bluuebunny/crossref_metadata_2025 huggingface dataset:
Structural basis for substrate recognition mechanism of ER glucosidase II
Authors: Satoh Tadashi , Toshimori Takayasu , Yamaguchi Takumi , Tong Zhu , Kato Koichi
Published: November 2015

The endoplasmic reticulum (ER) possesses a sophisticated quality control system to proofread newly synthesized proteins. A series of N-linked oligosaccharide intermediates attached on the nascent proteins serves as specific tags for the quality control system. In this system, glucosidase II is involved in trimming of non-reducing terminal glucose residue of N-glycan intermediates. Glucosidase II consists of approximately 110 kDa catalytic α subunit (GIIα) and 60 kDa non-catalytic regulatory β subunit (GIIβ). It has been shown that GIIα alone can hydrolyze a small α-glycosidase model substrate (pNP-glucose), while it cannot catalyze deglucosylation of the N-linked oligosaccharide substrates unless it makes a complex with GIIβ. In this study, we determined the first crystal structure of GIIα in the absence and presence of its inhibitor 1-deoxynojirimycin at 1.6-Å resolution. The crystal structure revealed that GIIα has a characteristic segment at the N-terminus as compared with the cognate glycoside hydrolases (GH31). Interestingly, the N-terminal segment was accommodated on the substrate-binding pocket. Based on these results, we suggest that the N-terminal segment of GIIα undergoes structural rearrangement through interaction with GIIβ, thereby promoting the substrate-binding capacity for the N-linked oligosaccharide substrates.
Structural analysis of GNAT acetyltransferases and protein acetylation
Authors: Kuhn Misty , Majorek Karolina , Filippova Ekaterina , Minasov George , Wolfe Alan , Minor Wladek , Anderson Wayne
Published: November 2015

The Center for Structural Genomics for Infectious Diseases (CSGID) applies structural genomics approaches to biomedically relevant proteins from human pathogens and provides the infectious disease community with a high throughput pipeline for structure determination. Target proteins include drug targets, essential enzymes, virulence factors and vaccine candidates. Bacterial species generally have many acetyl-coenzyme A dependent GCN5-like Acetyl Transferases (GNATs), however, the substrates of most of them are unknown. Proteomic analysis has also revealed extensive post-translational modification of bacterial proteins, especially acetylation of lysine Nε. These observations led the CSGID to develop a high throughput substrate screen and initiate characterization of bacterial GNATs. One of the bacterial GNATs that acetylates lysine residues, is the Pseudomonas aeruginosa protein PA4794, that acetylates both peptides having a C-terminal lysine and the drug, chloramphenicol. Surprisingly, the acetylation of these two substrates by PA4794 is catalyzed by the enzyme using different active site residues and different kinetic mechanisms. Although it was expected that the GNATs would play a major role in protein acetylation, much of the lysine acetylation observed in bacteria is actually due to the metabolite acetylphosphate (1,2). Crystal structures and proteomics experiments revealed what makes some lysine residues particularly sensitive to acetylphosphate dependent lysine acetylation and what is required for subsequent enzymatic deacetylation. CSGID is funded with Federal funds from the National Institute of Allergy and Infectious Diseases, National Institutes of Health, Department of Health and Human Services, under Contracts No. HHSN272200700058C and HHSN272201200026C and Midwest Center for Structural Genomics by grant GM094585
Generation, Recognition, and Erasure of 5-methylcytosine and its Oxidative Derivatives
Authors: Cheng Xiaodong , Hashimoto Hideharu , Olanrewaju Yusuf Olatunde , Hong Samuel , Zhang Xing
Published: November 2015

During the development, mammalian germ line cells and brains undergo a series of cellular and molecular events that lead to the erasure and re-establishment of epigenetic programs. It is possible that the active erasure and the re-establishment of 5-methylcytosine (5mC) marks during germ line differentiation and brain development from fetus to young adult involve dynamic changes of 5mC into oxidative marks, and that these modified cytosine residues in DNA are recognized by specific protein readers with distinct roles in the maintenance of epigenetic memory. Here we report our on-going biochemical and structural analyses of generation, recognition and erasure of these marks.
Research at the NSF STC for Biology with X-ray lasers
Authors: Spence John
Published: November 2015

