bluuebunny/crossref_metadata_2025 huggingface dataset:
Electron Diffraction vs aperiodic crystal: from indexation to structure solution
Authors: Boullay Philippe , Mouillard Gwladys , Barrier Nicolas , Perez Olivier , Palatinus Lukas
Published: November 2015

Can we solve aperiodic structures using intensities from electron diffraction? Yes! How? No mystery about it: the data analysis and the tools used for structure solution are essentially the same as the ones used in X-ray crystallography. The trick actually lies in new approaches used in electron crystallography. In analogy to X-ray diffraction, the so-called Electron Diffraction Tomography (EDT) [1] corresponds to a phi-scan data collection on a single crystal. There lies one major advantage of this technique: a powder sample is easily converted to infinitely large number of single crystals for electron diffraction. In case of aperiodic crystals this makes the difference over X-ray or neutron powder diffraction where, often, the lack of peaks clearly assignable to satellite reflections prevents any indexation and further analysis of the structure [2]. EDT allows for an accurate estimation of the modulation vector and a good guess of the super space group. These informations can be advantageously used as an input for X-ray or neutron powder diffraction. Not limited to indexation, EDT combined with Precession Electron Diffraction (PED) [3], offers a unique tool for solving modulated structures when crystals suitable for X-ray diffraction are missing. By limiting the paths for multiple scattering, PED makes the diffracted intensities closer to kinematical approximation so that they can be used efficiently for structure solution. Regarding aperiodic crystals, the superspace electron density map, generated as an output of the charge flipping algorithm used in Superflip, can be interpreted to obtain a structural model. This will be illustrated on a series of layered materials closely related to the Aurivillius phases belonging to the pseudo-binary Bi5Nb3O15-ABi2Nb2O9 (A=Pb, Sr, Ca, Ba). Limitations and possible combination with powder diffraction patterns will be discussed.
High pressure crystallization and crystallography of glucose isomerase
Authors: Suzuki Yoshihisa , Tsukamoto Masayuki , Fujiwara Takahisa , Uehara Yuuta
Published: November 2015

Crystallization of protein is still a "bottleneck" for the three-dimensional (3D) structure analysis of a protein molecule. Therefore, the study on the acceleration of the crystallization is important. Visuri et al. repoted, for the first time, that the crystallization of glucose isomerase (GI) crystals was significantly enhanced with increasing pressure [1], while they did not discuss further effects of pressure on the crystallization. We measured solubilities, growth rates of a crystal face, step velocities and two-dimensional (2D) nucleation rates, three-dimensional (3D) nucleation rates of the GI crystal under high pressure. From these results, we found the negative molar volume change of crystallization delta V, the decrease in a step ledge surface energy and the increase in a step kinetic coefficient with increasing pressure. Activation volume of crystallization delta V‡ was discussed and the absolute value of it was almost half of that of delta V [2]. X-ray crystallography is also very important to study how the three dimensional structure of protein is related to its function under high pressure at atomic level. Kundrot et al. analyzed the crystal structure of lysozyme at 100 MPa using a beryllium (Be) cell. The crystal was prepared under 0.1 MPa. Charron et al. reported that thaumatin crystals grown under 150 MPa were analyzed under 0.1 MPa. Although Kundrot et al. concluded that the protein structure under high pressure was compressed, they could not estimate the influence of the bond structure of the crystal at 0.1 MPa on the compressed structure. In Charron's case, the thaumatin structures in crystals prepared under 0.1 MPa and 150 MPa were almost identical, when they did X-ray analyses of both crystals at 0.1 MPa. From these two results, we considered that compression of the crystals was reversible. We conducted both Kundrot's and Charron's methods for GI using a stand-alone type Be vessel [3]. We found that (1) GI molecule was reversibly compressed under high pressure, (2) the distributions of water sites around GI molecules were almost identical in the range of pressures from 0.1 to 100 MPa.
Development of a new special environment powder neutron diffractomter, SPICA
Authors: Yonemura Masao , Mori Kazuhiro , Kamiyama Takashi , Fukunaga Toshiharu , Ishikawa Yoshihisa , Tomiyasu Ryoko , Idemoto Yasushi , Kanno Ryoji , Sulistyanintyas Dyah , Arai Hajime , Uchimoto Yoshiharu , Ogumi Zempachi
Published: November 2015

