bluuebunny/crossref_metadata_2025 huggingface dataset:
Optimal data collection using photon-counting Hybrid Pixel Detectors
Authors: Mueller Marcus , Wang Meitian , Schulze-Briese Clemens
Published: November 2015

The data collection parameters used in a diffraction experiment have a strong impact on the quality of the acquired data. A careful choice of parameters leads to better data and can make the difference between success and failure in phasing attempts and better data will also result in a more accurate atomic model. The selection of data acquisition parameters has to account for the application of the data in various phasing methods or high-resolution refinement. Furthermore, experimental factors like crystal characteristics and the properties of X-ray source and detector have to be considered. Hybrid Pixel Detectors are now for several years in use in macromolecular crystallography and an increasing number of synchrotron beamlines as well as laboratory instruments are equipped with such detectors. Photon-counting Hybrid Pixel Detectors have fundamentally different characteristics and offer various advantages over other detector technologies. To fully exploit the advantages of Hybrid Pixel Detectors, different data collection strategies than those established for other detector types have to be applied. Fine φ-slicing is a strategy particularly well suited because of the fast readout time and the absence of readout noise. This strategy was systematically investigated collecting a large number of data sets from crystals of four different proteins to investigate the benefit of fine φ-slicing on data quality with a noise-free detector in practice. The results show that fine φ-slicing can substantially improve scaling statistics and anomalous signal. Furthermore, when collecting data in continuous rotation at high frame rates up to hundreds of images per second, quality might be impaired by detector readout. Results on the influence of readout time on data quality will be presented and strategies to easily avoid detrimental effects of detector readout will be discussed.
The use of workflows for macromolecular crystallography experiments at the ESRF.
Authors: Svensson Olof , Brockhauser Sandor , Bowler Matthew , Nanao Max , Guijarro Matias , de Santis Daniele , Popov Alexander , Mueller-Dieckmann Christoph , Gordon Leonard , McCarthy Andrew
Published: November 2015

The high performance of modern synchrotron facilities means there is an increasing reliance on automated data analysis and collection methods. The EMBL and ESRF are actively involved in designing and implementing such automated methods. However, as these methods are evolving there is also a need to continually integrate newer and more sophisticated data analysis and collection protocols with experimental control. This integration often poses a challenge, requiring a high level software environment to automatically coordinate beamline control with data acquisition and analysis. This is why we have extended the Eclipse RCP version of the workflow tool Passerelle into a user friendly GUI for experiment design by scientists and programmers [1], which is now part of the Data Analysis WorkbeNch (DAWN) collaboration (http://www.dawnsci.org). The execution of several complex workflows using this technology are now fully integrated in the new version of MxCuBE [2] and deployed on the ESRF macromolecular crystallography beamlines. Here, I will present their current implementation and the data quality improvements that can be achieved. In particular we have developed automated crystal re-orientation workflows that can improve the success of ab initio phasing experiments and help mitigate against radiation damage effect [3]. Other protocols implemented include a 3D diffraction based centring routine, a dehydration protocol and the automated measurement of a crystals radiation sensitivity. Lastly I will present our future plans for other new advanced diffraction based workflow routines, including automated crystal screening and data collection protocols.
Current status of protein micro-crystallography at SPring-8
Authors: Hirata Kunio , Kawano Yoshiaki , Yamashita Keitaro , Ueno Go , Hikima Takaaki , Murakami Hironori , Hasegawa Kazuya , Baba Seiki , Kumasaka Takashi , Yamamoto Masaki
Published: November 2015

Protein micro-crystallography is one of the most advanced technologies for protein structure analysis. In order to realize this, an undulator beamline, named BL32XU, was constructed at SPring-8. The beamline can provide beam with size of 0.9 x 0.9 µm and photon flux of 6E10 photons/s. The beam size can be easily changed by users from 1 to 10 µm square with the same flux density. Through three years user operation, we have established several key systems for efficient protein micro-crystallography. One of them is the software for precise positioning of micro-crystals in `raster scan'. SHIKA is a program with GUI which searches diffraction spots in a plenty of low dose diffraction images obtained in raster scan. Finally, it generates 2D map of crystal positions based on the number of spots or spot intensities. Parameters and thresholds in peak search have been empirically optimized for LCP crystals and it provides robust results. Another system is for the data collection strategy. Almost all successful data collections were conducted via `helical data collection' on BL32XU using the line-focused beam. The GUI software, named KUMA, enables estimation of an accumulated dose and suggests suitable experimental conditions for helical data collection. The system is proven to be useful for experimental phasing using tiny LCP crystals of membrane proteins[1-3]. Based on them, the rapid and automatic data collection system using protein micro-crystals is under development. The new CCD detector, Rayonix MX225HS, was installed for faster data acquisition in 10 Hz with the pixel size of 78 µm square. The new SHIKA using GPUs is under development for faster and more accurate crystal alignment. Following this step, KUMA system can suggest experimental conditions for each crystal found on the loop. We also report about the effects of higher dose rate in protein crystallography up to the order of 100 MGy/s. This work was supported by Platform for Drug Discovery, Informatics, and Structural Life Science from the Ministry of Education, Culture, Sports, Science and Technology, Japan.
Improved direct-method aided iterative dual-space model completion
Authors: He Yao , Yao Deqiang , Huang Sheng , Zhang Tao , Lin Zhengjiong , Gu Yuanxin , Fan Haifu
Published: November 2015