"The NSF BioXFEL Science and Technology Center (STC) is a new consortium of six research campuses devoted to the application of x-ray free-electron lasers (XFELs) to structural biology. Over the last four years a variety of approaches have been made to the observation of protein structure and dynamics for various classes of proteins. The Linac Coherent Light source at SLAC, the first hard-Xray EXFEL, provides intense coherent hard X-ray pulses at 120 Hz which vaporize protein when focussed to a sub-micron beam. Atomic-resolution Bragg diffraction patterns are nevertheless obtained using 50 fs pulses prior to the onset of significant damage, in this ""diffract-then-destroy"" mode, which outruns radiation damage. This use of short pulses instead of freezing samples to reduce radiation damage therefore opens the way to the study of protein dynamics at room temperature in a native environment. I'll review the work of several groups using a range of approaches to different types of sample, including the following: 1. Differences between the frozen sychrotron structure of GPCR proteins and the RT XFEL structure [1]. 2. Pump-probe dynamic structures in Photosynthesis [2]. 3. XFEL study of 2D protein crystals [3]. 4. Prospects for improved resolution in XFEL imaging from single particles such as viruses, where patterns can be obtained from a single virus. 5. New ideas - the Lipid Cubic Phase injector (which allows protein nanocrystals to be studied also at sychrotrons) [4], prospects for fast Laue diffraction using coherent attosecond X-ray lasers, ab-initio phasing [5], the use of angular correlation functions for analysis of fast solution scattering, and two-color opportunites for serial femtosecond crystallography (SFX). See [6] for a recent review of the field. 1. W.Liu et al Science 342, 1521 (2013) 2. A.Aquila et al Optics Express 20, 2706 (2012) 3. M.Frank et al IUCrJ (2014) In press. 4. U.Weierstall et al Nature Comms. (2014) In press. 5. J. Spence et al Optics Express 19, 2866 (2011). 6. J. Spence et al Rep. Prog. Phys. 75, 102601 (2012)."
Feasibility study of visibility-based X-ray photon correlation spectroscopy
Authors: Inoue Ichiro , Shinohara Yuya , Watanabe Akira , Amemiya Yoshiyuki
Published: November 2015

When coherent X-rays impinge upon a disordered system, a grainy scattering pattern called speckle pattern is observed. If the system evolves with time, the corresponding speckle pattern also changes. Temporal changes in the speckle patterns therefore provide information on the dynamics of the system. This technique, which is called X-ray photon correlation spectroscopy (XPCS) [1], has shown the potential to access dynamic properties of various materials, such as colloidal suspensions, block copolymer, supercooled liquids, alloys, and antiferromagnetic materials. Although XPCS is a powerful technique for material science as recent studies show, it has a limitation of time resolution: dynamics faster than the frame rate of detector cannot be measured. When a two-dimensional (2D) detector is used in XPCS, the time resolution is limited to the order of milliseconds. For improving the time resolution of XPCS, we have extended speckle visibility spectroscopy (SVS) in the region of visible light [2] to the region of X-rays (X-ray SVS; XSVS) [3]. Since the minimum exposure time of the scattering patterns determines the time resolutions of XSVS and SVS, micro- or nano- second dynamics can be measured even with a 2D detector. Thus, XSVS has potential to bridge the time gap between XPCS and inelastic neutron/X-ray scattering techniques, and will be one of the promising tools for various science with the next generation synchrotron X-ray sources, such as diffraction limited storage rings and energy recovery linac based X-ray sources. In this presentation, we will describe the principle of XSVS and show the result of the application of XSVS to Brownian colloidal suspensions. This study was performed under the approval of JASRI (2011A1112, 2011B1131). We acknowledge Drs. N. Yagi and N. Ohta for their kind support in performing experiments.
Start-to-End XFEL experiment simulation:A framework and coherent imaging example
Authors: Mancuso Adrian , Yoon Chun Hong , Yurkov Mikhail , Schneidmiller Evgeny , Samoylova Liubov , Jurek Zoltan , Ziaja Beata , Buzmakov Alexey , Loh Duane , Tschentscher Thomas
Published: November 2015

The advent of newer, brighter, and more coherent X-ray sources, such as X-ray Free Electron Lasers (XFELs), represents a tremendous growth in the potential to apply coherent X-rays to determine the structure of materials from the micron-scale down to the Angstrom-scale. We present a framework for Start-to-End (S2E) simulations of a coherent X-ray experiment, including source parameters, propagation of the coherent X-rays though optical elements, interaction of the photons with matter, and their subsequent detection and analysis. To demonstrate this framework, we show a single-particle structure determination example using parameters of the Single Particles, Clusters and Biomolecules (SPB) instrument [1] at the under-construction European XFEL [2, 3]. We use cross platform wave optics software [4] for the propagation of the coherent beams, a molecular dynamics treatment of real space dynamics of atoms, ions and free electrons to account for radiation damage [5], and the Expansion-Maximization-Compression (EMC) algorithm [6] for assembling the simulated data before subsequent phasing and structure determination. It is hoped such simulations can provide an insight into the critical regions of parameter space for the single-particle imaging problem, and hence direct efforts to best utilize these next generation light sources.
Extracting Extremely Localized Molecular Orbitals from X-ray Diffraction Data
Authors: Genoni Alessandro
Published: November 2015

The accurate determination of electron densities in crystals from high-resolution X-ray diffraction data has become more and more important over the years. The existing techniques to accomplish this task can be subdivided into two great families: the multipole models and the wave function-based strategies. The former, which are the most widely used, are essentially linear scaling and allow an easy chemical interpretation of the obtained molecular charge densities, but they are also characterized by some drawbacks, such as the possible presence of unphysical negative regions in the resulting electron distributions. On the contrary, the latter always provide quantum mechanically rigorous electron densities, but they are more computationally expensive and, above all, the ease of chemical interpretation is almost completely lost. In this context, in order to combine the easy chemical interpretability of the multipole models with the quantum mechanical rigor of the wave-function based methods, we have recently extended the X-ray constrained wave function approach proposed by Jayatilaka in the framework of a quantum chemistry technique for the a priori determination of Extremely Localized Molecular Orbitals (ELMOs), namely we have developed a new strategy that allows to extract from X-ray diffraction data a single Slater determinant built up wit Molecular Orbitals strictly localized on small molecular fragments (e.g., atoms, bonds or functional groups). Preliminary tests have shown that the determination of X-ray constrained ELMOs is really straightforward. Furthermore, given the reliable transferability of the obtained Molecular Orbitals, we are constructing new ELMOs databases that can be used as alternative to the existing pseudo-atoms libraries for refining crystallographic structures and electron distributions of large systems. A detailed comparison between the new technique and the multipole models is also currently under investigation.
Experimental charge density studies: Discard valid data and overfit?
Authors: Herbst-Irmer Regine
Published: November 2015

