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Macromolecular refinement is an optimization process that aims to produce the most likely macromolecular structural model in the light of experimental data. As such, macromolecular refinement is one of the most complex optimization problems in wide use. Macromolecular refinement programs have to deal with the complex relationship between the parameters of the atomic model and the experimental data, as well as a large number of types of prior knowledge about chemical structure. This paper draws attention to areas of unfinished business in the field of macromolecular refinement. In it, we describe ten refinement topics that we think deserve attention and discuss directions leading to macromolecular refinement software that would make the best use of modern computer resources to meet the needs of structural biologists of the twenty‐first century. [ABSTRACT FROM AUTHOR]

Human porphobilinogen deaminase (PBGD), the third enzyme in the heme pathway, catalyzes four times a single reaction to convert porphobilinogen into hydroxymethylbilane. Remarkably, PBGD employs a single active site during the process, with a distinct yet chemically equivalent bond formed each time. The four intermediate complexes of the enzyme have been biochemically validated and they can be isolated but they have never been structurally characterized other than the apo- and holo-enzyme bound to the cofactor. We present crystal structures for two human PBGD intermediates: PBGD loaded with the cofactor and with the reaction intermediate containing two additional substrate pyrrole rings. These results, combined with SAXS and NMR experiments, allow us to propose a mechanism for the reaction progression that requires less structural rearrangements than previously suggested: the enzyme slides a flexible loop over the growing-product active site cavity. The structures and the mechanism proposed for this essential reaction explain how a set of missense mutations result in acute intermittent porphyria. [ABSTRACT FROM AUTHOR]

Glycoproteins traversing the eukaryotic secretory pathway begin life in the endoplasmic reticulum (ER), where their folding is surveyed by the 170-kDa UDP-glucose:glycoprotein glucosyltransferase (UGGT). The enzyme acts as the single glycoprotein folding quality control checkpoint: it selectively reglucosylates misfolded glycoproteins, promotes their association with ER lectins and associated chaperones, and prevents premature secretion from the ER. UGGT has long resisted structural determination and sequence-based domain boundary prediction. Questions remain on how this single enzyme can flag misfolded glycoproteins of different sizes and shapes for ER retention and how it can span variable distances between the site of misfold and a glucoseaccepting N-linked glycan on the same glycoprotein. Here, crystal structures of a full-length eukaryotic UGGT reveal four thioredoxinlike (TRXL) domains arranged in a long arc that terminates in two β-sandwiches tightly clasping the glucosyltransferase domain. The fold of themolecule is topologically complex, with the first β-sandwich and the fourth TRXL domain being encoded by nonconsecutive stretches of sequence. In addition to the crystal structures, a 15-Å cryo-EM reconstruction reveals interdomain flexibility of the TRXL domains. Double cysteine point mutants that engineer extra interdomain disulfide bridges rigidify the UGGT structure and exhibit impaired activity. The intrinsic flexibility of the TRXL domains of UGGT may therefore endow the enzyme with the promiscuity needed to recognize and reglucosylate its many different substrates and/or enable reglucosylation of N-linked glycans situated at variable distances from the site of misfold. [ABSTRACT FROM AUTHOR]

Charge density studies utilise a multipolar expansion of the atomic density (and the associated atomic scattering factor) in order to model asphericity. Contributions of the individual multipoles to the atomic density are then refined as multipole population coefficients. Refinement of these coefficients pertaining to odd-order multipoles that are invariant under the crystal point-group symmetry is often problematic, with ill-defined values and correlations plaguing the convergence to a good model. These difficulties have been discussed in generic terms in the literature, but never explicitly analysed in detail. In this communication, we show that the charge density multipolar atomic scattering factor can be partitioned in three contributions that differ in their behaviour under the point group symmetry of the crystal. This partitioning rationalises and predicts the conditions that give rise to ill-conditioning of the charge density refinement of these multipoles. [ABSTRACT FROM AUTHOR]

The article discusses research by the authors into the structure of the microbial alkaline phosphatase enzyme PhoX and its role in microorganisms' phosphate acquisition. Topics include the presence of an iron-calcium cofactor similar to certain metal complexes, details of the catalytic mechanism involved, and the possibility that microbial phosphate acquisition is limited by the bioavailability of iron.

Background Numerous epidemiologic studies have linked the presence of chronic obstructive pulmonary disease ( COPD) to coronary artery disease ( CAD). However, prevalence, pathological processes, clinical manifestations and therapy are still debated, as progress towards uncovering the link between these two disorders has been hindered by the complex nature of multimorbidity. Methods Articles targeting CAD in patients with COPD were identified from the searches of MEDLINE and EMBASE databases in July 2013. Three authors reviewed available evidence, focusing on the latest development on disease prevalence, pathogenesis, clinical manifestations and therapeutic strategies. Both clinical trial and previous reviews have been included in this work. Results The most accredited hypothesis asserts that the main common risk factors, that is, cigarette smoke and ageing, elicit a chronic low-grade systemic inflammatory response, which affects both cardiovascular endothelial cells and airways/lung parenchyma. The development of CAD in patients with COPD potentiates the morbidity of COPD, leading to increased hospitalizations, mortality and health costs. Moreover, correct diagnosis is challenging and therapies are not clearly defined. Conclusions Evidence from recently published articles highlights the importance of multimorbidity in patient management and future research. Moreover, many authors emphasize the importance of low-grade systemic inflammation as a common pathological mechanism and a possible future therapeutic target. [ABSTRACT FROM AUTHOR]