It has been proved that direct methods are efficient in providing phase constraints within the dual-space phase-model iterative framework [1]. The program OASIS is used for the direct-method implementation. Two kinds of iterative direct methods are performed by OASIS. One involves the use of SAD/SIR information [2], while the other dose not [3]. Improvements have been made on both kinds of iterative direct methods. First, the Srinivasan's weighting function replaces previously used Sim's weighting function in the direct-method phase derivation leading to better estimation of phases. This affects both kinds of direct-method iteration. Second, the process of the second kind direct-method iteration has been made multi-threaded and parallel. This significantly speeds up the iteration in multi-CPU systems. Finally, a new figure of merit combining the R factor and Srinivasan's weighting function is used to pick up the best partial structure instead of using the R factor alone. The improved procedure has been tested using a set of SIRAS data from the protein LegC3N with Hg-derivative at 5.0Å resolution and native at 2.1Å resolution. With the previous version of OASIS, SAD phasing at 5.0Å resolution was successful but phases were failed to extend to the 2.1Å resolution native data as is described in §3.2 of [1]. However the new version of OASIS has succeeded in extending 5.0Å resolution SAD phases to 2.1Å resolution native phases leading to a nearly complete structure model.
New tools for automated model completion and refinement
Authors: Echols Nathaniel , Morshed Nader , Moriarty Nigel , Afonine Pavel , Terwilliger Thomas , Adams Paul
Published: November 2015

Although macromolecular crystallography has been greatly accelerated by the development of automated software for data processing, phasing, and model building, most structures require significant manual intervention to yield a truly final model. In addition to missing individual protein or nucleic residues, this may include the addition of alternate conformations, ligands (both free and covalently bound), elemental ions, or modified amino acids. We have developed a number of tools to streamline several of these steps within the Phenix software suite (Adams et al. 2010): 1) an automated pipeline for the determination of ligand-bound structures by molecular replacement (Echols et al. 2014a); 2) placement of elemental ions during refinement (Echols et al. 2014b), as an extension of solvent placement; 3) fitting of additional conformations of protein residues into difference density. These tools reliably reproduce published structures in a majority of test cases, and in several instances identify details omitted by the original authors. Their low false positive rate makes them suitable for use in high-throughput workflows.
Optimizing Data Collection in the Home Lab
Authors: Benning Matthew
Published: November 2015

Many of the projects currently under investigation in biological research labs focus on macromolecules that are difficult to crystallize such as: complexes, multi-domain and membrane proteins. Typically, crystallization trials can produce small, weakly diffracting crystals that may also have other challenging attributes. Recent hardware and software developments have improved in-house data quality on a wide range of samples. Small and highly focused x-ray beams allow one to select the best diffracting portion of a larger crystal and reduce background scatter for much smaller samples. Shutterless data collection helps to reduce instrument error resulting from shutter jitter and allows fine slicing of data runs without frame to frame dead time penalties while the practice of dealing with multiple, cracked or twinned crystals has improved greatly due to software enhancements. Results from in-house data collection including: shutterless operation, optimization of crystal orientation and collection parameters will be discussed.
Towards User Operations of SONICC at GM/CA@APS
Authors: Becker Michael , Stepanov Sergey , Dettmar Christopher , Toth Scott , Newman Justin , Fischetti Robert , Simpson Garth
Published: November 2015

Second order nonlinear optical imaging of chiral crystals (SONICC), based on femtosecond laser scanning microscopy, has been implemented at GM/CA@APS undulator beamline 23ID-B for rapid protein crystal localization and centering. The technique is based on infrared laser light impinging on non-centrosymmetric crystals of proteins, which selectively may yield a frequency-doubled, visible signal generated by the anharmonic response of the electron cloud of the protein in response to the laser field. One aim of this method is to locate small crystals grown in opaque crystallization media for centering in X-ray beams of only a few microns or less in cross-section [1]. The optical system implemented at the beamline includes `trans' and `epi' detection of Second Harmonic Generation (SHG) signals [2]. In addition, scanning visible laser light across the sample and detecting two-photon excited UV fluorescence (TPE-UVF) provides complementary contrast based on the native fluorescence of proteins. An update on progress towards offering a user-friendly system to users will be provided. Different factors that influence imaging signals and the practice of successfully locating and accurately positioning a crystal via SONICC will be discussed.
Improving the diffraction of glypican-1 crystals by controlled dehydration
Authors: Awad Wael , Birkedal Gabriel , Thunnissen Marjolein , Mani Katrin , Logan Derek
Published: November 2015

