bluuebunny/crossref_metadata_2025 huggingface dataset:
Energy Storage in crystalline Materials based on multivalent Ions
Authors: Nestler Tina , Förster William , Braun Stefan , Münchgesang Wolfram , Meutzner Falk , Zschornak Matthias , Cherkouk Charaf , Leisegang Tilmann , Meyer Dirk
Published: November 2015

Energy conversion and storage has become the main challenge to satisfy the growing demand for renewable energy solutions as well as mobile applications. Nowadays, several technologies exist for the conversion of electric energy into e. g. heat, light and motion or vice versa. Among a large variety of storage concepts, the conversion of electrical in chemical energy is of great relevance in particular for location-independent use. Main factors that still limit the use of electrochemical cells are the volumetric and gravimetric energy density, cyclability as well as safety. The concept for a new thin-film rechargeable battery that possibly improves these properties is presented. In contrast to the widespread lithium-ion technology, the discussed battery is based on the redox reaction of multivalent Al-ions and their migration through solid electrolytes. The ion conduction and insertion processes in the crystalline materials of the suggested cell are discussed under a crystallographic point of view to identify suitable electrode and separator materials. A multilayer-stack of all-solid-state batteries is synthesized by pulsed laser deposition and investigated in situ, i. e. during charge and discharge, by X-ray reflection and diffraction methods. The correlation between crystal structure, morphology and electrical performance is investigated in order to characterize the ion diffusion and insertion process.
A system for time-resolved XRD measurements on hydrogen absorption processes
Authors: Machida Akihiko , Higuchi Kensuke , Katayama Yoshinori , Sakaki Kouji , Kim Hyunjeong , Nakamura Yumiko
Published: November 2015

"To improve performance of hydrogen storage materials, it is essential to understand detailed mechanism of hydrogenation and dehydrogenation reactions. In-situ powder diffraction measurements provide direct information about structural changes accompanying the reactions. We therefore installed a time-resolved x-ray diffraction (XRD) system at a beamline BL22XU at the SPrign-8, a synchrotron radiation facility in Japan. The system was equipped with two area detectors, a flat panel sensor for precise structural analyses and a high speed video camera connected to an x-ray image intensifier for observation of rapid phase changes. Maximum frame rate for the flat panel sensor and high-speed video camera was 2 fps and 125 fps (effective), respectively. A sample cell was connected to a hydrogen supply system. Opening of upstream valve of the sample cell or a change of the pressure at the sample triggered the recording of the diffraction patterns. The pressure of hydrogen gas was limited to 1 MPa. To demonstrate the performance of the system, we have performed time-resolved XRD experiments for LaNi4.5Al0.5. LaNi5 exhibits the significant broadening of the diffraction peaks by hydrogen absorption; however, LaNi4.5Al0.5 shows the no significant broadening. We have succeeded in the measurements of the structural change from the solid solution phase to the hydride phase and have found the formation of the transient intermediate phase on this reaction process. The system is currently used to study several materials. This work was partly supported by New Energy and Industrial Technology Development Organization (NEDO) under ""Advanced Fundamental Research Project on Hydrogen Storage Materials""."
Phase transition of nanoparticulate LiFePO4during high rate cycling
Authors: Liu Hao , Strobridge Fiona , Borkiewicz Olaf , Wiaderek Kamila , Chapman Karena , Chupas Peter , Grey Clare
Published: November 2015

A fundamental understanding of an electrode material requires the elucidation of its phase transformation mechanism during charge and discharge. Ex situ methods, which are carried out under equilibrium condition, have been widely used in charactering the thermodynamic phases at different states of charge, from which a thermodynamic phase transformation pathway can be constructed. However, ex situ measurements do not always reflect the process occurred in an operating battery as the non-equilibrium operating condition might result in deviations from the thermodynamic process, especially for high-rate materials, such as LiFePO4, which is predicted to exhibit a fundamentally different phase transformation process at high rates [1,2]. To probe the process at high rate, an in situ method with reasonable temporal resolution must be employed. In this work, the high rate galvanostatic cycling process of LiFePO4 nanoparticle electrode in a customised AMPIX cell [3] was investigated in situ by time-resolved synchrotron X-ray powder diffraction. Formation of continuous non-equilibrium solid solution phases between LiFePO4 and FePO4 was observed at 10 C rate. The in situ diffraction patterns were analysed by a refinement strategy that accounts for the asymmetrical diffraction peak profiles due to Li composition variations.
Structural Basis for Emergence of Superionic Conductivity by an Ion Exchange
Authors: Kato Kenichi , Kasai Hidetaka , Hori Akihiro , Takata Masaki , Kitagawa Susumu , Tanaka Hiroshi , Kobayashi Akira , Ozawa Nobuki , Kubo Momoji , Arikawa Hidekazu , Takeguchi Tatsuya , Sadakiyo Masaaki , Yamauchi Miho
Published: November 2015