Molecules that simultaneously inhibit independent or co-dependent proinflammatory pathways may have advantages over conventional monotherapeutics. OmCI is a bifunctional protein derived from blood-feeding ticks that specifically prevents complement (C)-mediated C5 activation and also sequesters leukotriene B4 (LTB4) within an internal binding pocket. Here, we examined the effect of LTB4 binding on OmCI structure and function and investigated the relative importance of C-mediated C5 activation and LTB4 in a mouse model of immune complex-induced acute lung injury (IC-ALI). We describe two crystal structures of bacterially expressed OmCI: one binding a C16 fatty acid and the other binding LTB4 (C20). We show that the C5 and LTB4 binding activities of the molecule are independent of each other and that OmCI is a potent inhibitor of experimental IC-ALI, equally dependent on both C5 inhibition and LTB4 binding for full activity. The data highlight the importance of LTB4 in IC-ALI and activation of C5 by the complement pathway C5 convertase rather than by non-C proteases. The findings suggest that dual inhibition of C5 and LTB44 may be useful for treatment of human immune complex-dependent diseases. [ABSTRACT FROM AUTHOR]

The human lectin complement pathway activation molecules comprise mannose-binding lectin (MBL) and ficolin-1, -2, and -3 in complex with associated serine proteases MASP-1, -2, and -3 and the non-enzymatic small MBL associated protein or sMAP. Recently, a novel plasma protein named MBL/ficolin associated protein-1 (MAP-1) was identified in humans. This protein is the result of a differential splicing of the MASP1 gene and includes the major part of the heavy chain but lacks the serine protease domain. We investigated the direct interactions of MAP-1 and MASP-3 with ficolin-3 and MBL using surface plasmon resonance and found affinities around 5 nM and 2.5 nM, respectively. We studied structural aspects of MAP-1 and could show by multi-angle laser light scattering that MAP-1 forms a calcium-dependent homodimer in solution. We were able to determine the crystal structure of MAP-1, which also contains a head-to-tail dimer ∼146 Å long. This structure of MAP-1 also enables modeling and assembly of the MASP-1 molecule in its entirety. Finally we found that MAP-1 competes with all three MASPs for ligand binding and is able to mediate a strong dose-dependent inhibitory effect on the lectin pathway activation, as measured by levels of C3 and C9. [ABSTRACT FROM AUTHOR]

Tetartohedral crystal twinning is discussed as a particular case of (pseudo)merohedral twinning when the number of twinned domains is four. Tetartohedrally twinned crystals often possess pseudosymmetry, with the rotational part of the pseudosymmetry operators coinciding with the twinning operators. Tetartohedrally twinned structures from the literature are reviewed and the recent structure determination of tetartohedrally twinned triclinic crystals of human complement factor I is discussed. [ABSTRACT FROM AUTHOR]

The complement system is a key component of innate and adaptive immune responses. Complement regulation is critical for prevention and control of disease. We have determined the crystal structure of the complement regulatory enzyme human factor I (fI). FI is in a proteolytically inactive form, demonstrating that it circulates in a zymogen-like state despite being fully processed to the mature sequence. Mapping of functional data from mutants of fI onto the structure suggests that this inactive form is maintained by the noncatalytic heavy-chain allosterically modulating activity of the light chain. Once the ternary complex of fI, a cofactor and a substrate is formed, the allosteric inhibition is released, and fI is oriented for cleavage. In addition to explaining how circulating fI is limited to cleaving only C3b/C4b, our model explains the molecular basis of disease-associated polymorphisms in fI and its cofactors. [ABSTRACT FROM AUTHOR]

Complement receptor 1-related protein Y (CrrY) is an important cell-surface regulator of complement that is unique to rodent species. The structure of rat CrrY domains 1-4 has been determined in two distinct crystal forms and reveals a 70° bend between domains 3 and 4. Comparisons of this structure with those of other complement regulators suggests that rearrangement of this interface may occur on forming the regulatory complex with C3b. [ABSTRACT FROM AUTHOR]

Abstract: Complement component C8 plays a pivotal role in the formation of the membrane attack complex (MAC), an important antibacterial immune effector. C8 initiates membrane penetration and coordinates MAC pore formation. High-resolution structures of C8 subunits have provided some insight into the function of the C8 heterotrimer; however, there is no structural information describing how the intersubunit organization facilitates MAC assembly. We have determined the structure of C8 by electron microscopy and fitted the C8α-MACPF (membrane attack complex/perforin)-C8γ co-crystal structure and a homology model for C8β-MACPF into the density. Here, we demonstrate that both the C8γ protrusion and the C8α-MACPF region that inserts into the membrane upon activation are accessible. [ABSTRACT FROM AUTHOR]

SoxB is an essential component of the bacterial Sox sulfur oxidation pathway. SoxB contains a di-manganese(II) site and is proposed to catalyze the release of sulfate from a protein-bound cysteine S-thiosulfonate. A direct assay for SoxB activity is described. The structure of recombinant Therm us thermophilus SoxB was determined by x-ray crystallography to a resolution of 1.5 Å. Structures were also determined for SoxB in complex with the substrate analogue thiosulfate and in complex with the product sulfate. A mechanistic model for SoxB is proposed based on these structures. [ABSTRACT FROM AUTHOR]