Glypicans are heparan sulfate proteoglycans that are attached to the cell membrane surface by glycosylphosphatidylinositol anchorage. Glypican-1 (Gpc-1) is the predominant heparan sulphate proteoglycan in the developing and adult human brain and is involved in developmental morphogenesis, growth factor and cytokine signalling. We determined the crystal structure of N-glycosylated human glypican-1 core protein at 2.55 Å resolution, which revealed a cylindrical, all α-helical fold (dimensions 120 x 30 x 30 Å), decorated with three major loops and containing the 14 cysteine residues conserved in all members of the glypican family (1). The Gpc-1 crystals were delicate, highly fragile plates, which displayed poor isomorphism, with cell dimensions varying between different crystals. These crystals also diffracted anisotropically, reflected in a Wilson B factor that was twice as large in the c* direction as in the a* and b* directions, which limited the effective resolution to 2.9 Å in the c* direction. Recently we have shown Gpc-1 crystals to be a successful case for improvement in diffraction properties by controlled crystal dehydration using the humidity control device (HC1b), which delivers a humidified air stream of a precise relative humidity that can be used to alter the solvent content inside the crystals (2). The optimal dehydration protocol was developed by investigation of the parameters: final relative humidity RHf, dehydration rate and total incubation time Tinc. Of these, the most important was shown to be Tinc. After dehydration using the optimal protocol, the diffraction quality of the Gpc-1 crystals was clearly improved, with significant reduction in the anisotropy. This generated better, less noisy electron density maps, which allowed the building of previously disordered parts of the model and displayed well-defined side chains.
Coping with translational non-crystallographic symmetry in molecular replacement
Authors: Read Randy , Adams Paul , McCoy Airlie
Published: November 2015

In translational noncrystallographic symmetry (tNCS), two or more copies of a component are present in a similar orientation in the asymmetric unit of the crystal. This causes systematic modulations of the intensities in the diffraction pattern, leading to problems with methods that assume, either implicitly or explicitly, that the distribution of intensities is a function only of resolution. To characterize the statistical effects of tNCS accurately, it is necessary to determine the translation relating the copies, any small rotational differences in their orientations, and the size of random coordinate differences caused by conformational differences. An algorithm has been developed to estimate these parameters and refine their values against a likelihood function. By accounting for the statistical effects of tNCS, it is possible to unmask the competing statistical effects of twinning and tNCS and to more robustly assess the crystal for the presence of twinning. Modified likelihood functions that account for the statistical effects of tNCS have been developed for use in molecular replacement and implemented in Phaser. With the use of these new targets, it is now possible to solve structures that eluded earlier versions of the program. Pseudosymmetry and space group ambiguities often accompany tNCS, but the new version of Phaser is less likely to fall into the traps that these set.
Crystallographic evidence for oxidative Met→Asp conversion in human hemoglobin
Authors: Soman Jayashree , Strader Michael , Hicks Wayne , Kassa Tigist , Singleton Eileen , Olson John , Weiss Mitchell , Mollan Todd , Wilson Michael , Alayash Abdu
Published: November 2015

The mutants HbA Bristol-Alesha (βV(E11)67M) and HbF Toms River (γV(E11)67M) [1,2] are examples of a `silent' posttranslational modification in which the side chain of the substituted amino acid is chemically modified (Met→Asp) resulting in a disparity between the DNA and protein sequences. In both cases the patients' hemolysate contained both V67M and V67D isoforms. But in the analogous α subunit mutant, Hb Evans αV(E11)62M, the conversion to Asp was not identified and DNA sequencing confirmed the Met replacement [3]. Our crystal structures of the three (ferrous) CO-bound recombinant V(E11)M mutants show the MetE11 side chain in similar conformations. But the air-oxidized β mutant crystals clearly showed a `bifurcated' and smaller electron density pattern for the E11 side chain, indicating the appearance of Asp. Also, the ligand electron-density at the iron atom in the oxidized β subunit appears to be an oxoferryl Fe4+=O rather than a Fe3+OH2 ferric complex. In contrast, there was little change in the electron density for αMetE11 in oxidized αV62M crystals. The ligand in the ferric α subunit is clearly a coordinated water molecule. But again, a ferryl Fe4+=O complex appears to occur in the wild-type β subunit. This strongly suggest that β subunits have a greater propensity to form highly reactive ferryl species, and that the ferryl species play a role in the Met→Asp conversion. Our autoxidation and proteomics studies showed that although all three recombinant VE11M mutants had similar, high rates of autooxidation and a strong H2O2 dose dependence on sulfoxide and sulfone formation, no Asp formation was detected in α subunits whereas MetE11 is converted to Asp to levels as high as 15% in vitro in β and γ subunits. We propose that the Met→Asp conversion specifically involves H2O2 mediated oxidation of the ferrous heme to an oxoferryl state, and because the transient ferryl intermediates are much less stable in the α subunits, there is no oxidative conversion.
Structural-Functional Analysis of USP7
Authors: Saridakis Vivian
Published: November 2015

Ubiquitin Specific Protease 7 (USP7) catalyzes the deubiquitination of several substrate proteins including p53, Mdm2 and UbE2E1. Deubiquitination results in their rescue from 26S proteasome mediated degradation thus USP7 is critical in maintaining the cellular steady state levels of its substrates. USP7 is also known to interact with several viral proteins including herpes simplex virus Infected Cell Protein 0 (ICP0), Epstein Barr Nuclear Antigen 1 (EBNA1) and Kaposi's sarcoma-associated viral interferon regulatory factor 4 (vIRF4). USP7 possesses several domains including an N-terminal TRAF, a catalytic and five ubiquitin-like domains. The crystal structures of several complexes revealed that most substrate proteins interact with the N-terminal domain of USP7 which folds into an eight-stranded antiparallel beta sandwich. EBNA1 and MCM-BP also interact with USP7 through its N-terminal domain in an identical manner to its substrates p53, Mdm2 and UbE2E1 however these proteins are not deubiquitinated by USP7. Substrates that interact with USP7 through its N-terminal domain possess a P/AxxS binding motif. The identification, characterization and structure determination of novel USP7 substrates is ongoing.
An UBR-box from a non-N-recognin: Crystal Structure of the UBR domain of UBR6
Authors: Muñoz-Escobar Juliana , Kozlov Guennadi , Trempe Jean-François , Gehring Kalle
Published: November 2015

