The Escherichia coli RnlA – RnlB toxin – antitoxin complex: production, characterization and crystallization
The Escherichia coli rnlAB operon encodes a toxin – antitoxin module that is involved in protection against infection by bacteriophage T4. The full-length RnlA – RnlB toxin – antitoxin complex as well as the toxin RnlA were purified to homogeneity and crystallized. When the affinity tag is placed on RnlA, RnlB is largely lost during purification and the resulting crystals exclusively comprise RnlA. A homogeneous preparation of RnlA – RnlB containing stoichiometric amounts of both proteins could only be obtained using a His tag placed C-terminal to RnlB. Native mass spectrometry and SAXS indicate a 1:1 stoichiometry...
Source: Acta Crystallographica Section F - December 31, 2019 Category: Biochemistry Authors: Garcia Rodriguez, G. Talavera Perez, A. Konijnenberg, A. Sobott, F. Michiels, J. Loris, R. Tags: toxin – antitoxin bacterial stress response macromolecular complex Escherichia coli research communications Source Type: research
The RING domain of mitochondrial E3 ubiquitin ligase 1 and its complex with Ube2D2: crystallization and X-ray diffraction
Mitochondrial E3 ubiquitin ligase 1 (MUL1) is located in the mitochondrial outer membrane and regulates various biological processes, including apoptosis, cell growth, mitophagy and mitochondrial dynamics. The C-terminal region of MUL1 faces the cytoplasm and contains the RING domain (MUL1-RING) where the Ub~E2 thioester binds. Unlike most RING-type E3 enzymes, MUL1-RING alone does not have an additional region that recruits a substrate protein, yet is still able to ubiquitylate the substrate, the p53 protein. Nevertheless, the exact mechanism of the ubiquitylation of p53 by MUL1-RING has not yet been elucidated. In order ...
Source: Acta Crystallographica Section F - December 31, 2019 Category: Biochemistry Authors: Lee, S.-O. Lee, C.-K. Ryu, K.-S. Chi, S.-W. Tags: mitochondrial E3 ubiquitin ligase 1 MUL1 RING domain MUL1-RING Ube2D2 ubiquitylation crystallization research communications Source Type: research
Complexation of the nickel and cobalt transcriptional regulator RcnR with DNA
RcnR is a transcription factor that regulates the homeostasis of cobalt and nickel in bacterial cells. Escherichia coli RcnR was crystallized with DNA that encompasses the DNA-binding site. X-ray diffraction data were collected to 2.9   Å resolution. The crystal belonged to space group P6122 or P6522, with unit-cell parameters a = b = 73.59, c = 157.66   Å , α = β = 90, γ = 120 ° . (Source: Acta Crystallographica Section F)
Source: Acta Crystallographica Section F - December 24, 2019 Category: Biochemistry Authors: Li, C. Vavra, J.W. Carr, C.E. Huang, H.-T. Maroney, M.J. Wilmot, C.M. Tags: RcnR transcription factor nickel homeostasis cobalt homeostasis research communications Source Type: research
Crystallographic analysis of Eisenia hydrolysis-enhancing protein using a long wavelength for native-SAD phasing
In this study, EHEP was purified from the natural digestive fluid of A. kurodai and was crystallized using the sitting-drop vapor-diffusion method. Native and SAD (single-wavelength anomalous diffraction) data sets were successfully collected at resolutions of 1.20 and 2.48   Å using wavelengths of 1.0 and 2.1   Å , respectively, from crystals obtained in initial screening. The crystals belonged to space group P212121 and contained one EHEP molecule in the asymmetric unit. All 20 S-atom sites in EHEP were located and the phases were determined by the SAD method using the S atoms in the natural protein as anomalous sc...