Various superionic conductors have been examined in terms of the application to electrolytes for solid fuel cells [1]. Recently we demonstrated by impedance measurements that a simple two-step chemical reaction transformed an electronic conductor NaxCoO2into a superionic one. In the present study, we performed in situ synchrotron X-ray diffraction experiments to investigate a structural mechanism for the superionic conductivity driven by the chemical treatment of the layered oxide NaxCoO2. We developed a temperature- and humidity-controllable capillary cell under hydrogen and helium gas flow to install in the Debye-Scherrer camera at BL44B2 of SPring-8. This cell allows us to explore a structural transformation process by reduction and humidification treatments. Structural identifications and refinements with in situ diffraction data proved that Co vacancies formed by a CoO separation suppressed the electronic conductivity. Meanwhile it turned out from charge estimation in the Na layers that the superionic conductor transition originated from an ion exchange of H3O+for Na+, which was confirmed by Raman spectroscopy measurements. In addition, charge densities clearly visualized the H3O+ions disordering around the Na original sites, suggesting that the H3O+behave as a carrier source. Finally it was found from electrostatic potentials that the disordering H3O+sites were coupled through shallow potential barriers to trace a honeycomb-like ion pathway. In the presentation, I will discuss what a carrier is for the superionic-conductive phase from different viewpoints such as activation energies, concentration cell tests, and molecular dynamics simulations using the experimental structure information.
SHIKA: a fast and accurate spot finder for raster scan on microfocus beamlines
Authors: Yamashita Keitaro , Hirata Kunio , Kawano Yoshiaki , Ueno Go , Hasegawa Kazuya , Kumasaka Takashi , Yamamoto Masaki
Published: November 2015

On BL32XU, a microfocus beamline at SPring-8, oscillation data are collected with typical horizontal beam size of 1 μm. Hence it requires very accurate crystal centering, which is difficult especially for invisible crystals e.g. LCP crystals. Therefore, we perform raster diffraction scan to find crystal positions based on their diffractivity using low-dose exposure. It had been time consuming process due to two reasons; i) slow readout time of CCD, ii) manual inspection of hundreds of diffraction images. To tackle this problem, we installed new fast-readout CCD detector, MX225HS (Rayonix, L.L.C.), and developed support tool for raster scan based crystal centering. The tool visually shows possible crystal position on 2D map based on spot populations, and therefore it is named SHIKA (Spot-wo Hirotte Ichiwo Kimeru Application; a Japanese abbreviation which means the application for crystal positioning by picking up spots). SHIKA automatically detects new images when raster scan started and finishes just after raster scan ends. On GUI, user can find and pass the crystal position information to KUMA (a tool suggesting helical data collection strategy with predicted radiation damage) to start data collection immediately. User can also see picked spots on diffraction images with GUI. SHIKA has been developed based on DISTL [1] and modified to be faster and more accurate, especially for LCP crystal which is an important target on BL32XU. SHIKA picks up spots after subtracting smoothed pseudo-background which is a key for better separation of spots and ring-like diffuse background of lipids. Smoothing is time-consuming, but SHIKA now uses GPUs for almost all process including high-speed median filter [2] so that it can be done within ~100 msec. Further development is under way for faster processing. Now SHIKA can be also used on BL41XU, a high flux beamline at SPring-8 with some adjustment for PILATUS3 (Detectris Ltd.) detector.
Visualisation of membrane protein crystals using X-ray imaging
Authors: Warren Anna , Armour Wes , Axford Danny , Basham Mark , Connolley Thomas , Hall David , Horrell Sam , McAuley Katherine , Mykhaylyk Vitaliy , Wagner Armin , Evans Gwyndaf
Published: November 2015

The focus in macromolecular crystallography is moving towards even more challenging target proteins that often crystallise on much smaller scales and are frequently mounted in opaque or highly refractive materials.[1,2] It is therefore essential that X-ray beamline technology develops in parallel to accommodate such difficult samples. In this poster the use of X-ray microradiography and microtomography is reported as a tool for crystal visualisation, location and characterization on the macromolecular crystallography beamlines at the Diamond Light Source. The technique is particularly useful for microcrystals, and crystals mounted in opaque materials such as lipidic cubic phase. X-ray diffraction raster scanning can be used in combination with radiography to allow informed decision-making at the beamline prior to diffraction data collection. It is demonstrated that the X-ray dose required for a full tomography measurement is similar to a diffraction grid scan. However, for sample location and shape estimation alone, just a few radiographic projections may be required; hence reducing the dose the crystals will be exposed to prior to the diffraction data collection.[3]
"""Objective"" Evaluation of Macromolecular Models"
Authors: Ten Eyck Lynn , Perez Martin
Published: November 2015

"Evaluation of the accuracy and quality of molecular models from X-ray crystallography is a continuing problem. The refinement process as currently practiced is inherently biased through use of the model to provide phase information for the refinement process. To investigate the extent of model bias we are developing model-free criteria for detailed analysis of crystal structures. We present preliminary findings from comparing a set of high resolution, well-refined structures from the Joint Center for Structural Genomics repository to maps based solely on phases from selenomethionine anomalous scattering. Removal of atoms with little or no support in the ""objective"" map reliably improves or does not degrade the free R-factor. The selenomethionine map, or any other map produced without the assumption of a molecular model, can be used to identify features supported by diffraction data and those which come from the application of structural constraints."
EMBL beamlines for macromolecular crystallography at PETRA III
Authors: Schneider Thomas , Bourenkov Gleb , Cianci Michele , Kallio Johanna , Pompidor Guillaume , Karpics Ivars , Fiedler Stefan
Published: November 2015