In experimental charge density investigation it is indispensable to use the highest possible quality of data. Therefore the multiplicity should be as high as possible, but poor data should be omitted. To decide about resolution limit and discarding outlier data often limits for Rint or I/σ(I) are used. A better approach is the `paired refinement method' [1] comparing two data sets by the fit of the models derived by the same refinement protocol to both data sets. For macromolecular data sets it could be shown that a higher resolution should be used than normally derived from the above mentioned criteria. First results for charge density data seem to show the same tendency but of course on a different level. The paired refinement strategy can also be used to investigate the influence of different scaling methods. In a recent version of SADABS [2] a new error model and a 3λ correction is implemented. With the paired refinement strategy the improvement in data quality gets obvious. A further concern in charge density investigation is the question of overfitting. In macromolecular refinement this is answered by the Rfree concept [3]. Here a refinement protocol is developed by refining against a work set of reflections, e.g. 90 % of the data. The remaining reflections are untouched in the whole refinement process but an Rfree value is calculated using only this test set of reflections. An overfitting can clearly be identified by a decrease in Rwork but an increase in Rfree. This refinement protocol is then used for a final refinement against all data. It will be discussed how this method could support charge density studies.
Polarized Neutron Diffraction study of the molecular magnetic anisotropy
Authors: Ridier Karl , Gillon Béatrice , Gukasov Arsen , Chaboussant Gregory , Borta Ana , Iasco Olga , Luneau Dominique , Sakiyama Hiroshi , Mikuriya Masahiro , Handa Makoto
Published: November 2015

The magnetic anisotropy is a prerequisite for a metal complex to behave as a single-molecule magnet (SMM). Unfortunately, today we do not fully understand the relationships between the local structural parameters and the magnetic anisotropy that results at the molecular level. This is an issue that has become recursive in this area. Out of the synthesis work which is still important, but generates a multiplication of SMMs with frustrating properties, there are various studies to understand these relationships among which most are theoretical studies. In this context, we believe that polarized neutron diffraction (PND) can provide an experimental and complementary point of view to these theoretical studies. PND is indeed well known to allow an accurate determination of the spin density in magnetic compounds and in the field of molecular magnetism it has provided unique information on the pathways and the nature of intra- or intermolecular magnetic coupling [1]. In the case of highly anisotropic paramagnetic materials, where local magnetic moments cannot be aligned by an external magnetic field, that is more tricky, but a method based on local magnetic susceptibility tensor, has been recently developed that allows now analysing the data in this case and obtaining the magnetization distribution [2]. This approach was first used for inorganic compounds. Our idea has been to use this approach to go beyond the reconstruction of spin density to study the magnetic anisotropy in molecular systems. In this paper, we present the results of such an approach applied for the first time to metal complexes that are simple mono and dinuclear cobalt(II) complexes.
Spin glass state structure theory and its application to explain experiments
Authors: Warczewski Jerzy , Gusin Paweł , Wojcieszyk Daniel
Published: November 2015

The authors have found the general description of all the eight fundamental magnetic structures and their symmetry groups (including spin glass state - sgs) with the use of the fibre bundle approach [1]. The structure of sgs turns out to consist in the Gaussian-like randomness of the orientations of all the separate atomic magnetic moments in the sample. It means that the magnetization vector M is also subject to Gaussian distribution. Thus the structure of the sgs is represented by the magnetization vector situated along the generatrix of a certain cone whose axis coincides with the direction of an internal spontaneous magnetic field indispensable for stability of the sgs in a sample [2]. The axis of the 3-dimensional Gaussian distribution mentioned above coincides also with the same direction. Every precession angle of M around this field makes the symmetry operation of sgs once the angle between M and this field remains constant. Thus the symmetry group of sgs turns out to be SO(2). The temperature dependence of magnetic susceptibility of sgs in the alloys CuMn [3] corresponds when heating to the gradual degeneration of the Gaussian distribution of the magnetization vector, and when cooling to its gradual recovery with no hysteresis. In both cases an internal spontaneous magnetic field mentioned above plays an essential role. The interrelation of this field, of the Gaussian distribution mentioned above, of the average kinetic energy of magnetic atoms and of the frustration of orientations of the magnetic moments placed on these atoms will be discussed.
Magnetoelastic driven Negative Thermal Expansion in a Framework Antiferromagnet
Authors: Saines Paul , Barton Phillip , Jura Marek , Knight Kevin , Cheetham Anthony
Published: November 2015