The degradation of many short-lived proteins in eukaryotic cells is carried out by the Ubiquitin Proteasome System. The N-end rule pathway links the half-life of proteins to the identity of its N-terminal residue, also called N-degron. Destabilizing N-degrons, are recognized by E3 ubiquitin ligases termed N-recognins. N-degrons are grouped into type 1, composed of basic residues, and type 2, composed of bulky hydrophobic residues. In mammals, four N-recognins mediate the N-end rule pathway: UBR1, UBR2, UBR4 and UBR5. These proteins share a ~70-residue zinc finger-like motif termed the Ubiquitin Recognin (UBR) box, responsible for their specificity. The mammalian genome encodes at least three more UBR-box proteins: UBR3, UBR6/FBXO11 and UBR7. However, these UBRs cannot recognize any type of N-degrons. Our lab reported the crystal structures of the UBR boxes from the human UBR1 and UBR2, rationalizing the empirical rules for the classification of type 1 N-degrons. Despite the valuable information obtained from those structures there is not a clear explanation for the no recognition of N-degrons by other UBR-box proteins. Here we report the crystal structure of the UBR-box domain from UBR6 also known as FBXO11. UBR6 is a F-box protein of the SKP1-Cullin1-F-box (SCF) ubiquitin ligase complex and does not recognize any type of N-degrons. We crystallized a 77-residue fragment of the UBR-box of UBR6 and determined its structure at 1.7 Å resolution. Unexpectedly, this domain adopts an open conformation compared to UBR1-box, without any N-degron binding pockets. Its zinc-binding residues are conserved as in the N-recognins, but they are arranged in different zinc-binding motifs. Molecules form dimmers stabilized by zinc ions. The crystal had 4 molecules per asymmetric unit and space group P212121. For phasing we used Zn-SAD. With this structure we hope to obtain clues that explain the absence of N-degron recognition in some members of the UBR family.
Structural basis of Atg conjugation systems essential for autophagy
Authors: Noda Nobuo
Published: November 2015

Autophagy is an evolutionarily-conserved, intracellular degradation system for which two ubiquitin-like modifiers, Atg8 and Atg12, play essential roles. After processed by Atg4, the exposed C-terminal glycine of Atg8 is activated by Atg7 (E1) and is then transferred to Atg3 (E2), and is finally conjugated with a phospholipid, phosphatidylethanolamine (PE) through an amide bond. Whereas, Atg12 is activated by the same E1, Atg7, without processing, and is then transferred to Atg10 (E2), and is finally conjugated with Atg5 through an isopeptide bond. Atg12-Atg5 conjugates, together with Atg16, function as an E3-like enzyme to facilitate the conjugation reaction between Atg8 and PE. During autophagy, Atg8-PE conjugates play a critical role in selective cargo recognition in addition to autophagosome formation. We determined the structures of all these Atg proteins and their complexes mainly by X-ray crystallography, and performed structure-based biochemical analyses on them [1,2]. These studies established the molecular mechanisms of Atg8 and Atg12 modification reactions that have many unique features compared with canonical ubiquitin-like systems. Furthermore, we found a conserved motif named the Atg8-family interacting motif (AIM), through which Atg8 recognizes specific cargoes and selectively incorporates them into autophagosomes for degradation [3].
Serial snapshot crystallography with a non-monochromatic microbeam
Authors: Dejoie Catherine , McCusker Lynne , Baerlocher Christian , Abela Rafael , Patterson Bruce , Grolimund Daniel , Pattison Philip , Kunz Martin , Tamura Nobumichi
Published: November 2015

X-ray free-electron laser (XFEL) sources create X-ray pulses of unprecedented brilliance and open up new possibilities for the structural characterization of crystalline materials. By exposing a small crystallite (100nm-10μm) to a single ultrafast pulse, a diffraction pattern can be obtained before the crystal is damaged. If such single-pulse diffraction patterns, collected sequentially on many randomly oriented crystallites, are combined, it is possible to determine the structure of the material accurately [1]. One of the drawbacks of this approach is that only a single position of the Ewald sphere is accessed in each pattern, so, because reflections have a finite width, the diffraction condition is not satisfied completely for any of the reflections recorded. The new XFEL source that is being developed in Switzerland (SwissFEL) will provide a broad-bandpass mode with an energy bandwidth of about 4% [2]. By using the full energy range of the SwissFEL beam, a new option for structural studies of crystalline materials becomes possible. In a recent study based on simulated data, we showed that a diffraction experiment with stationary crystallites in such an `extra pink' beam not only increases the number of reflection intensities that can be collected in a single shot, but also overcome the problem of `partial reflection' measurement [3]. To test the viability of the data processing with experimental data, attempts to simulate this 4% bandpass have been carried out on SNBL at ESRF and on the microXAS beamline at SLS. On SNBL, a single crystal was rotated over 360° and a continuous scan of the monochromator over the 4% energy range was performed every 1°. At SLS, a mirror was used to cut off the higher energies of the undulator beam and the energy threshold of a Pilatus detector to eliminate the lower ones. With this setup, a series of randomly oriented crystallites were measured. A comparison of the analysis of these datasets will be presented.
Cryogenic coherent X-ray diffraction imaging of biological particles at SACLA
Authors: Takayama Yuki , Nakasako Masayoshi , Oroguchi Tomotaka , Sekiguchi Yuki , Yamamoto Masaki , Yonekura Koji , Takahashi Yukio , Suzuki Akihiro , Matsunaga Sachihiro , Tsujimoto-Inui Yayoi
Published: November 2015