Source: Acta Crystallographica Section F - December 24, 2019 Category: Biochemistry Authors: Sun, X. Ye, Y. Sakurai, N. Kato, K. Yuasa, K. Tsuji, A. Yao, M. Tags: EHEP phlorotannin binding solutionless crystal mount native-SAD biofuel research communications Source Type: research
Crystal structure of an oxidized mutant of human mitochondrial branched-chain aminotransferase
This study presents the crystal structure of a thiol variant of the human mitochondrial branched-chain aminotransferase protein. Human branched-chain aminotransferase (hBCAT) catalyzes the transamination of the branched-chain amino acids leucine, valine and isoleucine and α -ketoglutarate to their respective α -keto acids and glutamate. hBCAT activity is regulated by a CXXC center located approximately 10   Å from the active site. This redox-active center facilitates recycling between the reduced and oxidized states, representing hBCAT in its active and inactive forms, respectively. Site-directed mutagenesis of the re...
Source: Acta Crystallographica Section F - December 24, 2019 Category: Biochemistry Authors: Herbert, D. Gibbs, S. Riddick, A. Conway, M. Dong, M. Tags: human mitochondrial branched-chain aminotransferase redox regulation CXXC center N-terminal loop interdomain loop transaminases research communications Source Type: research
The adhesive PitA pilus protein from the early dental plaque colonizer Streptococcus oralis: expression, purification, crystallization and X-ray diffraction analysis
PitA is the putative tip adhesin of the pilus islet 2 (PI-2)-encoded sortase-dependent pilus in the Gram-positive Streptococcus oralis, an opportunistic pathogen that often flourishes within the diseased human oral cavity. Early colonization by S. oralis and its interaction with Actinomyces oris seeds the development of oral biofilm or dental plaque. Here, the PI-2 pilus plays a vital role in mediating adherence to host surfaces and other bacteria. A recombinant form of the PitA adhesin has now been produced and crystallized. Owing to the large size ( ∼ 100   kDa), flexibility and complicated folding of PitA, obtaining...
Source: Acta Crystallographica Section F - December 22, 2019 Category: Biochemistry Authors: Kumari Yadav, R. Krishnan, V. Tags: adhesins PitA PI-2 sortase-dependent pilus Streptococcus oralis proteolysis terbium crystallophore research communications Source Type: research
Intermediate-resolution crystal structure of the human adenovirus B serotype 3 fibre knob in complex with the EC2-EC3 fragment of desmoglein  2
The cryo-electron microscopy (cryo-EM) structure of the complex between the trimeric human adenovirus B serotype 3 fibre knob and human desmoglein 2 fragments containing cadherin domains EC2 and EC3 has been published, showing 3:1 and 3:2 complexes. Here, the crystal structure determined at 4.5   Å resolution is presented with one EC2-EC3 desmoglein fragment bound per fibre knob monomer in the asymmetric unit, leading to an apparent 3:3 stoichiometry. However, in concentrated solution the 3:2 complex is predominant, as shown by small-angle X-ray scattering (SAXS), while cryo-EM at lower concentrations showed a majority ...
Source: Acta Crystallographica Section F - November 26, 2019 Category: Biochemistry Authors: Vassal-Stermann, E. Hutin, S. Fender, P. Burmeister, W.P. Tags: virus receptors extracellular domains cell-surface glycoproteins desmosomes viral proteins research communications Source Type: research
Crystal structure of the Wheat dwarf virus Rep domain
The Rep domain of Wheat dwarf virus (WDV Rep) is an HUH endonuclease involved in rolling-circle replication. HUH endonucleases coordinate a metal ion to enable the nicking of a specific ssDNA sequence and the subsequent formation of an intermediate phosphotyrosine bond. This covalent protein – ssDNA adduct makes HUH endonucleases attractive fusion tags (HUH-tags) in a diverse number of biotechnological applications. Solving the structure of an HUH endonuclease in complex with ssDNA will provide critical information about ssDNA recognition and sequence specificity, thus enabling rationally engineered protein – DNA inter...