Metal-organic frameworks (MOFs) have recently attracted great attention for their multiferroic, magnetocaloric and low dimensional magnetic order. These properties depend on the precise magnetic interactions in frameworks and a deeper understanding of the coupling between the lattice of these materials and their magnetic order is required to underpin the future developments of these promising compounds. Neutron diffraction is the ideal technique for such studies but its application to studies of the magnetic structure of MOFs has been limited to date due to challenges in solving the structure of these dilute yet complex magnetic compounds. We have recently examined the magnetic properties and structure of a new dicarboxylate framework, cobalt adipate, Co(C6H8O4), which adopts P21/c monoclinic symmetry.[1] This compound has been found to order antiferromagnetically at low temperature and fits to neutron diffraction data have shown it adopts Pb21/c magnetic symmetry. Its magnetic structure features sheets of Co cations coupled antiferromagnetically in two dimensions through carboxylate groups. The emergence of this order is accompanied by magnetoelastic coupling, which drives anisotropic negative thermal expansion along the a-axis at low temperature. This is the first evidence for such behaviour in a MOF and we find that both this behaviour and the spin orientation in this material are controlled by the presence of weak ferromagnetic dipole-dipole coupling between the layers of tetrahedral cobalt. Variable temperature high resolution synchrotron X-ray and neutron powder diffraction have also revealed that the monoclinic angle of Co adipate decreases on cooling, passing through a metrically orthorhombic phase without any indication of a phase transition. This unusual behaviour has been rationalised by examining the thermal expansion of the framework along its principal axis, highlighting the importance of such analysis in low symmetry materials.
Effect of high pressure on the crystal structures of sarcosine and betaine
Authors: Kapustin Eugene , Minkov Vasliy , Boldyreva Elena
Published: November 2015

It is well known that infinite head to tail chains built of zwitterions linked to each other by N-H...O hydrogen bonds are common structural motifs in crystals of amino acids. These chains coincide with directions of the smallest compressibility of a crystal structure on cooling and increasing pressure and can even remain after structural phase transitions. However one should take into account the dual nature of these chains. From the one hand zwitterions of amino acids are linked by N-H...O hydrogen bonds formed from the head, amino group, and the tail, carboxylate group. From the other hand besides hydrogen bonding there is electrostatic interactions which occur between positively charged amino group and negatively charged carboxylate group. Being guided by an idea to distinguish electrostatic contribution from the charge assisted N-H...O hydrogen bonds and to understand their role in the crystal structure distortion on increasing pressure, two crystal structures of N-methyl derivatives of the simplest amino acid glycine are considered as a case study. N-methylglycine or sarcosine has two donors for hydrogen bonding and so forms two infinite head to tail chains in the structure whereas N,N,N-trimethylglycine or betaine has no hydrogen bonds at all, but its zwitterions are lined up resembling head to tail chains. The effect of increasing hydrostatic pressure is different for two crystals. The structure of betaine compresses anisotropically, but sarcosine undergoes a phase transition accompanying crystal fragmentation and changes in N-H...O hydrogen bonds. The phase transition is kinetically controlled and strongly depends on the rate of variation of pressure. Of special interest is distortion of head to tail chains on increasing pressure comparing with those observed in polymorphic modifications of glycine.
The effects of pressure on structural strain in crystals with acetamide groups
Authors: McGregor Lindsay , Zakharov Boris , Boa Callum , Goryainov Sergei , Boldyreva Elena , Pulham Colin
Published: November 2015

Polymorphism is an ever growing area of interest in chemistry. Many active pharmaceutical ingredients (APIs) have the potential to have polymorphic forms, which can subsequently be used to the advantage of the pharmaceutical industry. There are a variety of conditions in which polymorphism can be examined; one way which has sparked interest in recent years is the influence of pressure and its effect on the behaviour of intermolecular bonds. The polymorphs of paracetamol were the first solid drugs for which the properties were compared at different pressures [1-2]. Another interesting research direction involves the comparison of the structural response to pressure of a series of chemically different compounds with similar molecular fragments, but possessing different molecular packing and intermolecular interactions. This is important for crystal engineering and for understanding structure-properties relationships. In the present study, we compare the response to pressure of a series of organic compounds with a common acetamide fragment: two polymorphs of paracetamol, two polymorphs of acetotoluidine, polymorphs and a hydrate of metacetamol, methacetin, and phenacetin. Both single-crystal Raman spectroscopy and X-ray diffraction were used. The effects of various pressure media have also undergone examination. The study was supported by the Year Abroad Programme of the University of Edinburgh (LM, CB), Russian Ministry of Science and Education and Russian Academy of Sciences (BAZ, SVG, EVB).
Contrasting compressibility trends in layered structures Ag(tcm) and Ni(CN)2
Authors: Adamson Jasper , Hodgson Sarah , Hunt Sarah , Cliffe Matthew , Cairns Andrew , Thompson Amber , Tucker Matthew , Funnell Nicholas , Hriljac Joseph , Lucas Timothy , Goodwin Andrew
Published: November 2015