SPICA, a new special environment powder neutron diffractometer was built at BL09 in the Material and Life science Facility (MLF) of the Japan Proton Accelerator Research Complex (J-PARC). This is the first instrument dedicated solely to the study of next-generation batteries in J-PARC and is optimized for in situ measurements to clarify the structural changes of battery materials at the atomic level. Our approach with this diffractometer is to reveal the reactions in batteries and to determine factors of safety and degradation over long periods in practical battery systems. To make in situ measurements of real batteries more fruitful, we need high Δd/d resolution with wider d ranges to detect many phases during chemical reaction, high neutron intensity to know the specific reaction process in high speed charge/discharge, low background and large sample area to install big sample environment and a dedicated chemistry area to carry out long-term scheduled experiments with many sets of on-beam measurements and off-beam charge-discharge measurements. The in situ measurements can be performed in realistic environment with external variables such as temperature, electric field (current density, pulsed current, and etc.), and high pressure in time-resolved conditions by the 2 m sample space. The reliability of the diffraction data has achieved a sufficiently high level for the structural analysis of materials using the Rietveld method. In the beginning stage of the commissioning, the structural changes of the materials, which are dependent on the lithium content in a commercialized Li-ion battery, were clearly observed. The lattice parameters for the anode and cathode materials as a function of the lithium content were extracted from the diffraction patterns. The current status of SPICA will be reported. ACKNOWLEDGEMENT: This work was predominantly supported by the RISING project of NEDO.
A novel open tubular continuous crystalliser: design and evaluation
Authors: Robertson Karen , Wilson Chick
Published: November 2015

The ability to continuously manufacture products can be of huge benefit to industry as it can reduce waste and capital expenditure. Continuous crystallisation has received tepid interest for many years but has come to the fore recently as it holds the potential for a radical transformation in the way crystalline products are manufactured, leading to the development method being embraced by major industries such as pharmaceuticals. In addition to the financial benefits offered by continuous crystallisation over conventional batch methods, a higher level of control over the crystallisation process can also be achieved – allowing improved, more consistent particle attributes to be obtained in the crystallisation process. This control is in part a consequence of the smaller volumes involved in continuous crystallisation, which also has the advantage of reducing any hazards associated with the materials being processed. By using smaller volumes, the mixing efficacy is inherently increased which reduces any disparity between local environments, thereby allowing kinetics to dictate the nature of the products. The EPSRC Centre for Innovative Manufacturing in Continuous Manufacturing and Crystallisation (CMAC [1]) in the UK is a collaborative national initiative to further the knowledge base and understanding of all aspects relating to continuous crystallisation and its use in the manufacturing of crystalline particulate products. In this work we present the design and construction of a novel continuous crystalliser and its evaluation using various model systems such as calcium carbonate (polymorph control [2]) and Bourne reactions (mixing efficacy [3]). The crystalliser will then be used in the co-crystallisation of agrichemical and pharmaceutical compounds with co-formers in an effort to optimise the solid-state properties of these materials such as solubility. Various aspects of the evaluation of the design of the new crystalliser will be presented with reference to these trials, and assessed critically with respect to evolution of this design and potential implementation in manufacturing processes.
A New Electrochemical Cell for Rietveld Refinements of In-Situ Powder ND Data
Authors: Bianchini Matteo , Suard Emmanuelle , Croguennec Laurence , Masquelier Christian
Published: November 2015

In-situ techniques proved to be exceptionally useful tools to understand electrode materials for Li-ion batteries (1). However in-situ neutron diffraction (ND) knew a slow development, due to the intrinsic difficulties it held (2). The two main limiting factors are: the high amount of electrode active material required for ND, which makes electrochemistry challenging; and the strong scattering coming from the different parts of the battery (current collectors, protective case, organic solvent-based electrolytes...). To overcome these problems, we have designed a new electrochemical cell, manufactured with a completely neutron-transparent (Ti,Zr) alloy. Used with deuterated electrolytes, the cell was able to combine good electrochemical properties and the ability to collect ND patterns operando, with good statistics and no other Bragg peaks than those of the electrode material of interest. This allows detailed structural determinations by Rietveld refinement during operation. The cell was validated using well-known battery materials such as LiFePO4 and Li1.1Mn1.9O4 (3). Li batteries using our in-situ cell were assembled and measured on the D20 high flux neutron powder diffractometer (ILL Grenoble). Rietveld refinements were used to analyze the data and the results showed good agreement with the expected values (obtained from the same materials measured as powders on the D2B high-resolution powder diffractometer). A real operando experiment was also conducted (Figure), where the active material LiFePO4 was charged (Li+ extracted) to reach FePO4 while collecting ND patterns (1 hour each for 24h of charge). Details can be found in (3). The cell is now ready to be used to study new challenging materials. As an example, we measured the charge operando of a series of spinel materials Li1+xMn2-xO4 (x=0, 0.05, 0.1). From the data, we observed how the material evolution upon charge strongly depends on x. Results from the refinements of these data will be reported and discussed.
In-operando study of swelling and switching of thermo-responsive polymer films
Authors: Zhong Qi , Wang Weinan , Bivigou-Koumba Achille , Laschewsky Andre , Papadakis Christine , Cubitt Robert , Mueller-Buschbaum Peter
Published: November 2015