Source: Acta Crystallographica Section F - November 26, 2019 Category: Biochemistry Authors: Everett, B.A. Litzau, L.A. Tompkins, K. Shi, K. Nelson, A. Aihara, H. Evans III, R.L. Gordon, W.R. Tags: crystal structure Wheat dwarf virus Rep domain HUH motif HUH-tag ssDNA engineered protein – ssDNA complexes research communications Source Type: research
Crystal structure of the MIF4G domain of the Trypanosoma cruzi translation initiation factor EIF4G5
Kinetoplastida, a class of early-diverging eukaryotes that includes pathogenic Trypanosoma and Leishmania species, display key differences in their translation machinery compared with multicellular eukaryotes. One of these differences involves a larger number of genes encoding eIF4E and eIF4G homologs and the interaction pattern between the translation initiation factors. eIF4G is a scaffold protein which interacts with the mRNA cap-binding factor eIF4E, the poly(A)-binding protein, the RNA helicase eIF4A and the eIF3 complex. It contains the so-called middle domain of eIF4G (MIF4G), a multipurpose adaptor involved in diff...
Source: Acta Crystallographica Section F - November 19, 2019 Category: Biochemistry Authors: Camillo dos Santos, L.P. de Matos, B.M. de Maman Ribeiro, B.C. Zanchin, N.I.T. Guimar ã es, B.G. Tags: MIF4G domain translation initiation factor EIF4G5 Trypanosoma cruzi research communications Source Type: research
SpaB, an atypically adhesive basal pilin from the lactobacillar SpaCBA pilus: crystallization and X-ray diffraction analysis
The SpaB pilin is recognized as the basal subunit of the sortase-dependent SpaCBA pilus, which is known to be produced by the Gram-positive Lactobacillus rhamnosus GG, a gut-adapted commensal advocated to have health benefits. Despite seeming to function as an archetypal basal pilin by serving as the terminal subunit in pilus assembly, SpaB also assumes an atypical role as a mucoadhesive protein. To shed light on the structural factors that contribute to this dual functional behaviour, a recombinant form of the L. rhamnosus GG SpaB pilin was produced and purified for crystallization and X-ray diffraction experiments. The c...
Source: Acta Crystallographica Section F - November 19, 2019 Category: Biochemistry Authors: Kumar Megta, A. Palva, A. von Ossowski, I. Krishnan, V. Tags: adhesion probiotics basal pilin host – microbe interaction sortase-dependent pili Lactobacillus rhamnosus GG SpaB research communications Source Type: research
An extracellular domain of the EsaA membrane component of the type VIIb secretion system: expression, purification and crystallization
The membrane protein EsaA is a conserved component of the type VIIb secretion system. Limited proteolysis of purified EsaA from Staphylococcus aureus USA300 identified a stable 48   kDa fragment, which was mapped by fingerprint mass spectrometry to an uncharacterized extracellular segment of EsaA. Analysis by circular dichroism spectroscopy showed that this fragment folds into a single stable domain made of mostly α -helices with a melting point of 34.5 ° C. Size-exclusion chromatography combined with multi-angle light scattering indicated the formation of a dimer of the purified extracellular domain. Octahedral crysta...
Source: Acta Crystallographica Section F - November 19, 2019 Category: Biochemistry Authors: Mietrach, N. Schlosser, A. Geibel, S. Tags: ESAT-6-like secretion system ESS type VII secretion system EsaA extracellular domain Staphylococcus aureus USA300 research communications Source Type: research
Conformational heterogeneity in apo and drug-bound structures of Toxoplasma gondii prolyl-tRNA synthetase
In this study, structures of apo and FF-bound T. gondii (TgPRS) are revealed and the dynamic nature of the conformational changes that occur upon FF binding is unraveled. In addition, this study highlights significant conformational plasticity within two different crystal structures of apo PRSs but not within drug-bound PRSs. The apo PRSs exist in multi-conformational states and manifest pseudo-dimeric structures. In contrast, when FF is bound the PRS dimer adopts a highly symmetrical architecture. It is shown that TgPRS does not display extant fold switching, in contrast to P. Â falciparum PRS, despite having over 65% se...