Coherent X-ray diffraction imaging (CXDI) is a promising technique to visualize internal structures of whole biological cells without sectioning. Utilizing X-ray free-electron laser (XFEL) for CXDI has potential to collect huge number of projected electron densities of such samples at higher resolutions than that limited by radiation damage. For biological application of XFEL-CXDI, sample particles must be kept in a hydrated state to maintain their functional structures, but be placed in vacuum to obtain weak diffraction signals. In addition, we need to deliver fresh sample particles one after another for XFEL exposure because every particle explodes just after X-ray irradiation. To handle these problems, we have been developing a system to fulfill cryogenic XFEL-CXDI of frozen-hydrated specimens. For cryogenic XFEL-CXDI, we prepare frozen-hydrated specimens by plunge-freezing sample particles dispersed onto thin film with humidity-controlling [1]. The diffraction experiments are conducted by using the cryogenic X-ray diffractometer KOTOBUKI-1 [2]. In a vacuum chamber of KOTOBUKI-1, a cryogenic pot equipped on a goniometer is filled with liquid nitrogen, which cools the specimen via thermal contact. Thus, KOTOBUKI-1 allows data collection at a specimen temperature of ~66 K with a positional fluctuation of less than 0.4 μm. A small angle-resolution of better than 500 nm is attainable by using a pair of L-shaped Si-slits placed before the specimen, which eliminate almost all parasite scattering from upstream. Diffraction patterns recorded on two MPCCD detectors in tandem arrangement are automatically processed and phase-retrieved by program suite SITENNO [3]. In our recent experiments performed in Japanese XFEL facility SACLA, we were able to collect a large number of diffraction patterns from biological samples at a resolution of 50 - 30 nm at an XFEL hit-rate of 20 - 100%. We report details of the cryogenic XFEL-CXDI and introduce imaging of chloroplasts as an example.
Three-dimensional imaging of acoustic phonons using an X-ray laser
Authors: Clark Jesse , Robinson Ian
Published: November 2015

"Key insights into the behaviour of materials can be gained by observing their structure when they undergo lattice distortion. Laser pulses on the femtosecond time scale can be used to induce disorder in a ``pump-probe"" experiment with the ensuing transients being probed stroboscopically using femtosecond pulses of visible light, X-rays or electrons (Fig. 1 A). Here we report two- and three-dimensional imaging of the generation and subsequent evolution of coherent acoustic phonons on the picosecond time scale within single gold nanocrystals using an X-ray free electron laser (Fig. 1 B). Our results allow comparisons between experiment and predictive models based on continuum elasticity theory and molecular dynamics simulations (Fig. 1 B). *Full author list - J.N. Clark L. Beitra, G. Xiong, A. Higginbotham, D. M. Fritz, H. T. Lemke, D. Zhu, M. Cholle, G. J. Williams, M. Messerschmidt, B. Abbey, R. J. Harder, A. M. Korsunsky, J. S. Wark, D. Reis, I. K. Robinson."
Temperature dependence of Uiso constraints in riding hydrogen treatments
Authors: Luebben Jens , Grabowsky Simon , Edwards Alison , Morgenroth Wolfgang , Sheldrick George , Dittrich Birger
Published: November 2015

"Anisotropic parametrisation of the thermal displacements of hydrogen atoms in single-crystal X-ray structure refinement is not possible with independent atom model (IAM) scattering factors. This is due to the weak scattering contribution of hydrogen atoms. Only when aspherical scattering factors are used can carefully measured Bragg data provide such information. For conventional structure determinations parameters of ""riding"" hydrogen atoms are frequently constrained to values of their ""parent"" heavy atom. Usually values of 1.2 and 1.5 times X-U_eq are assigned to H-U_iso in these cases. Such constraints yield reasonable structural models for room-temperature data. However, todays small molecule X-Ray diffraction experiments are usually carried out at significantly lower temperatures. To further study the temperature dependence of ADPs we have evaluated several data sets of N-Acetyl-L-4-Hydroxyproline Monohydrate at temperatures ranging from 9 K to 250 K. Methods compared were HAR [1], Invariom refinement [2], time-of-flight Neutron diffraction and the TLS+ONIOM approach [3]. In the TLS+ONIOM approach non-hydrogen ADPs from Invariom refinement provided ADPs for the TLS-fit. Hydrogen atoms in all methods were grouped and analyzed according to their Invariom name. We reach a good agreement of the temperature dependence of H-U_iso/X-U_eq. At very low temperatures the ratio H-U_iso/X-U_eq can be as high as 4, e.g. for Hydrogen attached to a sp3 carbon atom with three non-Hydrogen atom neighbors. Since all methods consistently show that the H-U_iso/X-U_eq ratio is temperature dependent, this effect should be taken into account in conventional structure determinations."
Insights on spin-polarization via the spin density Source Function
Authors: Gatti Carlo , Orlando Ahmed , Lo Presti Leonardo
Published: November 2015