Thermo-responsive polymers can exhibit a demixing transition which is of the lower critical solution temperature (LCST) type. The collapse transition of polymers with such LCST behavior is of great interest for applications where a strong change of volume is desired even for small changes of an external stimulus such as temperature. Examples of applications are valves in micro-fluidics, the release of drugs in the body or sensors. A polymer which is a promising candidate in this context is poly(N-isopropylacrylamide), PNIPAM. It exhibits a LCST of about 320C that is attributed to alterations in the hydrogen-bonding interactions of the amide group. A polymer with a higher transition temperature as compared to PNIPAM is the thermosensitive acrylate poly(methoxydiethylene glycol acrylate) or PMDEGA. In thin film geometry, such thermoresponsive polymers are of particular interest for use as thermosensitive surfaces, artificial pumps and muscles, light modulation systems and optical switches. The strong volume change translates into a change of the film thickness due to the thin film geometry. We investigate the kinetics of chain collapse of thin thermosensitive films as a function of quench depth. Homopolymer films and amphiphilic block copolymer films are compared. With time resolved neutron reflectivity (NR) we follow the kinetics of the transition from a swollen to a collapsed thermoresponsive film. Within 15 seconds a full NR curve is probed, which allows to determine the evolution of film thickness and of the water content in real time. Thus, we investigate in-operando the switching behavior caused by a thermal trigger. The observed complex three-step switching of the films is discussed.
Towards in operando material process characterization over many length scales
Authors: Allen Andrew , Zhang Fan , Levine Lyle , Ilavsky Jan
Published: November 2015

The properties and performance of complex material systems are frequently controlled by phenomena that operate over many length-scales from sub-nanometers to millimeters. Understanding the behavior of such materials requires statistically-representative measurement of these effects on the structure and microstructure evolution across this entire length-scale range, over timescales that match those of the phenomena of interest. Small-angle X-ray and neutron scattering (SAXS and SANS) can address much of this need and reveal cause-and-effect phenomena acting across many length scales. This is especially true if SAXS or SANS are combined with wide-angle X-ray and neutron scattering (WAXS and WANS) diffraction measurements to follow the corresponding phase evolution. These concepts are demonstrated in several high-impact studies pursued with our collaborators, including in operando studies to measure: the effects of gas sorption on the structures and microstructures of new carbon sorbent materials [1]; precipitate formation and growth, together with associated phase transformations in advanced light-weight alloys during annealing or plastic deformation; real-time dissolution, clustering and agglomeration of silver nanoparticles in an acidic environment (relevant to environmental health and safety concerns) [2]; and even cement hydration phenomena related to concrete shrinkage. Many of these measurements were made at the ultra-small-angle X-ray scattering (USAXS) facility at the Advanced Photon Source where rapid combined USAXS/SAXS/WAXS studies are now possible under in operando conditions. Planned further development of the instrument capabilities will significantly enhance such in operando measurements, as can be demonstrated by the impact on these same studies [3].
Photosystem II assembly factor HCF136 from A. thaliana at 1.67 Å resolution
Authors: Bergdahl Roland , Grundström Christin , Storm Patrik , Schröder Wolfgang , Sauer Uwe
Published: November 2015

The High Chlorophyll Fluorescence 136 protein (HCF136) is essential for the assembly and repair of Photosystem II (PSII) and its central reaction centre (RC)[1]. HCF136 is an abundant protein in the thylakoid lumen and has been suggested to directly interact with subunits of the RC. The multi-subunit pigment-protein PSII complex is imbedded in the thylakoid membrane of the oxygenic photosynthetic organisms, and responsible for water splitting during oxygenic photosynthesis. PSII harbours more than 20 different integral and peripheral membrane proteins and its assembly requires a high level of coordination[2]. Two proteins D1 (psbA) and D2 (psbD) form the core of the complex and bind most of the redox-active co-factors. The PSII RC contains, in addition to D1 and D2, the intrinsic PsbI subunit and cytochrome b559. Light is a harmful substrate and subunits are damaged during the water-splitting reaction. The largest irreversible damage is experienced by the central D1 protein that has the highest turnover rate of all thylakoid proteins. Analysis of mutated A. thaliana has identified HCF136 as an essential factor for PSII RC assembly and RC turnover and repair[3]. In order to gain functional and structural insight in the way the HCF136 protein is involved in the PSII repair cycle, we have cloned, expressed, purified and crystallized the HCF136 protein from A. thaliana. Here we present the structure of this doughnut shaped WD40 domain family protein determined at 1.67 Å resolution. Biochemical and biophysical analysis of HCF136 and components of the PSII RC are under way.
Structure & acceptor recognition of CLG1_GBSS, a cyanobacterial starch synthase.
Authors: Cuesta-Seijo Jose , Nielsen Morten , Palcic Monica
Published: November 2015

