNMT2) and methyl-CpG binding domain protein (SmMBD) as query sequences, we illustrate that essential DNA methylation machinery components are well conserved throughout the phylum. Secondly, using both molecular (methylation specific amplification polymorphism, MSAP) and immunological (enzyme-linked immunoabsorbent assay, ELISA) methodologies, we demonstrate that representative species (Echinococcus multilocularis, Protopolystoma xenopodis, Schistosoma haematobium, Schistosoma japonicum, Fasciola hepatica and Polycelis nigra) within all four platyhelminth classes (Cestoda, Monogenea, Trematoda and 'Turbellaria') contain methylated cytosines within their genome compartments. Conclusions: Collectively, these findings provide the first direct evidence for a functionally conserved and enzymatically active DNA methylation system throughout the Platyhelminthes. Defining how this epigenetic feature shapes phenotypic diversity and development within the phylum represents an exciting new area of metazoan biology.}, language = {en} } @article{WenckerMarincolaSchoenfelderetal.2021, author = {Wencker, Freya D. R and Marincola, Gabriella and Schoenfelder, Sonja M. K. and Maaß, Sandra and Becher, D{\"o}rte and Ziebuhr, Wilma}, title = {Another layer of complexity in Staphylococcus aureus methionine biosynthesis control: unusual RNase III-driven T-box riboswitch cleavage determines met operon mRNA stability and decay}, series = {Nucleic Acids Research}, volume = {49}, journal = {Nucleic Acids Research}, number = {4}, doi = {10.1093/nar/gkaa1277}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-259029}, pages = {2192-2212}, year = {2021}, abstract = {In Staphylococcus aureus, de novo methionine biosynthesis is regulated by a unique hierarchical pathway involving stringent-response controlled CodY repression in combination with a T-box riboswitch and RNA decay. The T-box riboswitch residing in the 5′ untranslated region (met leader RNA) of the S. aureus metICFE-mdh operon controls downstream gene transcription upon interaction with uncharged methionyl-tRNA. met leader and metICFE-mdh (m)RNAs undergo RNase-mediated degradation in a process whose molecular details are poorly understood. Here we determined the secondary structure of the met leader RNA and found the element to harbor, beyond other conserved T-box riboswitch structural features, a terminator helix which is target for RNase III endoribonucleolytic cleavage. As the terminator is a thermodynamically highly stable structure, it also forms posttranscriptionally in met leader/ metICFE-mdh read-through transcripts. Cleavage by RNase III releases the met leader from metICFE-mdh mRNA and initiates RNase J-mediated degradation of the mRNA from the 5′-end. Of note, metICFE-mdh mRNA stability varies over the length of the transcript with a longer lifespan towards the 3′-end. The obtained data suggest that coordinated RNA decay represents another checkpoint in a complex regulatory network that adjusts costly methionine biosynthesis to current metabolic requirements.}, language = {en} } @article{ZahoGhirlandoAlfonsoetal.2015, author = {Zaho, Huaying and Ghirlando, Rodolfo and Alfonso, Carlos and Arisaka, Fumio and Attali, Ilan and Bain, David L. and Bakhtina, Marina M. and Becker, Donald F. and Bedwell, Gregory J. and Bekdemir, Ahmet and Besong, Tabot M. D. and Birck, Catherine and Brautigam, Chad A. and Brennerman, William and Byron, Olwyn and Bzowska, Agnieszka and Chaires, Jonathan B. and Chaton, Catherine T. and Coelfen, Helmbut and Connaghan, Keith D. and Crowley, Kimberly A. and Curth, Ute and Daviter, Tina and Dean, William L. and Diez, Ana I. and Ebel, Christine and Eckert, Debra M. and Eisele, Leslie E. and Eisenstein, Edward and England, Patrick and Escalante, Carlos and Fagan, Jeffrey A. and Fairman, Robert and Finn, Ron M. and Fischle, Wolfgang and Garcia de la Torre, Jose and Gor, Jayesh and Gustafsson, Henning and Hall, Damien and Harding, Stephen