The Source Function (SF) [1-3], enables one to view chemical bonding and other chemical paradigms from a new perspective and using only information from the electron density observable and its derivatives. We show how this tool may be straightforwardly applied to another important observable, the electron spin density, which analogously to the electron density may be locally interpreted in terms of a cause-effect relationship of contributions from the atoms of a molecular or crystalline system. Application of the spin density SF to molecules in vacuo and to slab or crystals, is made possible through an extension (SPINSF code) of our electron density SF code for molecules and through a progress-version of the TOPOND code, respectively. The latter has now been fully integrated in the CRYSTAL-14 code, where it provides, via the keyword TOPO of the properties section of CRYSTAL-14, a complete charge density topological analysis according to the Quantum Theory of Atoms in Molecules. Analysis of the SF for the electron spin density implies the study of its Laplacian scalar field, which may be locally positive or negative even if the two composing densities, ρα and ρβ, have both negative or positive Laplacian densities. When the latter bear the same sign, that of the spin density Laplacian depends on their relative magnitudes, that is on the relative concentration or dilution of ρα and ρβ. Hence, in general, the local source for the spin density, LSs, greatly differs from the analogous function for the density, leading to large differences in their integrated atomic SF contributions. The combined study of LSs and of the spin density neatly reveals which are the molecular or crystal regions that are "ferromagnetically" or "antiferromagnetically" coupled and the local strength of such coupling. Applications to crystals of metal-complexes where the ligands play an innocent or a non-innocent role and to crystals of iron spin-crossover complexes are discussed.
Organometallic Single-Ion Magnets
Authors: Wang Bing-Wu , Wang Zhe-Ming , Gao Song
Published: November 2015

The single-molecule magnets (SMMs) are attracting the increasing interesting due to their potential applications in high density information storage, quantum computing, molecular spintronics, and magnetic refrigeration. This field provides scientists a possible access into the crossover of the classical and quantum world, and a wonderful model to study the fascinating magnetic properties between microscopic and macroscopic materials, such as slow magnetization relaxation and quantum tunneling effect. After the milestone discovery of the first single-molecule magnets (SMMs) Mn12ac, many new SMMs were structurally and magnetically characterized. The most studied systems are mainly traditional coordination compounds with polynuclear structures. However, for the difficulties in the control of magnetic anisotropy and exchange coupling interactions of the cluster-type molecules, Mn12ac molecule is still one of the most important SMMs with the high relaxation barrier. From 2011 [1-3], we explored an organometallic sandwich molecule, Cp*ErCOT(Cp* = pentamethylcyclopenta-dienide; COT = cyclooctatetraenide), which behaves as a single-ion magnets, into the field of molecular nanomagnets. It opened a door of SMMs to the chemists in organometallic chemistry. Recently, we found some new sandwich or half-sandwich lanthanide organometallic molecules could also show the slow relaxation of magnetization. We hope these systems can provide new understandings of slow magnetic relaxation and new clues on the design and synthesis of molecular nanomagnets. This work was supported by NSFC, the National Basic Research Program of China.
Temperature variable high-pressure volumetric studies of diethylene glycol
Authors: Dziubek Kamil , Mahgoub Mohamed , Alsoghier Hesham , Katrusiak Andrzej
Published: November 2015

It was demonstrated that direct compression experiments in a piston-cylinder press provide precise information on volume data and phase transition pressure complementary to X-ray diffraction. The experimental setup, described in detail previously [1], is capable of compressing liquid and solid samples up to ca. 2 GPa. We have continued these studies adding temperature control of the sample. Piston and cylinder chamber was placed in an thermally isolated mantle. The constant temperature was maintained by circulating hot air. As demonstrated by the experiments on diethylene glycol, it is a relatively quick and simple, yet efficient method for exploring phase diagrams and recording volume reduction at phase transition in different thermodynamic conditions.
Pressure-induced phase transitions in energetic materials
Authors: Pulham Colin , Coster Paul , Henderson Craig , Hunter Steven , Kleppe Annette , Marshall Wiilliam , Morrison Carole
Published: November 2015