Since 2012, EMBL Hamburg operates two new beamlines for macromolecular crystallography - P13 and P14 - at PETRA III at DESY (Hamburg, Germany). We exploit the high brilliance and the wide energy range offered by PETRA III to offer a wide range of conditions to fit the experimental conditions to the challenges posed by the samples. P13 provides high photon flux down to 4 keV. With a helium cone and a kappa goniostat, this allows optimized data collection for SAD phasing. Using adaptive mirrors, the focus size (H x V) can be adjusted between 30 x 20 μm^2 and 150 x 100 μm^2 to match the size of the sample. A MARVIN sample changer is in operation for rapid loading and unloading of samples. P14 offers a high photon flux (>10^12 ph/sec at 12 keV into 5 x 5 µm^2). The beamsize can be varied between 1 x 1.5 mm^2 (unfocused) and 5 x 5 µm^2 (fully focused) in less than a minute by moving the KB mirrors in and out of the beam. For small crystals, an MD3 vertical diffractometer with a sphere of confusion smaller than 100 nm offers excellent conditions. Both beamlines are equipped with PILATUS 6M-F detectors for shutter-less data collection and dedicated data processing computers. The beamlines are embedded into the 'Integrated Facility for Structural Biology' offering facilities for sample preparation and characterization, a laboratory specifically equipped for the preparation of heavy atom derivatives, and downstream facilities for data evaluation We will report about the status of the beamlines and describe typical experimental situations (small crystals, large unit cells, serial crystallography, low-energy phasing, small molecules and others).
Radiation damage effects on protein conformation at room temperature and 100K
Authors: Russi Silvia , Kann Shawn , van den Bedem Henry , González Ana M.
Published: November 2015

Protein crystallography data collection at synchrotrons today is routinely carried out at cryogenic temperatures to mitigate radiation damage to the crystal. Although damage still takes place, at 100 K and below, the immobilization of free radicals increases the lifetime of the crystals by orders of magnitude. Increasingly, experiments are carried out at room temperature. The lack of adequate cryo-protectants, the induced lattice changes or internal disorders during the cooling process, and the convenience of collecting data directly from the crystallization plates, are some of the reasons. Moreover, recent studies have shown that flash-freezing affects the conformational ensemble of crystal structures [1], and can hide important functional mechanisms from observation [2]. While there has been a considerable amount of effort in studying radiation damage at cryo-temperatures, its effects at room temperature are still not well understood. We investigated the effects of data collection temperature on secondary local damage to the side chain and main chain from different proteins. Data were collected from crystals of thaumatin and lysozyme at 100 K and room temperature. To carefully control the total absorbed dose, full data sets at room temperature were assembled from a few diffraction images per crystal. Several data sets were collected at increasing levels of absorbed dose. Our analysis shows that while at cryogenic temperatures, radiation damage increases the conformational variability, _x0004_at room temperature it has the opposite effect_x0005_. We also observed that disulfide bonds appear to break up at a different relative rate at room temperature, perhaps because of a more active repair mechanism. Our analysis suggests that elevated conformational heterogeneity in crystal structures at room temperature is observed despite radiation damage, and not as a result thereof.
Intentional crystal-contact-free space in protein crystal
Authors: Matsuoka Rei , Komuro Yasuaki , Sugita Yuji , Kohda Daisuke
Published: November 2015

To understand the function of proteins, it is essential to perform the structural analysis of the protein complexes with ligands, such as substrates or partner molecules. The motions of ligands are restricted by the contacts with neighbor protein molecules in the crystal lattice. Here, we propose a new technique to analyze dynamics of a ligand in the bound state preserved in the crystal-contact-free space, which is intentionally created in protein crystals. We used Tom20 as a target protein. Tom20 functions as a general protein import receptor, by recognizing N-terminal signal sequences (presequences) of mitochondrial matrix proteins. Our working hypothesis is that the promiscuous specificity of Tom20 is attributed to the large mobility of the presequneces in the binding groove of Tom20 (1,2). Our aim is to obtain electron density that reflects the large mobility of a presequence in the crystal-contact-free space. In order to create the crystal-contact-free space, we took advantage of a protein fused with maltose binding protein (MBP). The key of the design is the connection of the two proteins firmly. We fused the C-terminal α-helix of MBP and the N-terminal α-helix of Tom20 seamlessly. After a systematic model building study, we decided to use a design with four residues inserted in the linker region. We found smeared electron density in the binding site of presequences in the difference Fourier electron-density map. We attached an iodine atom at the N-terminus of the presequence and confirmed the N-terminal position in the smeared electron density. We performed molecular dynamics simulation without the tethering in solution (3). The electron density simulated from the MD trajectory was fully consistent with the smeared electron density in the crystal contact-free space. We concluded that the smeared electron density corresponded to the partially overlapping region of the multiple states of the bound presequence.
A new generation of CCP4 monomer library based on Crystallography Open Database
Authors: Long Fei , Grazulis Saulius , Merkys Andrius , Murshudov Garib
Published: November 2015