Starch synthesis was thought to occur exclusively in archaeplastida, which include green algae and land plants. Recently, amylopectin-like polymers have been identified in group V cyanobacteria[1]. In particular, a newly isolated cyanobacterium, CLG1, synthetizes granules containing both amylose and amylopectin essentially identical to plant starch[2]. These cyanobacteria are believed to have contributed some of the key starch synthesizing enzymes to plants. Starch synthases are the enzymes responsible for elongation of the maltooligosaccharide chains that compose the starch granule, working in concert with many other enzymes to create the complex structures of amylopectin and amylose. Here we report the crystal structure, refined to 2.2 Å, of GBSS, the granule bound starch synthase responsible for amylose synthesis in CLG1, in complex with ADP and either acarbose or glucose in the acceptor binding site. The structure reveals different conformational states of the ternary complex in three copies of GBSS in the asymmetric unit. The variations between monomers shed light on changes on the protein upon substrate recognition. In particular it clarifies the effect of acceptor binding in the conformation of the active site. This structure also illustrates the conformation of parts of the primary sequence that were absent from all plant starch synthase structures to date. Features in this structure are compared to both glycogen synthase and starch synthase structures. Both the similarities and the differences advance our knowledge on the necessary components of a starch synthase and point the way to their targeted structural and functional modification. The world-wide demand of cereals is expected to double from its current values by 2050 (FAO). Modification of proteins involved in starch synthesis, be it via traditional breeding or via genetic engineering, will likely be crucial to meeting the caloric intake needs of the human population in the coming decades.
Structural Insights into Bak Activation and Oligomerisation
Authors: Brouwer Jason , Robin Adeline , Thompson Geoff , Wardak Ahmad , Kluck Ruth , Colman Peter , Czabotar Peter
Published: November 2015

Apoptotic stimuli activate and oligomerise the pro-apoptotic proteins Bak and Bax resulting in mitochondrial outer membrane permeabilisation and subsequent cell death. This activation can occur when certain BH3-only proteins directly interact with Bak and Bax. A recent crystal structure by Czabotar et al. (2013) revealed a novel conformational change for Bax upon activation by BH3-only peptides. Distinguishing characteristics of BH3-only proteins capable of directly activating Bax were also elucidated. Here we describe complementary studies on the related protein Bak. We identify specific BH3-only peptides capable of inducing Bak dimerisation and describe crystal structures that provide key insights into Bak activation and oligomerisation. These structures demonstrate that Bak undergoes similar conformational changes upon activation to those observed with Bax. Altogether our results confirm an analogous mechanism for activation and dimerization of Bak and Bax in response to BH3-only peptides.
Crystal structure of human TAF5/TAF6/TAF9 complex at 2.7 A resolution
Authors: Haffke Matthias , Berger Imre
Published: November 2015

Human general transcription factor TFIID is a megadalton sized multiprotein complex containing the TATA-box binding protein (TBP) and 13 TBP Associated Factors (TAFs). Structural studies by cryo-electron microscopy (cryo-EM) have uncovered the overall architecture of TFIID, providing valuable insight into subunit assembly and promoter recognition at medium resolution. Despite considerable effort, high resolution structural information of TAFs to date have been largely limited to structures of individual domains and assemblies of TAF histone-fold pairs, which are prevalent in TFIID. We used our MultiBac baculovirus/insect cell technology to obtain recombinant TFIID complexes including holo-TFIID in unprecedented quality and quantity, setting the stage for high resolution structural analysis by X-ray crystallography. We identified stable subassemblies of the TFIID complexes by limited proteolysis, and utilized a rapid combinatorial co-expression approach to produce and purify specimens for high-throughput crystallization. We crystallized a thus defined complex formed by TAF5, TAF6 and TAF9 and determined the structure to a resolution of 2.7 Å, revealing intricate molecular interactions at the core of human TFIID.
Structural basis of substrate channeling in the PutA flavoenzyme
Authors: Tanner John
Published: November 2015

Proline utilization A (PutA) is a high-hanging fruit of X-ray crystallography. PutA is a membrane-associated bifunctional flavoenzyme that catalyzes the 4-electron oxidation of proline to glutamate by the sequential activities of proline dehydrogenase and aldehyde dehydrogenase domains. PutAs are challenging crystallography targets because of their long polypeptide chain length (1000-1300 residues) and multidomain architecture. In this talk, I will present new crystal structures and SAXS analysis of two PutAs. Seven high resolution crystal structures of a 1004-residue minimalist PutA were determined using Hg SIRAS phasing, and the oligomeric state and quaternary structure were determined with SAXS [1]. The structures reveal an elaborate and dynamic tunnel system featuring a 75-Å long tunnel that links the two active sites. Also, a novel mechanism-based inactivation strategy allowed the trapping of the elusive PutA-quinone complex in the crystalline state. These structures provide insight into the mechanism of substrate channeling and how the enzyme changes conformation during the catalytic cycle. I will conclude by describing the first structure of a new type of PutA that contains an additional C-terminal domain of unknown function (CTDUF) that is not present in the smaller minimalist enzyme [2]. This larger PutA reveals an unexpectedly different structural solution to the problem of sequestering the reaction intermediate.
Anaerobic crystallization for protein crystallography
Authors: Senda Miki , Senda Toshiya
Published: November 2015