Ag(tcm) (tcm = tricyanomethanide) and Ni(CN)2 are layered structures. Both these materials exhibit area negative thermal expansion (area-NTE) due to 'rippling' of the layers - a displacement pattern that causes the interlayer separation to increase and the effective layer area to decrease (Fig. 1a). We have shown that Ag(tcm) shows negative area compressibility (NAC) under hydrostatic pressure. The latter can be attributed to the rippling phenomenon: when hydrostatic pressure is applied, the effective layer area increases (Fig. 1a). On the contrary, Ni(CN)2 shows shows positive linear compressibility in all directions, albeit much more strongly along the stacking axis than in any direction parallel to the square-grid sheets. This is attributed to transverse phonon modes within the Ni(CN)2 sheets, which can be visualised as 'tilting' of the rigid unit modes (RUMs) (Fig. 1b). A discussion of relating such behaviour to the Grüneisen parameters is undertaken in this study.
Pressure-induced normal-incommensurate-commensurate phase transitions in TiPO4
Authors: Bykov Maxim , Bykova Elena , Dubrovinsky Leonid , Hanfland Michael , Liermann Hanns-Peter , Kremer Reinhard , van Smaalen Sander
Published: November 2015

The complex interplay between spin, charge, orbital, and lattice degrees of freedom has made low-dimensional quantum spin magnets with strong antiferromagnetic (AF) spin-exchange coupling prime candidates for studying unusual magnetic phenomena. A progressive spin-lattice dimerization in one-dimensional AF Heisenberg chains, which occurs below a critical temperature and induces a singlet ground state with a magnetic gap, is commonly referred to as spin-Peierls (SP) transition. Recently, the compounds TiOX (X = Cl, Br) and TiPO4have been intensively investigated due to their unconventional behavior [1,2]. Unlike standard SP systems, TiOX and TiPO4undergo a sequence of normal-incommensurate-commensurate phase transitions on cooling at remarkably high transition temperatures. The transition temperatures are related to the direct exchange interactions between Ti ions, which increases strongly with decreasing the distance between the Ti ions, and therefore is very sensitive to the applied hydrostatic pressure. We have performed pressure-dependent single-crystal X-ray diffraction of TiPO4using synchrotron radiation. TiPO4undergoes a pressure-induced pahse transiton towards an incommensurate phase already below 10 GPa. This transformation is followed by the lock-in phase transition to the dimerized SP phase. Both structures are analogous to those at low temperatures, but reveal significantly larger modulation amplitudes. In this contribution we will present the detailed discussion of the high-pressure structures of TiPO4and their behavior on compression. Furthermore, similarities and differences of high-pressure phase diagrams of TiOCl and TiPO4and discrepancies between predicted and observed structures will be considered.
Designing next-generation negative compressibility materials.
Authors: Cairns Andrew , Goodwin Andrew
Published: November 2015

Negative compressibility is a rare but desirable property whereby a material's crystal structure actually expands in one (negative linear compressibility, NLC) or two (negative area compressibility, NAC) principal directions against application of increasing hydrostatic pressure. The performance of such materials–for use in, for e.g., sensitive interferometric or ferroelectric pressure sensing devices, advanced actuators, or prototype artificial muscle–critically depends on the magnitude of intrinsic negative response. NLC and NAC have been previously reported in a diverse range of materials: from the elemental forms of selenium and tellurium, to transition metal oxides, halides and chalcogenides [1], to more recent reports of NLC in molecular materials, and metal–organic and metal–cyanide frameworks. We explore, using examples from our work [2,3] as well as that of others, how understanding known NLC and NAC materials can inform material design, and how the versatile chemistry of molecular frameworks–the connecting of cationic metal nodes with anionic molecular linkers in one or more dimensions–allows for the targeting, enhancing and coupling of functionalities. By analysis of the negative response across all known NLC and NAC materials we develop new understanding into the underlying mechanisms of these unusual responses. Structural motifs identified point towards strategies for designing the next-generation of these materials, including the simple "wine-rack," "honeycomb" and "spring" mechanisms, where hinging about nodes requires volume reduction by simultaneous expansion and contraction in perpendicular directions (Figure 1). We discuss the first report of "giant" NLC in zinc(II) dicyanoaurate(I), Zn[Au(CN)2]2, where the crystal structure expands >10% over 1.8 GPa [2], the unprecedented prolonged NAC in silver (I) tricyanomethanide, Ag(C4N3) [3], and conclude with our perspective on the challenges and opportunities that remain in the quest for even more extreme responses.
Twinning and Pseudosymmetry at High Pressures
Authors: Friese Karen , Grzechnik Andrzej
Published: November 2015