Dr adhesins are expressed on the surface of uropathogenic and diffusely adherent strains of Escherichia coli. The major adhesin subunit (DraE/AfaE) of these organelles mediates attachment of the bacterium to the surface of the host cell and possibly intracellular invasion through its recognition of the complement regulator decay-accelerating factor (DAF) and/ or members of the carcinoembryonic antigen (CEA) family. The adhesin subunit of the Dr haemagglutinin, a Dr-family member, additionally binds type IV collagen and is inhibited in all its receptor interactions by the antibiotic chloramphenicol (CLM). In this study, previous structural work is built upon by reporting the X-ray structures of DraE bound to two chloramphenicol derivatives: chloramphenicol succinate (CLS) and bromamphenicol (BRM). The CLS structure demonstrates that acylation of the 3-hydroxyl group of CLM with succinyl does not significantly perturb the mode of binding, while the BRM structure implies that the binding pocket is able to accommodate bulkier substituents on the N-acyl group. It is concluded that modifications of the 3-hydroxyl group would generate a potent Dr haemagglutinin inhibitor that would not cause the toxic side effects that are associated with the normal bacteriostatic activity of CLM. [ABSTRACT FROM AUTHOR]

Abstract: Many Gram-negative pathogenic bacteria use a complex macromolecular machine, known as the type 3 secretion system (T3SS), to transfer virulence proteins into host cells. The T3SS is composed of a cytoplasmic bulb, a basal body spanning the inner and outer bacterial membranes, and an extracellular needle. Secretion is regulated by both cytoplasmic and inner membrane proteins that must respond to specific signals in order to ensure that virulence proteins are not secreted before contact with a eukaryotic cell. This negative regulation is mediated, in part, by a family of proteins that are thought to physically block the entrance to the secretion apparatus until an appropriate signal is received following host cell contact. Despite weak sequence homology between proteins of this family, the crystal structures of Shigella flexneri MxiC we present here confirm the conservation of domain topology with the homologue from Yersinia sp. Interestingly, comparison of the Shigella and Yersinia structures reveals a significant structural change that results in substantial domain re-arrangement and opening of one face of the molecule. The conservation of a negatively charged patch on this face suggests it may have a role in binding other components of the T3SS. [Copyright &y& Elsevier]

Abstract: The complement (C) system is a potent innate immune defence system against parasites. We have recently characterised and expressed OmCI, a 16 kDa protein derived from the soft tick Ornithodoros moubata that specifically binds C5, thereby preventing C activation. The structure of recombinant OmCI determined at 1.9 Å resolution confirms a lipocalin fold and reveals that the protein binds a fatty acid derivative that we have identified by mass spectrometry as ricinoleic acid. We propose that OmCI could sequester one of the fatty acid-derived inflammatory modulators from the host plasma, thereby interfering with the host inflammatory response to the tick bite. Mapping of sequence differences between OmCI and other tick lipocalins with different functions, combined with biochemical investigations of OmCI activity, supports the hypothesis that OmCI acts by preventing interaction with the C5 convertase, rather than by blocking the C5a cleavage site. [Copyright &y& Elsevier]

Bacteria expressing type III secretion systems (T3SS) have been responsible for the deaths of millions worldwide, acting as key virulence elements in diseases ranging from plague to typhoid fever. The T3SS is composed of a basal body, which traverses both bacterial membranes, and an external needle through which effector proteins are secreted. We report multiple crystal structures of two proteins that sit at the tip of the needle and are essential for virulence: IpaD from Shigella flexneri and BipD from Burkholderia pseudomallei. The structures reveal that the N-terminal domains of the molecules are intramolecular chaperones that prevent premature oligomerization, as well as sharing structural homology with proteins involved in eukaryotic actin rearrangement. Crystal packing has allowed us to construct a model for the tip complex that is supported by mutations designed using the structure. [ABSTRACT FROM AUTHOR]

A construct consisting of residues 10–310 of BipD, a component of the Burkholderia pseudomallei type III secretion system (T3SS), has been overexpressed as a GST fusion, cleaved from the GST tag and purified. Crystals were grown of native and selenomethionine-labelled BipD. The crystals grow in two different polymorphs from the same condition. The first polymorph belongs to space group C222, with unit-cell parameters a = 103.98, b = 122.79, c = 49.17 Å, a calculated Matthews coefficient of 2.4 Å3 Da−1 (47% solvent content) and one molecule per asymmetric unit. The second polymorph belongs to space group P21212, with unit-cell parameters a = 136.47, b = 89.84, c = 50.15 Å, and a calculated Matthews coefficient of 2.3 Å3 Da−1 (45% solvent content) for two molecules per asymmetric unit (analysis of the self-rotation function indicates the presence of a weak twofold non-crystallographic symmetry axis in this P21212 form). The native crystals of both forms give diffraction data to 2.7 Å resolution, while the SeMet-labelled P21212 crystals diffract to 3.3 Å resolution. A K2PtCl4 derivative of the P21212 form was also obtained and data were collected to 2.7 Å with radiation of wavelength λ = 0.933 Å. The Pt-derivative anomalous difference Patterson map revealed two self-peaks on the Harker sections. [ABSTRACT FROM AUTHOR]