Crystallization has been a bottleneck in protein crystallography. Major problems in protein crystallization are 1) to find crystallization conditions effectively at the initial crystallization screening and 2) to improve the reproducibility of protein crystallization. To overcome these problems, we have proposed some techniques such as the immediate observation method (1). Recently, we realized that films and precipitates of oxidized proteins hampered the crystal formation, leading to poor reproducibility of the crystallization. To avoid oxidation of proteins, we examined anaerobic crystallization in an anaerobic chamber. The anaerobic chamber (Anaerobic `HARD', Hirasawa) was designed to carry out controlled anaerobic experiments for electron-transfer proteins. We have so far established typical procedures for the anaerobic crystallization (2). On the basis of our earlier experiences, the anaerobic crystallization was tested for various proteins. We found obvious differences between aerobic and anaerobic crystallization in some cases; some proteins could crystallize only under anaerobic conditions. Furthermore, the anaerobic crystallization improved reproducibility of crystallization as expected. We will report some examples of the anaerobic crystallization.
Rapid sample delivery by acoustic injection for serial protein crystallography
Authors: Roessler Christian , Allaire Marc , Orville Allen , Soares Alexei
Published: November 2015

Continuous irradiation of a single protein crystal alters its physical structure. This typically manifests as photoelectric chemical transformations and a loss of diffraction, which can be counterproductive to structure determination. Serial crystallography methods overcome these effects by recording partial datasets from multiple crystals, effectively spreading the dose over many crystals. As the number of crystals per dataset increases, however, new methods are needed to rapidly replenish samples in the X-ray beam. Acoustic droplet ejection (ADE) technology offers a robust and reliable method for transferring crystals. Acoustic injectors based on ADE technology use focused sound waves to deliver crystals contained within picoliter and nanoliter droplets, and can generate hundreds of droplets each second. Acoustic injectors have been tested at the National Synchrotron Light Source, where crystal-containing droplets were deposited on a goniometer-mounted conveyor belt. The conveyor belt enabled translation and rotation in the X-ray beam, permitting small wedges of data to be collected on each crystal. Crystals were also directly injected into oncoming X-ray pulses at the Linac Coherent Light Source, producing diffraction patterns from several samples under conditions of ambient temperature and pressure. Development is underway to incorporate acoustic injectors into the microfocus and automated macromolecular crystallography beamlines at the National Synchrotron Light Source II.
Optimization of Cryptic Leads from Trace Fluorescent Labeling Screening
Authors: Pusey Marc , Morris Michelle , Barcena Jorge , Yuan Qunying , Ng Joseph
Published: November 2015

We use trace fluorescent labeling (TFL) as a means of rapidly identifying crystals in the screening plate. The method involves the covalent labeling of between 0.1 to 0.2 % of the protein molecules with a fluorescent probe. Our standard labeling process uses the amine reactive dye 5(6)-carboxyrhodamine 6G succinimidyl ester (Invitrogen, C-6157), with reaction conditions (pH) adjusted to label random side chain amines. Previous results had shown that labeling below 1% does not affect the nucleation rate or diffraction data quality. Identification of crystalline outcomes is based on intensity; (labeled) protein packing density is highest in the crystalline form which will fluoresce more brightly than other precipitated forms. We found that there were many outcomes where the fluorescent images had regions of high intensity, but no corresponding crystalline structures were apparent using white light transmission microscopy. Under the governing paradigm, that intensity = structure, we hypothesized that these were likely lead conditions and tested that hypothesis with optimization screening using capillary counter diffusion (CCD). Four CCD experiments are set up for each lead condition experiment having different ratios of the stock screening cocktail components. The capillaries (40 mm long, 0.3, 0.2, or 0.1 mm ID) are filled with protein at 2.5X the sitting drop screening experiment concentration, sealed at one end using soft wax, then inserted open end down into 40 uL of the precipitant solution in a 1.2 mL titer tube (E&K Scientific, cat.# 684510-R), which is then sealed with a stopper (E&K Scientific, cat.# 64108-P). The capillaries can be examined using a microscope through the walls of the tube, using either white light or fluorescence illumination. We are currently finding ~40% of the identified lead conditions yield crystals upon optimization, representing a significant increase in the success rate for the screening experiment.
Protein Crystal Staining for Second Harmonic Generation Imaging
Authors: Newman Justin , Scarborough Nicole , Pogranichnyi Nicholas , Closser Richard , Simpson Garth
Published: November 2015

Recently, second harmonic generation (SHG) microscopy has become a useful tool in the field of structural biology for the detection of protein crystals. SHG, or the frequency doubling of light, is a process specific to crystalline media lacking inversion centers. Through theoretical models and experimental data, it is estimated that ~84% of the known protein crystal structures give detectable SHG signal using current SHG microscopy instrumentation. Extending this coverage could be extremely useful to structural biologists who routinely screen entire 96 well plates, with hundreds of crystallization conditions, in search of diffraction-quality protein crystals. A series of SHG active dyes, including Malachite Green (MG) and trans-4-[4-(dimethylamino)styryl]-1-methylpyridinium iodide (DMI) were investigated to assess their ability to enhance the nonlinear optical (NLO) response across a broad range of protein crystals with varying degrees of inherent SHG activity. MG and DMI were shown to enhance the SHG activity of tetragonal (P43212) lysozyme crystals, a protein that typically generates little to no SHG signal. SHG enhancements for lysozyme of approximately 16000x and 20x were achieved by intercalation of MG and DMI, respectively. These results are consistent with predictions based on the differences in symmetry and structure for the two dyes. The kinetics of the dye intercalation and uptake were investigated with SHG time-lapse images taken of a lysozyme crystal after the addition of MG dye into the crystallization well. Kinetic results indicate that an increase in SHG activity becomes easily noticeable within minutes of exposure to the dyes. These results show a significant progress towards increasing the coverage of SHG microscopy for protein crystal detection.
Structure-based engineering of designed armadillo repeat proteins.
Authors: Hansen Simon , Reichen Christian , Madhurantakam Chaithanya , Grütter Markus , Plückthun Andreas , Mittl Peer
Published: November 2015