Twinning is a common known problem in the study of crystal structures from single-crystal data and often related to a high degree of pseudosymmetry of the structure with respect to a higher symmetrical parent one. With the rising popularity of in situ single-crystal diffraction studies under high pressure, the occurrence of twinned structures is more and more frequently reported. In this contribution, we review the available information on merohedral and pseudomerohedral twinning as well as pseudosymmetry under high pressures [1]. For twinning by merohedry type I (inversion twinning), a reliable characterization of the twin domains and volume fractions is difficult and largely depends on the experimental conditions, i.e., on the number of measured Friedel pairs and the chosen wavelength. For twinning by merohedry (type II) and for twinning by pseudomerohedry, twin volume fractions could be reliably determined from high-pressure data for several cases. In none of these, a significant influence of hydrostatic pressure on the volume fractions of the individuals was observed. Pressure-induced twinning has also been observed for compounds which undergo first-order phase transitions. It is remarkable that the twinning operation in such cases is related to the loss of rotational symmetry elements of the higher symmetrical polymorph, although the high- and low-pressure phases are not in the group-subgroup relationship. The analysis of pseudosymmetry of several compounds as a function of pressure suggests that this parameter can be used to predict the (in)stability of compounds. In particular, a decrease in pseudosymmetry seems to be strongly correlated with the occurrence of first-order phase transitions in which the crystals break or amorphize.
Crystal structure of the extracellular signaling complex of interleukin-18
Authors: Tsutsumi Naotaka , Kimura Takeshi , Arita Kyohei , Ariyoshi Mariko , Ohnishi Hidenori , Yamamoto Takahiro , Zuo Xiaobing , Kondo Naomi , Shirakawa Masahiro , Kato Zenichiro , Tochio Hidehito
Published: November 2015

Interleukin (IL)-18 [1], a proinflammatory cytokine belonging to the IL-1 superfamily, plays important roles in both innate and adaptive immune system, which is involved in not only the function of host defense mechanism but also allergic reactions. IL-18 is secreted by various types of cells and strongly augments the production of interferon-γ. IL-18 is synthesized as a biologically inactive precursor (proIL-18), which is matured by the action of caspase-1 in the cell upon stimulation. The matured IL-18 is subsequently secreted extracellularly and binds to IL-18 receptor α (Rα) and IL-18 receptor β (Rβ) in a stepwise manner, forming the IL-18/Rα/Rβ ternary complex. The complex initiates the signaling that finally activates NF-κB via the MyD88 dependent pathway [2]. Here, we show the crystal structure of IL-18 (Fig a), the IL-18/Rα binary complex (Fig b) and the IL-18/Rα/Rβ signaling ternary complex (Fig c, d) at 2.33, 3.10 and 3.10 Å resolution, respectively. Overall, the recognition manner of IL-18 by the receptors was similar to that of IL-1β [3], although some remarkable differences such as the orientation of Ig-like domains of IL-18Rβ were revealed. We also demonstrate that carbohydrate chains on IL-18Rα contributes to the recognition of IL-18. Biochemical experiments based on the structure identify amino acid residues that are important in forming the ternary complex and the signal transduction. Our results not only show the common extracellular signaling architecture of IL-1 family cytokines but also unveil unique recognition mechanism of IL-18 with the detailed atomic structures. The structure of the signaling ternary complex of IL-18 would contribute to both understanding the pathogeneses of the IL-18 related diseases and designing new efficient therapeutic agents.
A molecular basis for citrullination dependent rheumatoid arthritis
Authors: Scally Stephen , Petersen Jan , Law Soi Cheng , Dudek Nadine , Nel Hendrick , Loh Khai Lee , Purcell Anthony , Reid Hugh , Thomas Ranjeny , Rossjohn Jamie
Published: November 2015

Rheumatoid arthritis (RA) is a chronic and debilitating autoimmune disease, characterized by inflammation of synovial tissue, joint pannus and bone erosion. The human leukocyte antigen (HLA) locus plays a vital role in immunity, encoding highly polymorphic molecules that present peptides to T cell lymphocytes. RA has a strong association with a region of the HLA-DRB1 locus known as the `shared epitope' (SE) and the presence of autoantibodies specific for citrullinated proteins. The SE maps to a highly polymorphic N-terminal region of the HLA-DRβ chain around amino acids 70-74. This region encodes a positively charged residue at position 71 that is thought to dictate the amino acid that is accommodated in the P4 pocket of the antigen-binding groove. Citrullination, the conversion of positively charged arginine to polar citrulline, is a physiological process catalyzed by peptidyl arginine deiminase. Previous studies have shown that citrullination of self-antigens can significantly increase the affinity of epitopes for SE alleles. Here we provide a molecular basis for how citrullinated vimentin and aggrecan epitopes can be presented by the SE alleles, HLA-DRB1*0401 and HLA-DRB1*0404. Citrulline was accommodated in the electropositive P4 pocket of HLA-DRB1*0401/04, whilst arginine was not. In addition, the RA resistant HLA-DRB1*0402 allomorph was capable of binding both arginine and citrulline in its electronegative P4 pocket. Peptide elution studies revealed that arginine was presented by HLA-DRB1*0402 but not by HLA-DRB1*0401/04. Moreover, citrullinated vimentin showed a greater sensitivity to proteolysis by cathepsin L, when compared to unmodified vimentin, indicating that citrullination can impact the repertoire of self-antigens presented. Using HLA Class II tetramers, we identified citrullinated vimentin and aggrecan specific CD4+ T cells from both HLA-DRB1*0401+ RA patients and healthy controls. In RA patients, the number of autoreactive T cells correlated with disease activity and were deficient in regulatory T cells compared to healthy controls. Together these findings provide significant insight into the role citrullination plays in the pathogenesis of RA[1].
Structural studies of nucleic acid sensing Toll-like receptor
Authors: Ohto Umeharu , Tanji Hiromi , Shibata Takuma , Krayukhina Elena , Uchiyama Susumu , Miyake Kensuke , Shimizu Toshiyuki
Published: November 2015