IpaD, the putative needle-tip protein of the Shigella flexneri type III secretion system, has been overexpressed and purified. Crystals were grown of the native protein in space group P212121, with unit-cell parameters a = 55.9, b = 100.7, c = 112.0 Å, and data were collected to 2.9 Å resolution. Analysis of the native Patterson map revealed a peak at 50% of the origin on the Harker section v = 0.5, suggesting twofold non-crystallographic symmetry parallel to the b crystallographic axis. As attempts to derivatize or grow selenomethionine-labelled protein crystals failed, in-drop proteolysis was used to produce new crystal forms. A trace amount of subtilisin Carlsberg was added to IpaD before sparse-matrix screening, resulting in the production of several new crystal forms. This approach produced SeMet-labelled crystals and diffraction data were collected to 3.2 Å resolution. The SeMet crystals belong to space group C2, with unit-cell parameters a = 139.4, b = 45.0, c = 99.5 Å, β = 107.9°. An anomalous difference Patterson map revealed peaks on the Harker section v = 0, while the self-rotation function indicates the presence of a twofold noncrystallographic symmetry axis, which is consistent with two molecules per asymmetric unit. [ABSTRACT FROM AUTHOR]

Type III secretion systems are essential virulence determinants for many Gram-negative bacterial pathogens. The type III secretion system consists of cytoplasmic, transmembrane, and extracellular domains. The extracellular domain is a hollow needle protruding above the bacterial surface and is held within a basal body that traverses both bacterial membranes. Effector proteins are trans-located, via this external needle, directly into host cells, where they subvert normal cell functions to aid infection. Physical contact with host cells initiates secretion and leads to formation of a pore, thought to be contiguous with the needle channel, in the host-cell membrane. Here, we report the crystal structure of the Shigella flexneri needle subunit MxiH and a complete model for the needle assembly built into our three-dimensional EM reconstruction. The model, combined with mutagenesis data, reveals that signaling of host-cell contact is relayed through the needle via intersubunit contacts and suggests a mode of binding for a tip complex. [ABSTRACT FROM AUTHOR]

Anisotropic displacement parameters for H atoms in molecular crystals are calculated with a simplified model that uses approximate vibrational modes, neglecting correlations between internal and external contributions to the mean square motions. These calculated values agree within a few esds with the ones obtained from neutron diffraction; used in charge density multipolar refinements against X-ray diffraction data, they yield electron densities and derived electrostatic properties that are more accurate than the ones obtainable with the traditional isotropic description. For example, electric field gradients at the H nuclei of L-Alanine are in quantitative agreement with Nuclear Quadrupole Resonance results. [Copyright &y& Elsevier]

A single antibody was shown to adopt different binding-site conformations and thereby bind unrelated antigens. Analysis by both x-ray crystallography and pre-steady-state kinetics revealed an equilibrium between different preexisting isomers, one of which possessed a promiscuous, low-affinity binding site for aromatic ligands, including the immunizing hapten. A subsequent induced-fit isomerization led to high-affinity complexes with a deep and narrow binding site. A protein antigen identified by repertoire selection made use of an unrelated antibody isomer with a wide, shallow binding site. Conformational diversity, whereby one sequence adopts multiple structures and multiple functions, can increase the effective sire of the antibody repertoire but may also lead to autoimmunity and allergy. [ABSTRACT FROM AUTHOR]

The experimental electron density distribution (EDD) of 1-methyluracil (1-MUR) was obtained by single crystal X-ray diffraction (XRD) experiments at 23 K. Four different structural models fitting an extensive set of XRD data to a resolution of (sinθ/λ)max = 1.143 Å−1 are compared. Two of the models include anharmonic temperature factors, whose inclusion is supported by the Hamilton test at a 99.95% level of confidence. Positive Fourier residuals up to 0.5 eÅ–3 in magnitude were found close to the methyl group and in the region of hydrogen bonds. Residual density analysis (RDA) and molecular dynamics simulations in the solid-state demonstrate that these residuals can be likely attributed to unresolved disorder, possibly dynamical and long–range in nature. Atomic volumes and charges, molecular moments up to hexadecapoles, as well as maps of the molecular electrostatic potential were obtained from distributed multipole analysis of the EDD. The derived electrostatic properties neither depend on the details of the multipole model, nor are significantly affected by the explicit inclusion of anharmonicity in the least–squares model. The distribution of atomic charges in 1-MUR is not affected by the crystal environment in a significant way. The quality of experimental findings is discussed in light of in-crystal and gas-phase quantum simulations. [ABSTRACT FROM AUTHOR]

Focuses on the first stages of extending the Bayesian program to accurate charge-density studies based on single-crystal x-ray diffraction data. Positional probability distribution of scatterers having maximum entropy relative to a given prior prejudice; Existence of non-nuclear density maxima at special positions for crystalline silicon and beryllium.