The use of prior chemical knowledge such as bond lengths, bond angles about constituent blocks of macromolecules and ligands is an essential part of macromolecular crystal structure analysis. One of the reliable sources of such chemical knowledge is small molecule database where small molecule crystal structures have been analysed against high-resolution, high-quality experimental data. Furthermore, vast amount of data in small molecule database provide comprehensive coverage of flexible chemical environment and enable proper statistical analysis to avoid biased representation of those chemical properties. This presentation describes our work on organization of the data from open-access and daily-updated small molecule database, Crystallography Open Database(COD) [1], into a new generation of CCP4 monomer library (Dictionary), a container of prior chemical knowledge [2]. In order to describe specific environment atoms are in, they are classified into different atomic types based on local graphs and some basic chemical properties of atoms. This scheme can be applied to any small molecule databases. The atom types, and values of bond lengths and bond associated with them, are further clustered into a hierarchical tree and an isomorphism-mapping algorithm is implemented to facilitate fast search among a large number of atom types (typically several millions). This also provides a mechanism to derive reliable values for bond lengths and angles of novel ligands. Metal and non-organic atoms are treated differently with organic ones. The original data in COD are curated using several criteria and further statistical analysis on derived values of bond lengths and angles are allow to extract reliable chemical information from such databanks as COD. There are several software tools associated with new dictionary including 1) generate "ideal" bond lengths and angles for unknown ligand; 2) generate starting coordinates to represent one of the conformation of the ligand under consideration.
aCHESYM: standardized placement of macromolecular models in the unit cell
Authors: Kowiel Marcin , Jaskolski Mariusz , Gzella Andrzej , Dauter Zbigniew
Published: November 2015

Unique choice of the unit cell and the asymmetric unit are well defined and described in the International Tables for Crystallography vol. A. Unfortunately, the placement of molecules within the unit cell is not standardized. Since structure solution programs often use random numbers in their algorithms, the selected set of atomic coordinates may be different even with successive runs of the same program. Although formally correct, an arbitrary choice of molecular placement within the unit cell is confusing and may lead to interpretation errors [1]. With the use of the anti-Cheshire unit cell introduced by Dauter [2], for all space groups without inversion symmetry, it is possible to transform the molecular model such that its center of gravity falls within the anti-Cheshire asymmetric unit cell. It means that for macromolecular crystal structures it should be possible to standardize the placement of the molecules within the unit cell. In consequence, it should be easier for crystallographers and non-crystallographers to compare similar or related crystal structures. An implementation of the anti-Cheshire concept has been programmed in Python as a web service, aCHESYM. The aCHESYM program takes a PDB file as input and transforms the macromolecular model into the desired anti-Cheshire region. The program can also handle structure factor CIF files if the transformation used requires reindexing of the reflection data. The unit cell, coordinates and displacement parameters of all atoms after transformation are saved in a new PDB file. All the calculated transformations are reversible, so there is no danger of data loss. Moreover, the program helps the user to find the most compact assembly of the molecules (chains) in the structure when there are several chains in the asymmetric unit.
Processing of Protein Crystals using by Deep-UV Pulsed Laser
Authors: Kawano Yoshiaki , Hikima Takaaki , Hirata Kunio , Baba Seiki , Murakami Hironori , Yamashita Keitaro , Kumasaka Takashi , Yamamoto Masaki
Published: November 2015

The absorption of X-rays which pass through the protein crystal is possibly the largest source of systematic errors in macromolecular crystallography. Therefore we are developing protein crystal processing system using Pulsed UV Laser Soft Ablation (PULSA) technique [1] to reduce the systematic error as well as background scattering from cryoprotectant agents. For high-quality diffraction data collection from organic material, crystals are usually processed to spherical shape in order to keep X-ray path length in crystal constant. This dramatically reduces systematic errors caused by `absorption of X-rays'. Although shaping crystal was thought to be effective for protein crystallography, there was no usual technique to achieve this because protein crystals are exceedingly fragile against mechanical stress. We are developing protein crystal processing system using PULSA technique. In this system, short pulsed UV-laser (maximum power: 1.0 μJ/pulse, wavelength: 193.4 nm, duration: less than 1.3 nsec) is raised by NSL-193L (Nikon Corporation) and focused on 4 μmφ (FWHM). The focused laser is controlled by galvanomic mirror system and irradiates a sample. Combining this mirror system with four-axis goniometer enables to process crystal to arbitrary shape that is easily defined on GUI. Several protein crystals have been successfully processed into spherical, column and square pole shape, etc. In the case of crystal processed into column shape (diameter is 50 μm), in addition to reducing absorption effects, signal-noise ratio of diffraction data can be increased by removing cryoprotectant agent surrounding the crystal. This work was supported by "Platform for Drug Discovery, Informatics, and Structural Life Science" from MEXT, Japan.
Room Temperature Serial Crystallography at Synchrotrons
Authors: Botha Sabine , Barends Thomas , Kabsch Wolfgang , Latz Beatrice , Nass Karol , Shoeman Robert , Dworkowski Florian , Panepucci Ezequiel , Wang Meitian , Schlichting Ilme , Doak R. Bruce
Published: November 2015