The specific recognition of macromolecules is key for many applications in biochemical research, medical diagnostics and disease treatment. Currently the development of new recognition molecules depends on the immunization of lab animals or combinatorial biochemistry techniques. Since both approaches are elaborate and require the availability of sufficient amounts of stable target molecules we are developing a modular system that allows a rational design of peptide recognition modules. This system is based on the armadillo repeat scaffold, because natural armadillo repeat proteins bind their targets in extended anti-parallel conformations with very regular binding topologies. The development of designed armadillo repeat proteins (dArmRPs) consisting entirely of identical internal repeats (full-consensus design) has been described [1]. Although dArmRP with 2nd generation capping repeats were predominantly monomeric in solution, crystal structures of dArmRP with different numbers of internal repeats revealed domain-swapped N-caps [2]. Redesign of the N-cap significantly improved thermodynamic stability and abrogated swapping of N-caps. These 3rd generation dArmRP recognize full-consensus peptides with nanomolar affinities. The dissociation constants depend on the lengths of the targeted peptides and the number of internal repeats. Three crystal structures of complexes between dArmRPs with five or six internal repeats and the targeted full-consensus (KR)5 peptide (either free or fused to the N- and C-terminii of globular proteins) confirm that the binding mode fulfills the expected regular topology. Further crystal structures of complexes between dArmRPs and the mismatch (RR)5 peptide revealed that the dArmRPs recognize their target peptides in a side-chain specific manner. Several crystal structures confirm that 3rd generation dArmRPs behave as stable and monomeric molecules that allow the selective recognition of the targeted peptide with the expected topology. Therefore, the rational assembly of binding modules from a pool of dipeptide-specific armadillo repeats to recognize peptides with given sequences should indeed be possible.
Protein Crystallization Controlled by Special Adhesion Molecules
Authors: Hasek Jindrich , Skalova Tereza , Dušková Jarmila , Koval Tomáš , Kolenko Petr , Fejfarová Karla , Stránský Jan , Dohnálek Jan
Published: November 2015

Protein precipitates from solution with decreasing amount of free water molecules available for protein hydration (water evaporation, binding water on precipitant molecules, etc.). However, a uniform stacking of protein molecules on the surface of a growing crystal requires an absolute dominance of a single protein-protein adhesion mode. Existence of any "mutually incompatible protein-protein adhesion mode" in the same crystal leads to stacking faults, leading to low quality crystals, or to a crystallization failure. As proteins possessing large molecular surface have usually more adhesion modes, we need substances that are able to modify kinetics of protein-protein adhesion in different adhesion modes. It can be done by "Protein Surface Active Molecules" (PSAM) [1] binding selectively and temporarily the protein surface. The PSAM are used either to block some adhesion modes, or to form crosslinks built-in permanently into the growing crystals. Thus, a combination of more "successful additives" in solution can act positively in synergy but also can lead to a complete failure depending on the specificity of the protein-adhesion properties. The theory of PSAM describes protein crystallization as a regular deposition of short-life-time protein-PSAM complexes and stresses a role of "sticking molecules" exhibiting highly specific adhesion to different protein surface patches active in the crystallization process. The concept of crystallization of "protein adducts" falling apart during their stacking into the crystal lattice may be more complicated for imagination but it provides explanations of many enigmatic crystallization phenomena, provides better control over the crystal quality and over preparation of different crystal forms. Proper choice of the PSAM active in formation of temporary protein-PSAM adducts eliminates deposition of protein molecules on the growing crystal surface in "incompatible protein-protein adhesion modes" and it is decisive for a success of protein crystallization in many cases. The use of PSAM allows to: increase the number of crystallizable proteins, minimize probability of stacking faults, grow more polymorphs showing the same protein molecule in different environments, select the polymorph ensuring the highest accuracy of structure determination. The work is supported by BIOCEV CZ.1.05/1.1.00/02.0109 from ERDF, MSMT projects EE2.3.30.0029, LG14009, CSF P302/11/0855.
Crystallographic studies for the folding of an extending peptide
Authors: Hanazono Yuya , Takeda Kazuki , Miki Kunio
Published: November 2015