E. and Hernandez Cifre, Jose G. and Herr, Andrew B. and Howell, Elizabeth E. and Isaac, Richard S. and Jao, Shu-Chuan and Jose, Davis and Kim, Soon-Jong and Kokona, Bashkim and Kornblatt, Jack A. and Kosek, Dalibor and Krayukhina, Elena and Krzizike, Daniel and Kusznir, Eric A. and Kwon, Hyewon and Larson, Adam and Laue, Thomas M. and Le Roy, Aline and Leech, Andrew P. and Lilie, Hauke and Luger, Karolin and Luque-Ortega, Juan R. and Ma, Jia and May, Carrie A. and Maynard, Ernest L. and Modrak-Wojcik, Anna and Mok, Yee-Foong and M{\"u}cke, Norbert and Nagel-Steger, Luitgard and Narlikar, Geeta J. and Noda, Masanori and Nourse, Amanda and Obsil, Thomas and Park, Chad K and Park, Jin-Ku and Pawelek, Peter D. and Perdue, Erby E. and Perkins, Stephen J. and Perugini, Matthew A. and Peterson, Craig L. and Peverelli, Martin G. and Piszczek, Grzegorz and Prag, Gali and Prevelige, Peter E. and Raynal, Bertrand D. E. and Rezabkova, Lenka and Richter, Klaus and Ringel, Alison E. and Rosenberg, Rose and Rowe, Arthur J. and Rufer, Arne C. and Scott, David J. and Seravalli, Javier G. and Solovyova, Alexandra S. and Song, Renjie and Staunton, David and Stoddard, Caitlin and Stott, Katherine and Strauss, Holder M. and Streicher, Werner W. and Sumida, John P. and Swygert, Sarah G. and Szczepanowski, Roman H. and Tessmer, Ingrid and Toth, Ronald T. and Tripathy, Ashutosh and Uchiyama, Susumu and Uebel, Stephan F. W. and Unzai, Satoru and Gruber, Anna Vitlin and von Hippel, Peter H. and Wandrey, Christine and Wang, Szu-Huan and Weitzel, Steven E and Wielgus-Kutrowska, Beata and Wolberger, Cynthia and Wolff, Martin and Wright, Edward and Wu, Yu-Sung and Wubben, Jacinta M. and Schuck, Peter}, title = {A Multilaboratory Comparison of Calibration Accuracy and the Performance of External References in Analytical Ultracentrifugation}, series = {PLoS ONE}, volume = {10}, journal = {PLoS ONE}, number = {5}, doi = {10.1371/journal.pone.0126420}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-151903}, pages = {e0126420}, year = {2015}, abstract = {Analytical ultracentrifugation (AUC) is a first principles based method to determine absolute sedimentation coefficients and buoyant molar masses of macromolecules and their complexes, reporting on their size and shape in free solution. The purpose of this multi-laboratory study was to establish the precision and accuracy of basic data dimensions in AUC and validate previously proposed calibration techniques. Three kits of AUC cell assemblies containing radial and temperature calibration tools and a bovine serum albumin (BSA) reference sample were shared among 67 laboratories, generating 129 comprehensive data sets. These allowed for an assessment of many parameters of instrument performance, including accuracy of the reported scan time after the start of centrifugation, the accuracy of the temperature calibration, and the accuracy of the radial magnification. The range of sedimentation coefficients obtained for BSA monomer in different instruments and using different optical systems was from 3.655 S to 4.949 S, with a mean and standard deviation of (4.304\(\pm\)0.188) S (4.4\%). After the combined application of correction factors derived from the external calibration references for elapsed time, scan velocity, temperature, and radial magnification, the range of s-values was reduced 7-fold with a mean of 4.325 S and a 6-fold reduced standard deviation of \(\pm\)0.030 S (0.7\%). In addition, the large data set provided an opportunity to determine the instrument-to-instrument variation of the absolute radial positions reported in the scan files, the precision of photometric or refractometric signal magnitudes, and the precision of the calculated apparent molar mass of BSA monomer and the fraction of BSA dimers. These results highlight the necessity and effectiveness of independent calibration of basic AUC data dimensions for reliable quantitative studies.}, language = {en} }