Serial Femtosecond Crystallography (SFX) is the most commonly used method for the emerging structure determination at X-ray free-electron lasers (FELs). The high peak brilliance of the FEL and the possibility of using femtosecond pulses afford use of nano-to-micron sized crystals in a diffraction-before-destruction approach for the acquisition of high-resolution undamaged diffraction data [1]. The crystals are obliterated upon exposure to an FEL X-ray pulse so only a single snapshot can be collected per crystal, necessitating a constant supply of fresh crystals. The crystals are therefore injected in a liquid microjet [2], [3]. We show that this serial method of data collection and the associated data analysis can be successfully adapted to serial crystallography (SX) measurements at synchrotrons, enabling room temperature studies using the unattenuated beam. Given the continuous supply of fresh crystals, the full tolerable dose can be used for each single crystal exposure, permitting analysis of small or weakly scattering crystals. FEL X-ray pulses are much shorter than the fraction of a second exposure time at a synchrotron, so SFX injection conditions are modified in SX such as to slow down the typically fast travelling crystals. By embedding the crystals in a viscous material the crystals remain in the beam long enough to yield measurable diffraction and smearing out of the diffraction peaks due to crystal tumbling is avoided. We demonstrate the successful application of room temperature SX at the Swiss Light Source at ambient pressure. Our experimental setup allows collection of both still and rotation data. Recent progress using model systems will be presented, establishing this high throughput, high dose rate approach as a new route to structure determination of macromolecules in their native environment and at room temperature.
ARP/wARP for crystallographic model building and drug discovery
Authors: Wiegels Tim , Pereira Joana , Vancea Ioan , Carolan Ciaran , Beshnova Daria , Heuser Philipp , Lamzin Victor
Published: November 2015

The ARP/wARP software project combines automated model building and refinement into an unified approach for macromolecular crystal structure determination. The project is based on two decades of extensive research and development in the areas of macromolecular X-ray crystallography, informatics, data mining and statistical pattern recognition. ARP/wARP collects a vast amount of computationally efficient methods and provides easy-to-use pipelines for building models of proteins, nucleotides, ligands, as well as their complexes. All methods are intuitively accessible from the ArpNavigator [1], which grants direct visualisation and real-time interaction with model building results. Structures determined using ARP/wARP include histones, hsp70, viral proteases, an insect antifreeze protein, transferases, deadenylases, synthases, kinases, photolyases and the spliceosome. The novel release of ARP/wARP, version 7.4, comes with notable innovations for determining structures at medium-to-low resolution such as exploitation of non-crystallographic symmetry, improved protocols for model update and estimation of validity of built models. Joint releases with the CCP4 suite improve software development and integration, and make the installation and updates fast and convenient for the user. A novel procedure for the automatic identification of ligands in electron density maps is introduced. It is based on the sparse parameterisation of density clusters and the matching of the pseudo-atomic grids thus created to conformationally variant ligands using mathematical descriptors of molecular shape, size and topology. The integration of the ViCi web-server for in-silico ligand-based drug design and updated stereo-chemical restraints for ligand fitting make ARP/wARP an asset for crystallographic drug discovery pipelines.
ESRF/DBT/EMBL:BM14-2: MX station for in situ, dehydration and MAD experiments
Authors: Manjasetty Babu , Khadrouche Akim , Lavault Bernard , Felissaz Franck , Mairs Trevor , Barret Ray , Siebrecht Ralf , Márquez Jose-Antonio , Cipriani Florent , Belrhali Hassan
Published: November 2015

BM14 was the first dedicated macromolecular crystallography (MX) beamline to come into operation at the ESRF during 1995. This experimental station has been designed specifically to produce MX diffraction data using Single/Multiple-wavelength Anomalous Diffraction (SAD/MAD) methods (www.bm14.eu). Currently, beamline is operated by a consortium between the ESRF, the EMBL-Grenoble and the Department of Biotechnology (DBT), India. Recently, the beamline optics was fully upgraded (hence the new denomination BM14-2). A four-time increase in beam brilliance is achieved with the concomitant reduction in average exposure time (~5s today versus 20s before), leading to a substantial gain in the screening capacity. Upgrade includes a new channel-cut crystal equipped with a second crystal pusher to reject high-energy harmonics. The latter device turned to be crucial for Sulphur-SAD experiment success rate. The special goniometer head is installed on microdiffractometer (MD2) goniometer which is capable of handling the CrystalDirect (CD) nanocrystallisation plates [1] so that any crystallisation hit can be exposed to the beam. The plate characteristics are optimal for in situ data collection and they produce diffraction patterns with significantly reduced background. A newly designed dehydration-to-cryogenic nozzle-changer device is also installed in order to avoid the down time between the experiments (such as, In situ screening, humidity control device (HC1), standard cryogenic data collection). European users may apply for access via the Biostruct-X program (www.biostruct-x.eu) both for CD plate nanocrystallisation trials and for beamtime. Users from India are encouraged to apply from DBT-Portal (http://process.mbu.iisc.ernet.in/BM14/index.jsp) for beamtime. The optics hutch refurbishment and implementation of the novel devices in the experimental hutch opens promising perspectives for collecting data at room temperature and that are of importance to the MX users.
A novel method to stabilize weak protein complexes for crystallographic studies
Authors: Almawi Ahmad , Matthews Lindsay , Guarné Alba
Published: November 2015