Full-length proteins can fold into thermodynamically stable structures at an exceptionally fast rate as shown by in vitro experiments. In contrast, it takes much more time to finish nascent protein folding than full-length protein folding, because nascent protein folding depends on the rate of ribosome biosynthesis in the living cell. Therefore nascent polypeptide chains in vivo fold co-translationally in different manners from the full-length proteins. However, the transient structures and the co-translational folding pathway are not well understood. In order to reveal the atomic details of nascent protein folding, we studied the hPin1 WW domain, which consists of two beta-hairpins between the three-stranded beta-sheets. Here we report a series of WW domain N-terminal fragment structures with increasing amino acid length by using circular dichroism spectroscopy and X-ray crystallography. In crystallization, maltose-binding protein was fused just behind the WW domain fragments to fix the C-terminus as nascent proteins are anchored to the ribosome. Co-translational folding of beta-sheet-rich proteins is discussed based on our finding that intermediate-length fragments unexpectedly take a helical conformation, even though the full-length protein has no helical regions. Furthermore, in a region of one of the loop structures of the full-length protein, these fragments take different formations. Our results suggest that the newly synthesized polypeptides adopt the most stable conformation during the course of peptide extension and fold into the native structures, eventually.
Detection of protein nanocrystals based on the reversibility of crystallization
Authors: Dörner Katerina , Martin-Garcia Jose , Kupitz Christopher , Wachter Rebekka , Fromme Petra
Published: November 2015

A prerequisite for conventional X-ray protein structure analysis is the growth of crystals with a sufficient size in the range of several µm. This is a time consuming and not always successful process, challenging especially when working with membrane proteins. The recently developed technique of femtosecond X-ray crystallography enables structure analysis of crystals with a size in the nm range, thus the process of growing large single crystals can be avoided. Moreover femtosecond X-ray nanocrystallography is a potential method to overcome the radiation damage problem and to perform time-resolved structure analysis (1, 2). Nanocrystals are too small to be detected with an optical microscope, hence crystal growth cannot be monitored with common methods used in crystallography. A powerful technique to screen for nanocrystals is Second Order Nonlinear Imaging of Chiral Crystals (SONICC). This method is based on the principle of second harmonic generation and detects noncentrosymmetric ordered crystals (3). However, the instrumentation is not generally accessible yet. In addition, proteins ordered in higher symmetry crystal classes do not necessarily lead to a positive SONICC signal. In this work, a new method is developed to screen for nanocrystals based on the reversibility of crystallization. We show that dilution experiments performed with a crystallization robot and monitored by a crystallization imaging system enable the distinction between precipitation comprised of nanocrystals and precipitation caused by aggregated protein.
A new protein crystal mounting method using humid air and glue coating
Authors: Baba Seiki , Kumasaka Takashi
Published: November 2015

Cryopreservation of protein crystals are a useful to reduce radiation damage for synchrotron experiments, the cryoloop mounting method using cryoprotectant agents is the widely used. However, Protein crystals are fragile, and they have often trouble to find a condition suitable for cryo-cooling. X-ray diffraction experiments at room temperature can evaluate the quality of the protein crystal and perform structural analysis without being affected shrink of the crystal by cryo-cooled and addition cryoprotectant agents. And, conventional humidity controlled method is possible to improve resolution of limited protein crystals [1]. However, protein crystals of these diffraction experiments cannot apply same mounting method. We developed a new crystal mounting method, the humid air and glue-coating (HAG) mounting method, which involves a combination of controlled adjustable humid air and water-soluble polymer glue for crystal coating [2]. By coating with the water-soluble polymer glue, most crystals exposed to the controlled humid air were stable at room temperature and could be cryocooled under optimized humidity without additional cryoprotectant agents. For example, the crystals of the bacterial hydrolase RsbQ was mechanically very fragile and sensitive to environmental changes. Thus, RsbQ crystal cannot apply cryoloop mount with cryoprotectant agents. By using the HAG method, we were able to obtain 1.4 Å data and solve its structure. For another example, membrane protein crystal was improved resolution to optimize humidity. The crystals by using HAG method reproducibly showed crystal lattice transformation in response to a change in humidity, thus using this method a series of isomorphic crystals can be prepared. We introduce HAG method, and demonstrate its success with various protein crystals.
"From one seed a whole handful": homologous proteins as seeds in crystallisation
Authors: Abuhammad Areej , McDonough Michael , Brem Jürgen , Schofield Christopher , Garman Elspeth
Published: November 2015

Protein structures have significantly impacted and aided drug discovery efforts. However, it is not enough to know the structure of a protein; it must be the right structure. Small alteration in sequence can lead to different conformations and oligomerization states, cause changes which lead to different active site architecture and also which modify function. Protein crystallization is an essential prerequisite for the determination of protein structures by X-ray crystallography. We have obtained encouraging initial results for a hitherto unexplored crystallization method with the enzyme arylamine N-acetyltransferase from M. tuberculosis (TBNAT). Despite prolonged and varied trials to crystallize TBNAT, an important anti-tubercular drug target, no crystals were obtained. In an alternative approach, cross-seeding of TBNAT protein with micro-crystalline seeds from a homologous NAT from M. marinum (74 % sequence identity (SID)) surprisingly resulted in a single 20 micron sized TBNAT crystal that diffracted to 2.1 Å and allowed for TBNAT structure determination (Abuhammad et al., 2013). To our knowledge, cross-seeding crystallisation using homologous proteins has only been previously successful in cases with more than 85% SID. In this study, we have explored the effect of low sequence homology on cross seeding using β-lactamases with SID as low as 30%. Despite the low SIDs, the results show cross seeding leads to an increase in hits obtained, the identification of new crystallization conditions, shortening of crystallization time and an improvement in the quality of the crystals obtained.
Imogolite nanotubes: a 2D x-ray scattering study of films of oriented samples
Authors: Mariette Céline , Amara Mohamed-Salah , Paineau Erwan-Nicolas , Thill Antoine , Rouzière Stephan , Launois Pascale
Published: November 2015