Aims: The aims of the study were two-fold: i) examine the validity and reliability of high-speed kicking actions using foot-mounted inertial measurement unit's (IMU), ii) quantify soccer players within-microcycle and inter-positional differences in both the frequency and speed of technical actions. Methods: During the in-season phase (25 weeks) of the UK domestic season, 21 professional soccer player ball releases, high-speed ball releases and ball release index were analysed. Pearson product-moment correlation coefficient and confidence intervals were used to determine the validity between the systems, whilst a general linear mixed model analysis approach was used to establish estimated marginal mean values for total ball releases, high-speed ball releases and ball release index. Results: Good concurrent validity was observed for ball release velocity and high-speed kicks against a high-speed camera (r2- 0.96, CI 0.93–0.98). Ball releases, high-speed ball releases and ball release index all showed main effects for fixture proximity (p>0.001), playing positions (p>0.001) and across different training categories (p>0.001). The greatest high-speed ball releases were observed on a match-day (MD)+1 (17.6 ± 11.9; CI- 16.2 to 19) and MD-2 (16.8 ± 15; CI- 14.9 to 18.7), with MD+1 exhibiting the highest number of ball releases (161.1 ± 51.2; CI- 155.0 to 167.2) and ball release index (145.5 ± 45.2; CI- 140.1 to 150.9) across all fixture proximities. Possessions (0.3 ± 0.9; CI- 0.3 to 0.4) and small-sided games (1.4 ± 1.6; CI- 1.4 to 1.5), had the lowest values for high-speed ball releases with technical (6.1 ± 7.2; CI- 5.7 to 6.6) and tactical (10.0 ± 10.5; CI- 6.9 to 13.1) drills showing the largest high-speed ball releases. Conclusions: The present study provides novel information regarding the quantification of technical actions of professional soccer players. Insights into absolute and relative frequency and intensity of releases in different drill types, provide practitioners with valuable information on technical outputs that can be manipulated during the process of planning training programmes to produce desired outcomes. Both volume and speed of ball release actions should be measured, when monitoring the technical actions in training according to fixture proximity, drill type and player position to permit enhanced training prescription. [ABSTRACT FROM AUTHOR]

Mouse Double Minute 2 (MDM2) is a key negative regulator of the tumor suppressor protein p53. MDM2 overexpression occurs in many types of cancer and results in the suppression of WT p53. The 14-3-3 family of adaptor proteins are known to bind MDM2 and the 14-3-3σ isoform controls MDM2 cellular localization and stability to inhibit its activity. Therefore, small molecule stabilization of the 14-3-3G/MDM2 protein-protein interaction (PPI) is a potential therapeutic strategy for the treatment of cancer. Here, we provide a detailed biophysical and structural characterization of the phosphorylation-dependent interaction between 14-3-3σ and peptides that mimic the 14-3-3 binding motifs within MDM2. The data show that di-phosphorylation of MDM2 at S166 and S186 is essential for high affinity 14-3-3 binding and that the binary complex formed involves one MDM2 di-phosphorylated peptide bound to a dimer of 14-3-3σ. However, the two phosphorylation sites do not simultaneously interact so as to bridge the 14-3-3 dimer in a 'multivalent' fashion. Instead, the two phosphorylated MDM2 motifs 'rock' between the two binding grooves of the dimer, which is unusual in the context of 14-3-3 proteins. In addition, we show that the 14-3-3σ-MDM2 interaction is amenable to small molecule stabilization. The natural product fusicoccin A forms a ternary complex with a 14-3-3σ dimer and an MDM2 di-phosphorylated peptide resulting in the stabilization of the 14-3-3σ/MDM2 PPI. This work serves as a proof-of-concept of the drugability of the 14-3-3/MDM2 PPI and paves the way toward the development of more selective and efficacious small molecule stabilizers. [ABSTRACT FROM AUTHOR]

Endoplasmic reticulum (ER) retention of misfolded glycoproteins is mediated by the ER‐localized eukaryotic glycoprotein secretion checkpoint, UDP‐glucose glycoprotein glucosyl‐transferase (UGGT). The enzyme recognizes a misfolded glycoprotein and flags it for ER retention by re‐glucosylating one of its N‐linked glycans. In the background of a congenital mutation in a secreted glycoprotein gene, UGGT‐mediated ER retention can cause rare disease, even if the mutant glycoprotein retains activity ("responsive mutant"). Using confocal laser scanning microscopy, we investigated here the subcellular localization of the human Trop‐2‐Q118E, E227K and L186P mutants, which cause gelatinous drop‐like corneal dystrophy (GDLD). Compared with the wild‐type Trop‐2, which is correctly localized at the plasma membrane, these Trop‐2 mutants are retained in the ER. We studied fluorescent chimeras of the Trop‐2 Q118E, E227K and L186P mutants in mammalian cells harboring CRISPR/Cas9‐mediated inhibition of the UGGT1 and/or UGGT2 genes. The membrane localization of the Trop‐2 Q118E, E227K and L186P mutants was successfully rescued in UGGT1−/−cells. UGGT1 also efficiently reglucosylated Trop‐2‐Q118E‐EYFP in cellula. The study supports the hypothesis that UGGT1 modulation would constitute a novel therapeutic strategy for the treatment of pathological conditions associated to misfolded membrane glycoproteins (whenever the mutation impairs but does not abrogate function), and it encourages the testing of modulators of ER glycoprotein folding quality control as broad‐spectrum rescue‐of‐secretion drugs in rare diseases caused by responsive secreted glycoprotein mutants. [ABSTRACT FROM AUTHOR]

The article discusses the case of a male patient with Mycobacterium marseillense-associated pulmonary disease in Italy. Topics discussed include the patient's history of hospital admissions in 2005, 2006 and 2007, computed tomography in 2010 which revealed the worsening condition of the patient's lungs, and treatment with ethambutol, azithromycin and rifampicin.