Most protein interactions mediating critical steps in cellular pathways are transient and, hence, are difficult to capture using structural approaches. An example from Saccharomyces cerevisiae is the interaction between checkpoint effector Rad53 kinase and replication initiation Dbf4-Cdc7 kinase. Dbf4 is the regulatory and Cdc7 the catalytic subunit of this kinase, which functions in activating replication origins. When a replication fork is damaged, the checkpoint response activates Rad53, which binds transiently, yet specifically, to Dbf4 and this, in turn, inhibits the activity of Dbf4-Cdc7. The outcome of this interaction prevents activation of late origins during replication stress. Our laboratory has extensively characterized the Dbf4-Rad53 interaction, thereby providing an excellent system to probe new ways to stabilize a weak protein complex. The N-terminal forkhead associated (FHA) domain of Rad53 mediates the interaction with the BRCA-1 C-terminus (BRCT) domain of Dbf4. FHA and BRCT domains are modular domains, in which their N- and C-termini lie on the same face. Thus, we decided to stabilize the Dbf4-Rad53 complex using linkers of different lengths to join the two proteins. We generated four different Dbf4-linker-Rad53 fusions and characterized them biochemically, as well as structurally using small angle X-ray scattering. Only one of the fusions yielded crystals suitable for crystallographic analysis, and we solved the structure by molecular replacement. The four copies of the fusion in the asymmetric unit showed identical Dbf4-Rad53 interfaces that were in agreement with previous studies characterizing the Dbf4-Rad53 interaction. Importantly, the interaction in the crystal structure occurs inter- rather than intra-molecularly suggesting that the linker increases local protein concentration but does not impose complex formation. In a broader sense, our work reveals general trends that can be used to design linkers to capture weak protein complexes.
BDamage: Quantifying radiation damage in MX structures
Authors: Gerstel Markus , Deane Charlotte , Garman Elspeth
Published: November 2015

Radiation damage is a limiting factor in macromolecular X-ray crystallography diffraction experiments. Global damage leads to a unit cell size increase and non-isomorphism, and to a loss of long range crystal order which is visible in the decay of the diffraction pattern and loss of high resolution information during data collection. Specific damage causes detectable changes at particularly susceptible sites in the protein structure [1,2], such as the reduction of metallo-centres, elongation and subsequent breaking of disulphide bonds and decarboxylation of aspartate and glutamate residues. Between and within these groups the decay does not happen uniformly at equal rates throughout the protein, leading to preferential specific damage. Specific damage can result in misleading biological conclusions on protein mechanism and function being drawn. We have defined a new atom-specific metric, BDamage, which facilitates the identification of protein regions susceptible to specific radiation damage as well as the quantification of the susceptibility, allowing further investigations into preferential specific damage. BDamage has been validated using a paired set of low-dose/high-dose protein structures [3]. Results show that BDamage successfully separates susceptible residues from stable parts of the protein. A non-redundant subset of previously refined structures submitted to the PDB was then analyzed for indications of specific radiation damage. BDamage indicates that the distribution of specific damage is independent of secondary protein structure or disulphide bond configuration, but shows a correlation with solvent accessibility. Results indicate a possible use of BDamage as a quality control metric for structure submission. Further research into an alternative quantification of real-space specific radiation damage, using the decay of electron density over multiple datasets, is outlined.
Serial crystallography using synchrotron radiation - novel strategies for microcrystallography
Authors: Gati Cornelius , Bourenkov Gleb , Klinge Marco , Rehders Dirk , Stellato Francesco , Oberthuer Dominik , Betzel Christian , Schneider Thomas , Chapman Henry , Redecke Lars
Published: November 2015

Protein crystallography continues to be one of the most frequently used techniques to obtain structural information of biomacromolecules to atomic resolution. Since protein crystals of delicate target systems are often limited in size, one of the main goals in the design of modern beamlines is the construction of highly intense X-ray beams with small focal size to obtain high resolution diffraction images of microcrystals. However, this development has led to the situation, that the full intensity of the beam can destroy a protein crystal within fractions of a second. Therefore often only a small number of diffraction patterns can be obtained from one single crystal. Here we describe the adaptation of the serial crystallography approach, which has first been developed at X-ray Free-Electron Lasers (Chapman et al. 2011) to the usage of a microfocus synchrotron beamline, using a standard cryogenic loop for sample delivery. We proved this concept with in vivo grown cathepsinB microcrystals (TbCatB, Koopmann et al. 2012, Redecke et al. 2013) (average of 9 μm3), a medically and pharmaceutically relevant protein, involved in the life cycle of T. brucei. In these experiments it was possible to show that serial crystallography enables the utilization and outcome of the above described bottlenecks and features of modern 3rd generation synchrotron microfocus beamlines. Our strategy exploits the combination of a micron-sized X-ray beam, high precision diffractometry and shutterless data acquisition with a pixel-array detector. By combining the data of 80 TbCatB crystals, it was possible to assemble a dataset to 3.0 Å resolution. The data allow the refinement of a structural model that is consistent with that previously obtained using FEL radiation, providing mutual validation.
Structures of Biological Macromolecules from Multi-crystal Native SAD Phasing
Authors: Hendrickson Wayne , Liu Qun
Published: November 2015