Explosives and propellants, known generically as energetic materials, are widely used in applications that include mining, munitions, and automotive safety. Key properties of these materials include: reliable performance under a range of environmental conditions; long-term stability; environmental impact; processability; sensitivity to accidental initiation through stimuli such as impact, shock, friction, and electrostatic discharge. Many of these properties are affected by the crystal structure of the energetic material. Explosives experience elevated pressures and temperatures under detonation conditions – such conditions often induce phase transitions in the energetic material. Hence detailed studies of pressure-induced structural changes in these materials are essential in order to understand and model fully their behaviour. This presentation will describe some recent high-pressure studies (using a combination of X-ray and neutron diffraction techniques) on 2,4-dinitroanisole (DNAN), an insensitive explosive that is replacing TNT in some applications [1,2]. DNAN shows rich pressure-induced polymorphism, with at least four high-pressure forms having been identified to date. One of the structures provides insight into as to why DNAN is particularly insensitive to initiation by shock. The presentation will also describe the interplay between experiment and theory, which will be illustrated by experimental and computational high-pressure studies of 1,1-diamino-2,2-dinitroethene (DADNE or FOX-7). A very subtle phase transition has been identified at a pressure of ~2.0 GPa and the implications of this will be discussed in relation to the observed structural changes and properties of this material.
Structural insights into SraP-mediated S. aureus adhesion to host cells
Authors: Yang Yi-Hu , Jiang Yong-Liang , Chen Yuxing , Zhou Cong-Zhao
Published: November 2015

Staphylococcus aureus, a Gram-positive bacterium causes a number of devastating human diseases, such as infective endocarditis, osteomyelitis, septic arthritis and sepsis. S. aureus SraP, a surface-exposed serine-rich repeat glycoprotein (SRRP), is required for the pathogenesis of human infective endocarditis via its ligand-binding region (BR) adhering to human platelet. It remains unclear how SraP interacts with human host. Here we report the 2.05 Å crystal structure of the BR of SraP, revealing an extended rod-like architecture of four discrete modules. The N-terminal legume lectin-like module specifically binds to N-acetylneuraminic acid. The second module adopts a β-grasp fold similar to Ig-binding proteins, whereas the last two tandem repetitive modules resemble eukaryotic cadherins but differing in calcium coordination pattern. Small-angle X-ray scattering and molecular dynamic simulation indicated the three C-terminal modules function as a rigid stem to extend the N-terminal lectin module outward. Further structure-guided mutagenesis analyses showed that SraP binding to sialylated receptors promotes S. aureus adhesion to and invasion into host epithelial cells. Our findings have thus provided novel structural and functional insights into the SraP-mediated interaction of S. aureus with host epithelial cells.
Crystal structure of EndoS, an immunomodulatory endoglycosidase specific for human IgG antibodies
Authors: Trastoy Beatriz
Published: November 2015

In order to evade host immune mechanisms, many bacteria secrete immunomodulatory enzymes. Streptococcus pyogenes, one of the most common human pathogens, secretes a large endoglycosidase, EndoS, which removes carbohydrates in a highly specific manner from IgG antibodies. This modification renders antibodies incapable of eliciting host effector functions through either complement or Fc γ receptors, providing the bacteria with a survival advantage. On account of this antibody-specific modifying activity, EndoS is being developed as a promising injectable therapeutic for autoimmune diseases that rely on autoantibodies. Additionally, EndoS is a key enzyme used in the chemoenzymatic synthesis of homogenously glycosylated antibodies with tailored Fc γ receptor-mediated effector functions. Despite the tremendous utility of this enzyme, the molecular basis of EndoS specificity for, and processing of, IgG antibodies has remained poorly understood. Here, we report the X-ray crystal structure of EndoS and provide a model of its encounter complex with its substrate, the IgG1 Fc domain. We show that EndoS is composed of five distinct protein domains, including glycosidase, leucine-rich repeat, hybrid Ig, carbohydrate binding module, and three-helix bundle domains, arranged in a distinctive V-shaped conformation. Our data suggest that the substrate enters the concave interior of the enzyme structure, is held in place by the carbohydrate binding module, and that concerted conformational changes in both enzyme and substrate are required for subsequent antibody deglycosylation. The EndoS structure presented here provides a framework from which novel endoglycosidases could be engineered for additional clinical and biotechnological applications.
Structure of mouse Clr-g, a CTL ligand for NK receptor NKR-P1F
Authors: Skálová Tereza , Kotýnková Kristýna , Vaněk Ondřej , Dušková Jarmila , Hašek Jindřich , Koval' Tomáš , Kolenko Petr , Stránský Jan , Fejfarová Karla , Dohnálek Jan
Published: November 2015

Natural killer (NK) cells are large granular lymphocytes with innate immune reaction against tumor cells or cells affected by viral infection. They have a variety of receptors on their surface which mediate contact with cells tested by NK cells. Using X-ray crystallography, we determined a structure of an extracellular part of mouse C-type lectin related protein g (Clr-g, [1], PDB code 3RS1), a ligand for NK receptor NKR-P1F. The ligand and the receptor are both of C-type lectin like fold and this is the first determined structure of a CTL ligand of an NK receptor. The protein was produced in E. coli. The rod-like crystals appeared by spontaneous crystallization of the pure protein (2.5 mg/ml) on tube walls. Diffraction data of the vitrified crystal were measured at synchrotron BessyII of HZB in Berlin and were processed up to 1.95 Å. The protein was found in the form of dimers similar to that of CD69. The N-terminus of the chain (residues Met, Asn, Lys) is in the crystal bound to a neighbor dimer and shows thus that binding of a peptide to mouse Clr-g is possible, although not expected or confirmed by other experiments known to us. A model of interaction of Clr-g with NKR-P1F was designed based on electrostatic complementarity of both molecules. This work was supported by the Czech Science Foundation (grant No. P302/11/0855), Ministry of Education, Youth and Sports of the Czech Republic (grant No. LG14009) and by the project BIOCEV CZ.1.05/1.1.00/02.0109 from the ERDF.
Domain swapping in structure of mNKR-P1A: unique feature with unknown function
Authors: Kolenko Petr , Rozbeský Daniel , Skálová Tereza , Kovaľ Tomáš , Fejfarová Karla , Dušková Jarmila , Stránský Jan , Hašek Jindřich , Dohnálek Jan
Published: November 2015