In this article, the author focuses on the prevalence of molecular replacements (MR) in structural science and placing a homologous model in a crystal's unit cell based experimental model. He states that MR would become the technique of choice to obtain initial phases for the majority of future macromolecular crystal structures. He further highlights future prospects of course that will allow crystals and electron microscopy (EM) and tomography images of larger and larger macromolecular.

Borrelia burgdorferi, a spirochete transmitted to human hosts during feeding of infected Ixodes ticks, is the causative agent of Lyme disease. Serum-resistant B. burgdorferi strains cause a chronic, multisystemic form of the disease and bind complement factor H (FH) and FH-like protein 1 (FHL-1) on the spirochete surface. Here we report the atomic structure for the key FHL-1- and FH-binding protein BbCRASP-1 and reveal a homodimer that presents a novel target for drug design. [ABSTRACT FROM AUTHOR]

The possible occurrence of static/dynamic disorder at the Mg site in pyrope (Mg3Al2Si3O12), with or without anharmonic contribution to the thermal vibrations even at low temperatures, has been largely debated but conclusions were contrasting. Here a report is given on the experimental charge density distribution, ρEXP, of synthetic pyrope at T = 30 K, built through a Stewart multipolar expansion up to l = 5 and based on a very precise and accurate set of in-home measured single-crystal X-ray diffraction amplitudes with a maximum resolution of 0.44 Å. Local and integral topological properties of ρEXP are in substantial agreement with those of ρTHEO, the corresponding DFT-grade quantum charge density of an ideal pyrope crystal, and those derived from synchrotron investigations of chemical bonding in olivines. Relevant thermal atomic displacements, probably anharmonic in nature, clearly affect the whole structure down to 30 K. No significant (> 2.5σ) residual Fourier peaks are detectable from the ρEXP distribution around Mg, after least-squares refinement of a multipole model with anharmonic thermal motion at the Mg site. Experimental findings were confirmed by a full analysis of normal vibration modes of the DFT-optimized structure of the perfect pyrope crystal. Mg undergoes wide displacements from its equilibrium position even at very low temperatures, as it is allocated in a ∼ 4.5 Å large dodecahedral cavity and involved in several soft phonon modes. Implications on the interplay among static/dynamic disorder of Mg and lattice vibrational degrees of freedom are discussed. [ABSTRACT FROM AUTHOR]

Fusarium endophytes damage cereal crops and contaminate produce with mycotoxins. Those fungi overcome the main chemical defence of host via detoxification by a malonyl‐CoA‐dependent enzyme homologous to xenobiotic metabolizing arylamine N‐acetyltransferase (NAT). In Fusarium verticillioides (teleomorph Gibberella moniliformis, GIBMO), this N‐malonyltransferase activity is attributed to (GIBMO)NAT1, and the fungus has two additional isoenzymes, (GIBMO)NAT3 (N‐acetyltransferase) and (GIBMO)NAT2 (unknown function). We present the crystallographic structure of (GIBMO)NAT1, also modelling other fungal NAT homologues. Monomeric (GIBMO)NAT1 is distinctive, with access to the catalytic core through two "tunnel‐like" entries separated by a "bridge‐like" helix. In the quaternary arrangement, (GIBMO)NAT1 monomers interact in pairs along an extensive interface whereby one entry of each monomer is covered by the N‐terminus of the other monomer. Although monomeric (GIBMO)NAT1 apparently accommodates acetyl‐CoA better than malonyl‐CoA, dimerization changes the active site to allow malonyl‐CoA to reach the catalytic triad (Cys110, His158 and Asp173) via the single uncovered entry, and anchor its terminal carboxyl‐group via hydrogen bonds to Arg109, Asn157 and Thr261. Lacking a terminal carboxyl‐group, acetyl‐CoA cannot form such stabilizing interactions, while longer acyl‐CoAs enter the active site but cannot reach catalytic Cys. Other NAT isoenzymes lack such structural features, with (GIBMO)NAT3 resembling bacterial NATs and (GIBMO)NAT2 adopting a structure intermediate between (GIBMO)NAT1 and (GIBMO)NAT3. Biochemical assays confirmed differential donor substrate preference of (GIBMO)NAT isoenzymes, with phylogenetic analysis demonstrating evolutionary separation. Given the role of (GIBMO)NAT1 in enhancing Fusarium pathogenicity, unravelling the structure and function of this enzyme may benefit research into more targeted strategies for pathogen control. [ABSTRACT FROM AUTHOR]