Native metalloproteins have been routine subjects for mutli- and single-wavelength anomalous diffraction (respectively MAD and SAD) analyses for some time; however, despite notable early successes, native molecules without heavy atoms (Z ≥ 25) have only recently become routinely accessible. Crystals of native proteins and nucleic acids have substantial contents of light elements (P, S, Cl, K, Ca) of potential for use in SAD phasing. Anomalous signals from such elements can be enhanced by using a lower than usual x-ray energy; nevertheless, typical Bijvoet differences usually still remain at a level comparable to noise. We have devised robust SAD procedures to study native, light-atom-only biological structures. We have so far used a modestly low energy (6 - 7 keV), but we further enhance the signal-to-noise in anomalous diffraction by combining data from multiple crystals chosen to be statistically equivalent. We have applied our multi-crystal native SAD approach in several structure determinations (1,2) at sizes up to 1200 ordered residues per asymmetric unit and at resolutions so far as low as 3.2 Å. Our tested practices can be replicated readily, and we plan further improvements in computing protocols and in instrumentation.
Structural Basis for Interaction Between NO66 and Osterix
Authors: Wu Minhao , Tao Yue , Zhou Xing , Sinha Krishna , Zang Jianye
Published: November 2015

The differentiation of mesenchymal stem cells to osteoblasts is one of the critical steps of bone formation. Osterix (Osx) is an osteoblast-specific transcriptional factor required for bone formation and osteoblast differentiation, which has been shown to interact with other factors to control the expression of osteoblast-specific genes. A novel JmjC domain containing protein NO66 has been identified in the regulation network of Osx, which plays an important role in osteoblast differentiation through interaction with Osx. Here we report the crystal structure of NO66, showing it exists as a functional tetramer form. A hinge domain links N-terminal JmjC domain and C-terminal wHTH domain of NO66 and is essential for its tetrameric assembly. The oligomerization interface of NO66 provides the binding site for Osx, which interacts with a conserved fragment of Osx. Further work demonstrates that the hinge domain-dependent oligomerization is essential for NO66 to interact with Osx and controls Osx-dependent gene expression. Our finding reveals that homo-oligomerization of JmjC domain containing proteins plays a critical role in interaction with regulatory factors and may have significant physiological roles.
Bacterial flavin homeostasis: the role of an FAD pyrophosphatase/FMN transferase
Authors: Tomchick Diana , Deka Ranjit , Brautigam Chad , Liu Wei , Norgard Michael
Published: November 2015

Treponema pallidum, an obligate parasite of humans and the causative agent of syphilis, has evolved the capacity to exploit host-derived metabolites for its survival. Flavin-containing compounds are essential cofactors that are required for metabolic processes in all living organisms, and riboflavin is a direct precursor of the cofactors FMN and FAD. Unlike many pathogenic bacteria, Treponema pallidum cannot synthesize riboflavin; we recently described a flavin-uptake mechanism composed of an ABC-type transporter [1]. However, there is a paucity of information about flavin utilization in bacterial periplasms. We have identified the TP0796 lipoprotein as a previously uncharacterized Mg2+-dependent FAD pyrophosphatase/FMN transferase within the ApbE superfamily [2,3]. Biochemical and structural investigations revealed that the enzyme has a unique bimetal Mg2+ catalytic center. Furthermore, the pyrophosphatase activity is product-inhibited by AMP, indicating a possible role for this molecule in modulating FMN and FAD levels in the treponemal periplasm. The ApbE superfamily was previously thought to be involved in thiamine biosynthesis, but our characterization of TP0796 prompts a renaming of this superfamily as a periplasmic flavin-trafficking protein (Ftp). Treponemal Ftp (Ftp_Tp) is the first structurally and biochemically characterized metal-dependent FAD pyrophosphatase/FMN transferase in bacteria. We have shown in vitro and in vivo that Ftps from several types of pathogenic bacteria are capable of flavinylating proteins through covalent attachment of FMN via a phosphoester bond to threonine residues of an appropriate sequence signature. Progress on the structural characterization of a product of this post-translational modification will be presented. This new paradigm for a bacterial flavin utilization pathway may prove to be useful for future inhibitor design.
Expression and purification of human Afamin for structure/function analysis
Authors: Altamirano Alessandra , Fineder Linda , Berberich Ramona , Fürnrorh Barbara , Fresser Friedrich , Ritsch Andreas , Naschberger Andreas , Lenac Tihana , Jonjic Stipan , Baier Gottfried , Scheffzek Klaus , Dieplinger Hans , Rupp Bernhard
Published: November 2015

