TY - JOUR AB - Human tRNA (uracil-5-)-methyltransferase 2 homolog A (TRMT2A) is the dedicated enzyme for the methylation of uridine 54 in transfer RNA (tRNA). Human TRMT2A has also been described as a modifier of polyglutamine (polyQ)-derived neuronal toxicity. The corresponding human polyQ pathologies include Huntington's disease and constitute a family of devastating neurodegenerative diseases. A polyQ tract in the corresponding disease-linked protein causes neuronal death and symptoms such as impaired motor function, as well as cognitive impairment. In polyQ disease models, silencing of TRMT2A reduced polyQ-associated cell death and polyQ protein aggregation, suggesting this protein as a valid drug target against this class of disorders. In this paper, the 1.6 Å resolution crystal structure of the RNA-recognition motif (RRM) from Drosophila melanogaster, which is a homolog of human TRMT2A, is described and analysed. AU - Witzenberger, M. AU - Janowski, R. AU - Niessing, D. C1 - 69864 C2 - 55291 CY - 2 Abbey Sq, Chester, Ch1 2hu, England SP - 36-42 TI - Crystal structure of the RNA-recognition motif of Drosophila melanogaster tRNA (uracil-5-)-methyltransferase homolog A. JO - Acta Crystallogr. F-Struct. Biol. Cryst. Commun. VL - 80 PB - Int Union Crystallography PY - 2024 ER - TY - JOUR AB - Methanobactins (MBs) are ribosomally produced and post-translationally modified peptides (RiPPs) that are used by methanotrophs for copper acquisition. The signature post-translational modification of MBs is the formation of two heterocyclic groups, either an oxazolone, pyrazinedione or imidazolone group, with an associated thioamide from an X-Cys dipeptide. The precursor peptide (MbnA) for MB formation is found in a gene cluster of MB-associated genes. The exact biosynthetic pathway of MB formation is not yet fully understood, and there are still uncharacterized proteins in some MB gene clusters, particularly those that produce pyrazinedione or imidazolone rings. One such protein is MbnF, which is proposed to be a flavin monooxygenase (FMO) based on homology. To help to elucidate its possible function, MbnF from Methylocystis sp. strain SB2 was recombinantly produced in Escherichia coli and its X-ray crystal structure was resolved to 2.6 Å resolution. Based on its structural features, MbnF appears to be a type A FMO, most of which catalyze hydroxylation reactions. Preliminary functional characterization shows that MbnF preferentially oxidizes NADPH over NADH, supporting NAD(P)H-mediated flavin reduction, which is the initial step in the reaction cycle of several type A FMO enzymes. It is also shown that MbnF binds the precursor peptide for MB, with subsequent loss of the leader peptide sequence as well as the last three C-terminal amino acids, suggesting that MbnF might be needed for this process to occur. Finally, molecular-dynamics simulations revealed a channel in MbnF that is capable of accommodating the core MbnA fragment minus the three C-terminal amino acids. AU - Stewart, A.* AU - Dershwitz, P.* AU - Stewart, C.* AU - Sawaya, M.R.* AU - Yeates, T.O.* AU - Semrau, J.D.* AU - Zischka, H. AU - DiSpirito, A.A.* AU - Bobik, T.A.* C1 - 67819 C2 - 54297 CY - 2 Abbey Sq, Chester, Ch1 2hu, England SP - 111-118 TI - Crystal structure of MbnF: An NADPH-dependent flavin monooxygenase from Methylocystis strain SB2. JO - Acta Crystallogr. F-Struct. Biol. Cryst. Commun. VL - 79 IS - Pt 5 PB - Int Union Crystallography PY - 2023 ER - TY - JOUR AB - The stability and homogeneity of a protein sample is strongly influenced by the composition of the buffer that the protein is in. A quick and easy approach to identify a buffer composition which increases the stability and possibly the conformational homogeneity of a protein sample is the fluorescence-based thermal-shift assay (Thermofluor). Here, a novel 96-condition screen for Thermofluor experiments is presented which consists of buffer and additive parts. The buffer screen comprises 23 different buffers and the additive screen includes small-molecule additives such as salts and nucleotide analogues. The utilization of small-molecule components which increase the thermal stability of a protein sample frequently results in a protein preparation of higher quality and quantity and ultimately also increases the chances of the protein crystallizing. AU - Reinhard, L.* AU - Mayerhofer, H.* AU - Geerlof, A. AU - Müller-Dieckmann, J.* AU - Weiss, M.S.* C1 - 26220 C2 - 32129 SP - 209-214 TI - Optimization of protein buffer cocktails using Thermofluor. JO - Acta Crystallogr. F-Struct. Biol. Cryst. Commun. VL - 69 IS - 2 PB - Wiley-Blackwell PY - 2013 ER - TY - JOUR AB - Type V myosins constitute the main cargo-transporting class of myosin motors in higher eukaryotes. They are mainly defined by their C-terminal globular domain, which is required for cargo binding as well as for motor auto-inhibition in the absence of cargo. To date, high-resolution structures only exist for globular domains from yeast. Since the majority of cellular cargoes in yeast are very different from the cargoes in higher eukaryotes, structural insights into the domain organization of globular domains from human type V myosins are important. The globular domain of human Myo5a was cloned, expressed and crystallized and data sets were collected. The crystals belonged to space group P212121, with unit-cell parameters a = 75.04, b = 86.70, c = 131.41 Å, α = β = γ = 90°, and diffracted with data-collection quality to 2.5 Å resolution. AU - Velvarska, H. AU - Niessing, D. C1 - 28236 C2 - 33020 SP - 1220-1223 TI - Purification, crystallization and preliminary crystallographic analysis of the globular domain of the human type V myosin Myo5a. JO - Acta Crystallogr. F-Struct. Biol. Cryst. Commun. VL - 69 IS - 11 PB - Wiley-Blackwell PY - 2013 ER - TY - JOUR AB - Kar9p is required for correct positioning of the mitotic spindle in Saccharomyces cerevisiae. The in vivo function of Kar9p is well understood, but no structural information is available. Additionally, molecular details of how Kar9p interacts with other proteins are scarce. Full-length Kar9p was expressed in Escherichia coli, purified and crystallized. Diffraction data were collected and processed at 7 angstrom resolution. One crystal showed diffraction to 3 angstrom resolution. The crystals that diffracted to 7 angstrom resolution belonged to space group P3, with unit-cell parameters a = b = 195.02, c = 257.15 angstrom, alpha = beta = 90, gamma = 120 degrees. The crystal that diffracted to 3 angstrom resolution belonged to space group P222, with unit-cell parameters a = 46.37, b = 74.64, c = 133.63 angstrom, alpha = beta = gamma = 90 degrees. AU - Hüls, D. AU - Niessing, D. C1 - 10702 C2 - 30343 SP - 1251-1254 TI - Purification, crystallization and preliminary crystallographic analysis of Kar9p from Saccharomyces cerevisiae. JO - Acta Crystallogr. F-Struct. Biol. Cryst. Commun. VL - 68 IS - 10 PB - Wiley-Blackwell PY - 2012 ER -