Inorganic nanotubes represent an emerging class of nanobuilding blocks. Among them, imogolites are alumino-silicate or alumino-germanate nanotubes with well controlled diameter and helicity. As such, they constitute a model platform for the study of molecular interactions and confinement at the nanoscale, complementing the one constituted by carbon nanotubes. We focus here on double-walled alumino-germanate nanotubes, discovered very recently [1]. They are formed of two concentric tubes (figure inset), with respective internal diameters of 1.6 and 3.1nm and up to 1 micron in length [2]. We report the first experimental study, using wide angle x-ray scattering, performed on films of oriented nanotubes (figure). Structural changes of the nanotubes and behavior of the confined water under heating are investigated in-situ. The study of oriented samples gives new information that is not available with powder diffraction. Above all, the contribution to the scattering signal of internal and external tubes can be separated as well as the translational/rotational correlations. The use of wide image plate detectors allows one to access large area of the reciprocal space in a single image. Simulations of the two-dimensionnal scattering diagrams will be presented. A key question, the correlation between internal and external tube, which is of great interest for understanding friction properties at the nanoscale, will be discussed.
Retrieval of 3D deformations of single crystal defects by X-ray topo-tomography
Authors: Asadchikov Victor , Besedin Ilya , Buzmakov Alexey , Chukhovskii Felix , Zolotov Denis , Roshchin Boris
Published: November 2015

A challenge in X-ray topo-tomography is the issue of quantitatively determining the 3D-deformation-distribution field associated with single defects of crystal lattices. In the present report an endeavor is made to retrieve a 3D-deformation-distribution field around single line defects (dislocations) in crystals by using the X-ray topo-tomography method. The general layout of the X-ray topo-tomography experiment is depicted in Fig. 1[1]. For our purposes we have used plane-parallel samples of crystal Si with surface orientation (111), the sample thickness being 1 mm, in which the linear dislocations have been inserted according to [2]. The experiments were carried out at the X-ray wavelength of MoKα1 (λ=0.071 nm). Experimental series of the X-ray (2 ¯20)-reflection topography images with the rotation angle step 20around the diffraction vector, the total angular range 3600, have been got out. Furthermore, such the 2D- topographic images are used for getting 3D-images by means of the modified algebraic method SART developed in [3]. In parallel, for 3D reconstruction the corresponding 2D-topographic dislocation images are simulated by use of the Born-approximation analytical and numerical solutions based on Takagi-Taupin equations describing the two-beam X-ray diffraction by the deformed crystals. Certainly, all the above approaches are applied to the comparative analysis of opportunities of determining 3D-deformation-distribution field around the dislocations under consideration.
Algorithms for Two-dimensional XRD Data Evaluation
Authors: He Bob
Published: November 2015

The diffracted x-rays from a polycrystalline (powder) sample form a series diffraction cones in space since large numbers of crystals oriented randomly in the space are covered by the incident x-ray beam. Each diffraction cone corresponds to the diffraction from the same family of crystalline planes in all the participating grains. When a two-dimensional (2D) detector is used for x-ray powder diffraction, the diffraction cones are intercepted by the 2D detector and the x-ray intensity distribution on the sensing area is converted to an image-like diffraction pattern. The 2D pattern contains the scattering intensity distribution as a function of two orthogonal angles. One is the Bragg angle 2θ and the other is the azimuthal angle about the incident x-ray beam, denoted by γ. A 2D diffraction pattern can be analyzed directly or by data reduction to the intensity distribution along γ or 2θ. The γ-integration can reduce the 2D pattern into a diffraction profile analogs to the conventional diffraction pattern which is the diffraction intensity distribution as a function of 2θ angles. This kind of diffraction pattern can be evaluated by most exiting software and algorithms for conventional applications, such as, phase identification, structure refinement and 2θ-profile analysis. However, the materials structure information associated to the intensity distribution along γ direction is lost through γ-integration. The intensity distribution and 2θ variations along γ contain more information, such as the orientation distribution, strain states, crystallite size and shape distribution. In order to understand and analyze 2D diffraction data, new approaches and algorithms are necessary. The diffraction vector approach has been approved to be the genuine theory in 2D data analysis. The unit diffraction vector used for 2D analysis is a function of both 2θ and γ. The unit diffraction vector for all the pixels in the 2D pattern measured in the laboratory coordinates can be transformed to the sample coordinates. The vector components can then be used to derive fundamental equations for many applications, including stress, texture, crystal orientation and crystal size evaluation by γ-profile analysis. The unit diffraction vector is also used in polarization and absorption correction.