Toll-like receptors (TLRs) sense pathogen-associated molecular patterns originating from invading microorganism and evoke innate immune responses. Among TLRs, TLR3, TLR7, TLR8, and TLR9 are localized to endosomal membranes and are responsible for the recognition of nucleic acids. TLR7 and TLR8 recognize single stranded RNA. In addition, TLR7 and TLR8 are activated by small chemical compounds. TLR9 recognizes DNA containing Cytosine-phosphate-Guanine motif. Theses nucleic acid sensing TLRs are attractive therapeutic targets for the modulation of immune responses in the viral and bacterial infections and in the pathogenesis of autoimmune diseases. However, the structural basis for the nucleic acid recognition and signaling mechanisms remains to be elucidated. Therefore, we conducted crystallographic studies of these TLRs. Recently, we have determined the crystal structures of the extracellular domain of human TLR8 in the unliganded form and in the liganded forms with chemical ligands (Tanji et al., 2013). Both unliganded and liganded forms of TLR8 were dimer. Ligands were located at two equivalent positions in the dimerization interface. The ligand binding induced the reorganization of the preformed dimer to the activated dimer such that the C-terminal regions of the two protomers are in close proximity to enable the subsequent dimerization of the intracellular signaling domains and its interactions with adaptor proteins.
Structural studies of anti-IgE Fab fragments
Authors: Mitropoulou Alkistis , Beavil Andrew , Henry Alistair , Sutton Brian , Davies Anna
Published: November 2015

In recent decades, the incidence of allergy, an immune disorder mediated by immunoglobulin E (IgE), has increased [1]. In Europe alone, allergic rhinitis and asthma affect 113 and 65 million people respectively [2]. The symptoms of these diseases not only cause discomfort to patients but also may be fatal, especially in asthmatic sufferers. Although the aetiology of allergy is debated, it is widely accepted that the interaction of IgE with its high-affinity receptor (FcεRI) is directly implicated to the pathogenesis of allergy [1]. The treatments for allergy include antihistamines and/or bronchodilators, for symptomatic relief, or corticosteroids to prevent the onset of symptoms. However, none of these medications are direct modulators of allergy. The most effective therapeutic for asthma is omalizumab, a monoclonal anti-IgE antibody. Omalizumab, an IgG1 antibody, prevents the allergic response by inhibiting the binding of IgE to FcεRI. The exact mechanism of action of omalizumab is yet to be defined, but it inhibits the IgE/FcεRI protein-protein interaction. Studies on the interaction of omalizumab Fab with IgE are crucial to reveal the binding site. We report here the structures of omalizumab Fab and two different Fab mutants in different crystal forms, and examine the effect of packing on the structure. We observed conformational changes in the complementarity-determining regions (CDRs) between the mutants compared with wild-type Fab. Side-chain conformational changes associated with the different crystal packings were also observed among the six different crystal structures.
Studies of Human Interleukin 24
Authors: Lubkowski Jacek , Zhang Di , Kotenko Sergei , Wlodawer Alexander
Published: November 2015

In this report we present our progress in the functional and structural studies of human interleukin 24 (hIL-24). Interleukins and interferons (cytokines) together with their cognate receptors form a `front-end' of convoluted signaling networks, responsible for an immune-response to the presence of various pathogens. Cytokines are subjected to vigorous research related to their role in human physiology and disease and potential therapeutic uses. After many years of intense studies, information on some of cytokines is absent or limited, partly because new members are still being identified as well as due to difficulties of generating significant amounts of active preparations. A sub-family of `IL-10-related cytokines' also called the cytokine receptor type 2 family (CRF2) comprises nine members. One of CRF2 members is interleukin 24 (IL-24). The most interesting biological feature of human IL-24 (hIL-24) is its tumor inhibitory activity, observed in vitro for several cancer cell lines. IL-24 signals through the receptor complex comprising the high-affinity chain (IL-22R1 or IL-20R1) and the secondary, low-affinity chain (IL-20R2). Structure of IL-24 is currently unknown, although it is expected to be similar to those of other members of the CRF2 family. However, in silica analysis indicates several differences in the molecule of IL-24 when compared to its close homologues. One of more interesting is lack of two canonical disulfide bonds, found in all other interleukins from the CRF2 family, due to missing needed Cys residues. While an alternative, unique disulfide bond in IL-24 is possible, the appropriate experimental evidence is missing. Native IL-24 is N-glycosylated. Although this modification appears dispensable for biological activity of related cytokines, its role in IL-24 is not clear. We have expressed insoluble hIL-24 in bacteria and refolded it subsequently into a soluble, functional form. The mass spectrometry analysis confirmed presence of a single disulfide bond. Our non-glycosylated variant of hIL-24 activates the cognate receptor as efficiently as commercial preparations from eukaryotic sources. We observed, however, that stability of refolded hIL-24 is somewhat compromised. Subsequently, we plan investigating the structural properties of this cytokine.
Structural Studies of TREM-2 Mutants Linked to Neurodegenerative Diseases
Authors: Kober Daniel , Brett Tom
Published: November 2015