We report about the response of Arabidopsis thaliana to chronic and temporary Cd2+ stress, and the Cd2+ induced activation of ER stress and unfolded protein response (UPR). Cd2+-induced UPR proceeds mainly through the bZIP60 arm, which in turn activates relevant ER stress marker genes such as BiP3, CNX, PDI5 and ERdj3B in a concentration- (chronic stress) or time- (temporary stress) dependent manner. A more severe Cd-stress triggers programmed cell death (PCD) through the activation of the NAC089 transcription factor. Toxic effects of Cd2+ exposure are reduced in the Atbzip28/bzip60 double mutant in terms of primary root length and fresh shoot weight, likely due to reduced UPR and PCD activation. We also hypothesised that the enhanced Cd2+ tolerance of the Atbzip28/bzip60 double mutant is due to an increase in brassinosteroids signaling, since the amount of the brassinosteroid insensitive1 receptor (BRI1) protein decreases under Cd2+ stress only in Wt plants. These data highlight the complexity of the UPR pathway, since the ER stress response is strictly related to the type of the treatment applied and the multifaceted connections of ER signaling. The reduced sensing of Cd2+ stress in plants with UPR defects can be used as a novel strategy for phytoremediation. • Cadmium stress in not corrected perceived in A. thaliana bzip28/bzip60 mutant, with a consequent attenuate molecular response and increased tolerance to metal. [ABSTRACT FROM AUTHOR]

N-Chloro-N′-(p-fluorophenyl)-benzamidine (NCLBA) is a N-halamine derivative which can easily release chlorine, for example when stimulated by high-energy photons. Despite the rich chemistry performed by N-halamines, the chemical properties of the N–Cl bond are poorly investigated. In this work, we determine the accurate charge density distribution of NCLBA by single crystal X-ray diffraction. A very low temperature (T = 17.5 K), coupled with the low X-ray flux of a fine-focus conventional source, allowed the specimen to survive for longer than 750 h of data acquisition without appreciable diffraction deterioration. Electronic and electrostatic properties of NCLBA are not significantly affected by the crystal field, enabling the derivation of molecular properties from the X-ray experimental data. The N–Cl bond in NCLBA is one of the longest reported to date in available structural studies. In general, the longer the N-halogen bond, the lower the amount of electron sharing in the internuclear region, with the bond approaching its homolytic dissociation limit. The synergy between accurately measured high-order data and low temperature enabled modelling of the residual thermal motion anharmonicity of the molecule's halogen atoms with refinement of Gram–Charlier thermal cumulants at the expense of large parameter correlations, as the data extension is rather short of the Kuhs empirical rule. [ABSTRACT FROM AUTHOR]

Melanization contributes to arthropod-specific innate immunity through deposition of melanin at wound sites or around parasites, with concomitant release of microbicidal reactive oxygen species. Melanization requires sequential activation of proteolytic enzymes in the hemolymph, including the final enzyme pro-phenoloxidase. Black cells ( Bc ) is a mutation causing spontaneous melanization of Drosophila crystal cells, a hemocyte cell type producing phenoloxidases. Bc individuals exhibit circulating black spots but fail to melanize upon injury. Although Bc is widely used as a loss-of-function mutant of phenoloxidases, the mutation causing Bc remained unknown. Here, we identified a single point mutation in the pro-phenoloxidase 1 ( PPO1 ) gene of Bc flies causing an Alanine to Valine change in the C-terminal domain of PPO1, predicted to affect the conformation of the N-terminal pro-domain cleavage site at a distance and causing uncontrolled catalytic activity. Genomic insertion of a PPO1 A480V transgene phenocopies Black cells , proving that A480V is indeed the causal mutation of the historical Bc phenotype. [ABSTRACT FROM AUTHOR]

It has recently been shown that the biosynthetic route for both the d1-haem cofactor of dissimilatory cd1 nitrite reductases and haem, via the novel alternative-haem-synthesis pathway, involves siroheme as an intermediate, which was previously thought to occur only as a cofactor in assimilatory sulphite/nitrite reductases. In many denitrifiers (which require d1-haem), the pathway to make siroheme remained to be identified. Here we identify and characterize a sirohydrochlorin-ferrochelatase from P aracoccus pantotrophus that catalyses the last step of siroheme synthesis. It is encoded by a gene annotated as cbiX that was previously assumed to be encoding a cobaltochelatase, acting on sirohydrochlorin. Expressing this chelatase from a plasmid restored the wild-type phenotype of an E scherichia coli mutant-strain lacking sirohydrochlorin-ferrochelatase activity, showing that this chelatase can act in the in vivo siroheme synthesis. A Δ cbiX mutant in P . denitrificans was unable to respire anaerobically on nitrate, proving the role of siroheme as a precursor to another cofactor. We report the 1.9 Å crystal structure of this ferrochelatase. In vivo analysis of single amino acid variants of this chelatase suggests that two histidines, His127 and His187, are essential for siroheme synthesis. This CbiX can generally be identified in α-proteobacteria as the terminal enzyme of siroheme biosynthesis. [ABSTRACT FROM AUTHOR]

The electron paramagnetic resonance technique of double electron-electron resonance (DEER) was used to measure nanometre-scale distances between nitroxide spin labels attached to the complement regulatory protein CD55 (also known as decay accelerating factor) and the von Willebrand factor A (vWF-A) domain of factor B. Following a thorough assessment of the quality of the data, distances obtained from good-quality measurements are compared to predicted distances from a previously hypothesised model for the complex and are found to be incompatible. The success of using these distances as restraints in multi-body docking routines is presented critically. [ABSTRACT FROM PUBLISHER]