Natural killer (NK) cells, large granular lymphocytes, play an important role in the innate immune response against viruses, parasites and tumour cells. NK cells use a wide repertoire of surface receptors to modulate their activity [1]. The family of NKR-P1 surface receptors of NK cells belong to proteins with C-type lectin-like (CTL) fold. The overall architecture of other known CTL receptors (e.g. members of Ly49 family, NKG2D, CD94, mouse CLRg) is conserved [2]. The mechanism of ligand binding has been revealed by the crystal structure of Nkp65 bound to its keratinocyte ligand [3]. However, observation of domain swapping in crystal structure of mouse (m) NKR-P1A represents an unusual structural feature that might be involved in a new mechanism of ligand binding that would be specific for some members of NKR-P1 family. Nevertheless, our crystal structure of mNKR-P1A represents a unique structural observation that demands careful analysis. Even the latest structural studies do not answer the question of function or role of swapped domain of the receptor in potential ligand binding. We have generated new variants of mNKR-P1A of varied chain length that undergo biochemical and structural analysis including mass spectrometry.
Broad Neutralization of Viral Pathogens
Authors: Wilson Ian
Published: November 2015

Influenza, Hepatitis C, and HIV-1 continue to constitute significant threats to global health. We have structurally and functionally characterized several potent, broadly neutralizing antibodies (bnAbs) against HIV-1, influenza and hepatitis C viruses. The surface antigens of these viruses are the main target of neutralizing antibodies. However, most antibodies are strain-specific and protect only against highly related strains within the same subtype. Recently, a number of antibodies have been identified that are much broader and neutralize across multiple subtypes and types of these viruses through binding to functionally conserved sites, such as the receptor binding site or the fusion domain. For example, co-crystal structures of bnAbs with influenza hemagglutinin (HA) identified highly conserved sites in the fusion domain (stem) and in the receptor binding site (head) as target for broad neutralization[1]. HCV is also genetically diverse, but some antibodies have potent neutralizing activity across most genotypes of the virus. One family of these antibodies targets a conserved antigenic site on the HCV E2 envelope glycoprotein that overlaps with the CD81 receptor-binding site[2]. For HIV-1, structural and functional characterization of different families of bnAbs have led to identification of novel epitopes on HIV-1 Env, many of which involve glycans. These glycan-dependent Abs have unique features that enable them to penetrate the glycan shield and bind complex epitopes that consist of sugars and underlying protein segments on gp120 on HIV-1 Env. Recent x-ray[3] and EM structures of a soluble form of HIV-1 Env have revealed that the epitopes are more extensive and complex than previously appreciated. This structural information is now being used to aid in structure-assisted vaccine design for HIV-1, HCV and for a more universal flu vaccine. IAW is supported by NIH grants AI100663, AI082362, AI84817, AI099275 and GM094586 and the Crucell Vaccine Institute.
CD1d lipid-antigen recognition by the γδ TCR
Authors: Le Nours Jerome , Uldrich Adam , Pellicci Daniel , Gherardin Nicholas , McPherson Kirsty , Lim Ricky , Patel Onisha , Beddoe Travis , Gras Stephanie , Rossjohn Jamie , Godfrey Dale
Published: November 2015

The T lymphocytes repertoire is divided into two major lineages, αβ and γδ T cells, which are defined by their T cell receptor (TCR) gene-segment usage. To date, the key discoveries on human CD1d restricted T cells have focussed on the type I Natural Killer T cells (NKT) subset that express an invariant TCR α chain (Vα24Jα18) which pairs with a β chain (Vβ11). The structural basis for the recognition of CD1d-lipid antigen by type I NKT cells is also now well established [1, 2]. However, there are other subsets of NKT cells that exhibit reactivity towards lipid-antigen presenting molecules (CD1d) but that do no express the typical Vα24Jα18 TCR. We identify human NKT cell subsets that express Vδ1+ γδ TCRs that recognize CD1d presenting the lipid-antigen α-galactosylceramide (α-Galcer). Here, we describe the first crystal structure of a CD1d/γδ TCR ternary complex [3] and provide structural insights into the binding mode of a γδ TCR with CD1d-αGalcer. The γδ TCR binds orthogonally over the A' pocket of CD1d, that is in clear contrast with the typical type I parallel docking mode in which the αβ TCR is perched over the F' pocket of CD1d. The germ line-encoded CDR1δ loop dominates the contacts with the CD1d molecule while the CDR3γ loop represents the main structural determinant for the antigen specificity. These findings highlight the emergence of diverse populations of NKT TCRs that exhibit different binding mode and lipid antigen specificity.