Triggering Receptor Expressed on Myeloid Cells-2 (TREM-2) is an extracellular surface protein expressed on myeloid cells of the monocyte/macrophage lineage including dendritic cells, macrophages, osteoclasts, and microglia. Recent genetic studies have revealed point mutations in TREM-2 that correlate with a dramatically increased risk for the development of neurodegenerative diseases, including Alzheimer's disease, frontotemporal dementia, and Parkinson's disease. This represents the first molecular link between inflammatory processes and neurodegenerative disease. TREM-2 modulates the innate immune inflammatory response; however, the biological ligand for TREM-2 remains elusive. As a first step towards understanding the role of TREM-2 in neurodegenerative disease, we have undertaken structural and biophysical studies of wild-type and mutant TREM-2 proteins. We developed a mammalian-cell based expression system for the successful production of TREM-2 in quantities suitable for structural studies. We have crystallized the TREM-2 Ig domain and determined the structure at 3.3 Å resolution. Analysis of this structure reveals the location of disease-linked mutations and produces hypotheses about their involvement in structural stability and ligand binding. In addition, we are studying the affect these mutations have on the stability of the protein using biochemical stability and surface expression assays. We are also pursuing structural studies of the point mutants to elucidate any structural changes caused by mutation. These studies are crucial to understanding the functional consequences of TREM-2 point mutations linked to the development of neurodegenerative diseases and, ultimately, to develop patient-specific molecular therapies to treat them.
Towards a structural characterization of the IFIT antiviral complex
Authors: Dimech Christina , Nagar Bhushan
Published: November 2015

Our first line of defense against viral pathogens is the innate immune system. Interferon-induced proteins with tetratricopeptide repeats (IFITs) are innate immune effector molecules that are thought to confer antiviral defense through the formation of the IFIT `Interactome', a multiprotein complex made up of IFIT1, IFIT2, IFIT3 and several other host factors1. Through IFIT1, this complex has the ability to distinguish self from non-self nucleic acids such as virus-derived RNA bearing 5´-triphosphate or viral mRNA lacking 2´-O methylation on the first two nucleotides1,2. We have limited information on the architecture of this complex, its role in innate immunity, and its activity downstream of RNA binding remain unclear. To better understand the mechanisms of Interactome formation, we are investigating the structure of its core, namely the IFIT1-IFIT2-IFIT3 complex. Since it is challenging to crystallize the complex as a whole, likely due to its size and heterogeneity, we are also targeting the structure of individual components and co-crystals of interacting domains. A crystal structure of human IFIT2 is available, and our lab has solved the structure of N-terminal human IFIT1 and, more recently, N-terminal IFIT3. In this study, we aim to characterize the interaction between IFIT1 and IFIT2, and between IFIT3 and IFIT2, through gel-filtration binding assays, in vitro pull-downs and deletion mutations. Preliminary results on the expression and purification of IFIT2-deletion mutants will be presented, as well as purification of IFIT subcomplexes. Understanding the molecular mechanisms behind IFIT-mediated virus elimination will help us unravel the complexities of these interactions and significantly advance our fundamental knowledge of innate immunity, paving the way for designing novel immunotherapeutics, which could potentially complement anti-cancer strategies that rely on oncolytic RNA viruses.
TRIM protein domain topology and implications for antiviral immunity
Authors: Jacques David , Jeffries Cy , Caines Matthew , Lammers Michael , Mallery Donna , Price Amanda , McLaughlin Stephen , Johnson Chris , Svergun Dmitri , James Leo
Published: November 2015

The tripartite motif (TRIM) proteins are a large family of >100 members, several of which have important roles in antiviral immunity and innate immune signaling. TRIM5α associates with incoming HIV-1 capsids, interfering with controlled disassembly and targeting them for degradation by the proteasome. TRIM21 is a cytosolic antibody receptor, which also targets incoming viral capsids for proteasomal degradation. TRIM25 is also involved in innate immunity, being essential for the ubiquitination of RIG-I. Recent positive selection analysis has predicted another 10 TRIM proteins with antiviral activity. Despite the fact that TRIM5α, 21 and 25 play key roles in antiviral protection, their mechanism of action is incompletely understood. All three proteins share a similar domain architecture, comprising a RING, B Box, coiled coil and PRYSPRY domains. The RING domains are responsible for ubiquitin ligase activity, while the PRYSPRY domains determine target specificity. We have used a combination of crystallography and SAXS to generate the first complete model for a TRIM protein structure. Crystallographic studies of TRIM25 reveal a central elongated coiled-coil domain with an unusual right-handed twist. The dimer formed by the coiled-coil is antiparallel but is followed by additional helices that reverse the direction of the protein chain. This structure suggests that the N-terminal domains of each monomer are separated but the C terminal domains are maintained in proximity. Multi-angle light scattering (MALS), isothermal titration calorimetry (ITC) and SAXS analysis confirms that this dimer structure is present in solution. Furthermore, scattering studies on the tripartite motif of TRIM21, comprising RING, B Box and coiled-coil, demonstrate that the first two domains of each monomer are held 150-200 Å apart. Finally, SAXS measurement of a complex between intact TRIM21 and its ligand, IgG Fc, provides the first empirical structure of a complete TRIM protein.