Source: Acta Crystallographica Section F - November 6, 2019 Category: Biochemistry Authors: Mishra, S. Malhotra, N. Kumari, S. Sato, M. Kikuchi, H. Yogavel, M. Sharma, A. Tags: toxoplasmosis prolyl-tRNA synthetase enzyme – inhibitor complexes comparative crystallography drug design r.m.s.d. per residue profile apo holo transistions research communications Source Type: research
The non-swapped monomeric structure of the arginine-binding protein from Thermotoga maritima
Domain swapping is a widespread oligomerization process that is observed in a large variety of protein families. In the large superfamily of substrate-binding proteins, non-monomeric members have rarely been reported. The arginine-binding protein from Thermotoga maritima (TmArgBP), a protein endowed with a number of unusual properties, presents a domain-swapped structure in its dimeric native state in which the two polypeptide chains mutually exchange their C-terminal helices. It has previously been shown that mutations in the region connecting the last two helices of the TmArgBP structure lead to the formation of a variet...
Source: Acta Crystallographica Section F - November 4, 2019 Category: Biochemistry Authors: Smaldone, G. Ruggiero, A. Balasco, N. Abuhammad, A. Autiero, I. Caruso, D. Esposito, D. Ferraro, G. Gelardi, E.L.M. Moreira, M. Quareshy, M. Romano, M. Saaret, A. Selvam, I. Squeglia, F. Troisi, R. Kroon-Batenburg, L.M.J. Esposito, L. Berisio, R. Vitaglia Tags: domain swapping protein oligomerization protein structure dynamics – stability proline residue arginine-binding protein Thermotoga maritima research communications Source Type: research
Structural analysis of free and liganded forms of the Fab fragment of a high-affinity anti-cocaine antibody, h2E2
A high-affinity anti-cocaine monoclonal antibody, designated h2E2, is entering phase 1 clinical trials for cocaine abuse therapy. To gain insight into the molecular details of its structure that are important for binding cocaine and cocaine metabolites, the Fab fragment was generated and crystallized with and without ligand. Structures of the unliganded Fab and the Fab fragment bound to benzoylecgonine were determined, and were compared with each other and with other crystallized anti-cocaine antibodies. The affinity of the h2E2 antibody for cocaine is 4   nM, while that of the cocaine metabolite benzoylecgonine is 20 â€...
Source: Acta Crystallographica Section F - November 4, 2019 Category: Biochemistry Authors: Tan, K. Zhou, M. Ahrendt, A.J. Duke, N.E.C. Tabaja, N. Ball, W.J. Kirley, T.L. Norman, A.B. Joachimiak, A. Schiffer, M. Wilton, R. Pokkuluri, P.R. Tags: high-affinity anti-cocaine antibody antigen-binding fragment crystal structure benzoylecgonine complex h2E2 Fab fragment research communications Source Type: research
Major conformational changes in the structure of lysozyme obtained from a crystal with a very low solvent content
A new crystal form of lysozyme with a very low solvent content (26.35%) has been obtained in the orthorhombic space group P212121 (with unit-cell parameters a = 30.04, b = 51.68, c = 61.53   Å ). The lysozyme structure obtained from these crystals does not show the typical overall fold. Instead, major conformational changes take place in some elements of the secondary structure and in the hydrophobic core of the protein. At the end of the central α -helix ( α 2), Glu35 is usually buried in the catalytic site and shows an abnormally high pKa value, which is key to the activity of the enzyme. The high pKa value of this ...
Source: Acta Crystallographica Section F - November 4, 2019 Category: Biochemistry Authors: Salinas-Garcia, M.C. Plaza-Garrido, M. Alba-Elena, D. Camara-Artigas, A. Tags: low solvent content lysozyme folding intermediate research communications Source Type: research