Summary: Introduction: Chronic obstructive pulmonary disease (COPD) is characterized by a relatively irreversible airflow limitation caused by chronic inflammation, in most cases tobacco-related. The impact of COPD on morbidity and mortality at the single-patient level depends upon the severity of COPD symptoms and the existence of other types of systemic and/or pulmonary disease, also known as co-morbid conditions. Materials and methods: This review examines the pulmonary diseases commonly associated with COPD, in terms of their prevalence, clinical features, pathogenic mechanisms, prognoses, and implications for management of COPD. Results: The incidence and prevalence of various pulmonary diseases are significantly increased in patients with COPD. These conditions include symptomatic bronchiectasis, combined pulmonary fibrosis and emphysema, lung cancer, sleep-related respiratory disorders, and pulmonary embolism. Some of these concomitant respiratory diseases have an independent negative impact on the prognosis of COPD patients, and their presence has important implications for treatment of these patients. Conclusions: Physicians treating patients with COPD need to be aware of these coexisting pulmonary diseases. All patients with COPD should be carefully evaluated to identify pulmonary comorbidities, since they not only influence the prognosis but also have an impact on disease management. The treatment of COPD is no longer restricted exclusively to inhaled therapy. The therapeutic approach to this disease is becoming increasingly multidimensional in view of the fact that successful management of comorbidities might positively affect the course of COPD itself. [Copyright &y& Elsevier]

Abstract: Over the past few years, there have been substantial advances in our understanding of sarcoidosis immunopathogenesis. Conversely, the etiology of the disease remains obscure for a number of reasons, including heterogeneity of clinical manifestations, often overlapping with other disorders, and insensitive and nonspecific diagnostic tests. While no cause has been definitely confirmed, there is increasing evidence that one or more infectious agents may cause the disease, although the organism may no longer be viable. Here we present 2 cases, in which sarcoidosis preceded tuberculosis and non-Hodgkin lymphoma. Development of new lesions in a patient with chronic/remitting sarcoidosis should be looked at with suspicion and promptly investigated in order to rule out an alternative/concomitant diagnosis. In such cases, tissue confirmation from the most accessible site, and bone marrow biopsy—if lymphoma is in the differential diagnosis—should be performed. In conclusion, we strongly advise that physicians be ready to reconsider the diagnosis of sarcoidosis in the presence of atypical manifestations or persistent/progressive disease despite conventional therapy. [Copyright &y& Elsevier]

Enzymatic catalysis of oxygenation reactions in the absence of metal or organic cofactors is a considerable biochemical challenge. The CO-forming 1-H-3-hydroxy-4-oxoquinaldine 2,4-dioxygenase (HOD) from Arthrobacter nitroguajacolicus Rü6la and 1-H-3-hydroxy-4-oxoquinoline 2,4-dioxygenase (QDO) from Pseudomonas putida 33/1 are homologous cofactor-independent dioxygenases involved in the breakdown of N-heteroaromatic compounds. To date, they are the only dioxygenases suggested to belong to the a/p-hydrolase fold superfamily. Members of this family typically catalyze hydrolytic processes rather than oxygenation reactions. We present here the crystal structures of both HOD and QDO in their native state as well as the structure of HOD in complex with its natural 1 -H-3-hydroxy-4-oxoquinaldine substrate, its N-acetylanthranilate reaction product, and chloride as dioxygen mimic. HOD and QDO are structurally very similar. They possess a classical α/β-hydrolase fold core domain additionally equipped with a cap domain. Organic substrates bind in a preorganized active site with an orientation ideally suited for selective deprotonation of their hydroxyl group by a His/Asp charge-relay system affording the generation of electron-donating species. The "oxyanion hole" of the α/β-hydrolase fold, typically employed to stabilize the tetrahedral intermediate in ester hydrolysis reactions, is utilized here to host and control oxygen chemistry, which is proposed to involve a peroxide anion intermediate. Product release by proton back transfer from the catalytic histidine is driven by minimization of intramolecular charge repulsion. Structural and kinetic data suggest a nonnucleophilic general-base mechanism. Our analysis provides a framework to explain cofactor-independent dioxygenation within a protein architecture generally employed to catalyze hydrolytic reactions. [ABSTRACT FROM AUTHOR]

Macrophage scavenger receptor A (SR-A) is a multifunctional, multiligand pattern recognition receptor with roles in innate immunity, apoptotic cell clearance, and age-related degenerative pathologies, such as atherosclerosis and Alzheimer's disease. Known endogenous SR-A ligands are polyanionic and include modified lipoproteins, advanced glycation end products, and extracellular matrix proteins. No native plasma ligands have been identified, but it is known that SR-A recognition of unidentified serum components mediates integrin-independent macrophage adhesion, which may drive chronic local inflammation. In this study, we used a high-throughput fractionation and screening method to identify novel endogenous SR-A ligands that may mediate macrophage adhesion. SR-A was found to recognize the exchangeable apolipoproteins A-I and E (apo A-I and apo E, respectively) in both lipid-free and lipid-associated form, suggesting the shared amphipathic α-helix as a potential recognition motif. Adhesion of RAW 264.7 macrophages to surfaces coated with apo A-I and apo E4 proved to be integrin-independent and could be blocked by anti-SR-A antibodies. The presence of apo A-I and apo E in pathological deposits, such as atherosclerotic lesions and neurotoxic Alzheimer's plaques, suggests a possible contribution of SR-A-dependent adhesion of macrophages to an inflammatory microenvironment. [ABSTRACT FROM AUTHOR]