Protein glycosylation plays an important role in protein stability, folding, and secretion but presents a major challenge for protein crystallization. Crystallization of highly glycosylated proteins is often difficult because of conformational and chemical inhomogeneity of the glycan decorations. Therefore, it is almost always necessary to modify the material to obtain a conformational homogenous protein that can crystallize. (1) We have explored several avenues in the case of the human 87 kDa glycoprotein afamin. Afamin (AFM), a member of the albumin gene family, is mainly expressed in the liver and secreted into the bloodstream. (2) Elevated afamin plasma concentrations are associated with major diseases such as metabolic syndrome and cancer;(3) however, pathophysiological functions are largely unknown. Therefore, we are pursuing the crystal structure of various forms of recombinantly expressed human afamin (rhAFM) for structure guided exploration and analysis of its function. Multiple variants of rhAFM are pursued: 1) fully glycosylated wild-type rhAFM (expressed in CHO cell lines); 2) rhAFM complexed with Fab fragments of two anti-AFM mAbs, 3) enzymatic deglycosylation of rhAFM with PGNaseF, 4) partially glycosylated rhAFM (expressed in glycosylation-deficient Lec1-CHO cells); and 5) glycosylation-free rhAFM, obtained from HEK293 cells virally transfected with a cDNA mutant lacking all 5 glycosylation sites by replacement of ASN with ASP. Yields of up to 2 mg/mL glycosilation free mutant rhAFM in HEK273 cells have been comparable to native rhAFM. C-terminal His6-tagged rhAFM was captured by Ni-IMAC from serum-free cell culture supernatants. All variants and Fab-AFM complexes were polished via SEC yielding pure products per SDS-PAGE and immunoblot analysis. Crystals have been obtained from Fab fragments and crystalline spherulites which are useful for microseeding from Fab-AFM complexes.
KCTD proteins as Cullin3 E3 Ligase adapters
Authors: Ji Alan , Privé Gilbert
Published: November 2015

Cullin3 (Cul3) is an ubiquitin E3 ligase responsible for catalyzing the transfer of an ubiquitin moiety from an E2 enzyme to a target substrate protein. The C-terminal region of Cul3 binds RBX1/E2-ubiquitin, while, the N-terminal region interacts with various BTB domain proteins which serve as substrate adaptors. Previously, our group determined the crystal structures of the homodimeric BTB proteins SPOP and KLHL3 in complex with the N-terminal domain of Cul3, revealing the determinants responsible for the BTB/Cul3 interaction [1, 2]. A second class of BTB-domain containing proteins, the KCTD proteins, are also Cul3 substrate adaptors but these do not share many of the previously determined features for Cul3 binding. Furthermore, KCTD proteins form homotetramers and homopentamers via BTB oligomerization rather than the previously described homodimers. Despite these differences, many KCTD proteins interact with Cul3 with dissociation constants of approximately 50 nM. While the target substrates for many of the KCTD/Cul3 E3 ligase complexes are unknown, recent studies have implicated the GABAβ2 receptor as an interactor of KCTD 8, 12, 12b and 16. Here, we report the pentameric crystal structure of the KCTD9 BTB domain and our progress on the structural characterization of Cul3/KCTD/substrate complexes.
Lysine carbamylation for enzymatic function: metal and structural requirements
Authors: Hsieh Yin-Cheng , Chan Sunney , Yang Yuh-Shyong , Chen Chun-Jung
Published: November 2015

Lysine carbamylation, a post-translational modification, facilitates metal coordination for specific enzymatic activities. Carbamylation on lysine extends the residue length by ~2 Å and changes the side chain from a positive to negative charge at neutral pH. The proteins involved with lysine carbamylation are found to be related to the human diseases, such as type 2 diabetes, developmental delay, metabolic acidosis, mental retardation, hypotonia and seizures. We have determined structures of the vertebrate dihydropyrimidinase from Tetraodon nigroviridis (TnDhp) in various states: the apo enzyme as well as two forms of the holo enzyme with one and two metals at the catalytic site. The essential active-site structural requirements have been identified with possible existence of four metal-mediated stages of lysine carbamylation. Only one metal is sufficient for stabilizing lysine carbamylation; however, the post-translational lysine carbamylation facilitates additional metal coordination for the regulation of specific enzymatic activities through controlling the conformations of two dynamic loops, Ala69–Arg74 and Met158–Met165, located in the tunnel for the substrate entrance. The substrate/product tunnel is in the "open form" in the apo-TnDhp, in the "intermediate state" in the mono-metal TnDhp, and in the "closed form" in the di-metal TnDhp structure, respectively. Structural comparison also suggests that the C-terminal tail plays a role in the enzymatic function through interactions with the Ala69–Arg74 dynamic loop. In addition, the structures of the di-metal TnDhp in complexes with hydantoin, N-carbamyl-β-alanine and N-carbamyl-β-amino isobutyrate, as well as apo-TnDhp in complex with a product analog, N-(2-acetamido)-iminodiacetic acid, have been determined. These structural results illustrate how a protein exploits unique lysines and the metal distribution to accomplish lysine carbamylation as well as subsequent enzymatic functions.