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Protein science 32(2), e4567 (2023). doi:10.1002/pro.4567, DNA-binding proteins from starved cells (Dps) are small multifunctional nanocages expressed by prokaryotes in acute oxidative stress conditions or during the starvation-induced stationary phase, as a bacterial defense mechanism. Dps proteins protect bacterial DNA from damage by either direct binding or by removing precursors of reactive oxygen species from solution. The DNA-binding properties of most Dps proteins studied so far are related to their unordered, flexible, N- and C-terminal extensions. In a previous work, we revealed that the N-terminal tails of Deinoccocus grandis Dps shift from an extended to a compact conformation depending on the ionic strength of the buffer and detected a novel high-spin ferrous iron center in the proximal ends of those tails. In this work, we further explore the conformational dynamics of the protein by probing the effect of divalent metals binding to the tail by comparing the metal-binding properties of the wild-type protein with a binding site-impaired D34A variant using size exclusion chromatography, dynamic light scattering, synchrotron radiation circular dichroism, and small-angle X-ray scattering. The N-terminal ferrous species was also characterized by Mössbauer spectroscopy. The results herein presented reveal that the conformation of the N-terminal tails is altered upon metal binding in a gradual, reversible, and specific manner. These observations may point towards the existence of a regulatory process for the DNA-binding properties of Dps proteins through metal binding to their N- and/or C-terminal extensions., Published by Protein Society, Bethesda, Md.

Scientific reports 11(1), 22311 (2021). doi:10.1038/s41598-021-01774-6, During the last decades discussions were taking place on the existence of global, non-thermal structural changes in biological macromolecules induced by Terahertz (THz) radiation. Despite numerous studies, a clear experimental proof of this effect for biological particles in solution is still missing. We developed a setup combining THz-irradiation with small angle X-ray scattering (SAXS), which is a sensitive method for detecting the expected structural changes. We investigated in detail protein systems with different shape morphologies (bovine serum albumin, microtubules), which have been proposed to be susceptible to THz-radiation, under variable parameters (THz wavelength, THz power densities up to 6.8 mW/cm$^2$, protein concentrations). None of the studied systems and conditions revealed structural changes detectable by SAXS suggesting that the expected non-thermal THz-induced effects do not lead to alterations of the overall structures, which are revealed by scattering from dissolved macromolecules. This leaves us with the conclusion that, if such effects are present, these are either local or outside of the spectrum and power range covered by the present study., Published by Macmillan Publishers Limited, part of Springer Nature, [London]

Journal of synchrotron radiation 28(3), 812-823 (2021). doi:10.1107/S1600577521003404, Small-angle X-ray scattering (SAXS) is an established method for studyingnanostructured systems and in particular biological macromolecules in solution.To obtain element-specific information about the sample, anomalous SAXS(ASAXS) exploits changes of the scattering properties of selected atoms whenthe energy of the incident X-rays is close to the binding energy of their electrons.While ASAXS is widely applied to condensed matter and inorganic systems, itsuse for biological macromolecules is challenging because of the weak anomalouseffect. Biological objects are often only available in small quantities and areprone to radiation damage, which makes biological ASAXS measurements verychallenging. The BioSAXS beamline P12 operated by the European MolecularBiology Laboratory (EMBL) at the PETRA III storage ring (DESY, Hamburg)is dedicated to studies of weakly scattering objects. Here, recent developmentsat P12 allowing for ASAXS measurements are presented. The beamline control,data acquisition and data reduction pipeline of the beamline were adaptedto conduct ASAXS experiments. Modelling tools were developed to computeASAXS patterns from atomic models, which can be used to analyze the data andto help designing appropriate data collection strategies. These developments areillustrated with ASAXS experiments on different model systems performed atthe P12 beamline., Published by Wiley-Blackwell, [S.l.]

Crystallization of recombinant proteins in living cells is an exciting new approach for structural biology that provides an alternative to the time-consuming optimization of protein purification and extensive crystal screening steps. Exploiting the potential of this approach requires a more detailed understanding of the cellular processes involved and versatile screening strategies for crystals in a cell culture. Particularly if the target protein forms crystalline structures of unknown morphology only in a small fraction of cells, their detection by applying standard visualization techniques can be time consuming and difficult owing to the environmental challenges imposed by the living cells. In this study, a high-brilliance and low-background bioSAXS beamline is employed for rapid and sensitive detection of protein microcrystals grown within insect cells. On the basis of the presence of Bragg peaks in the recorded small-angle X-ray scattering profiles, it is possible to assess within seconds whether a cell culture contains microcrystals, even in a small percentage of cells. Since such information cannot be obtained by other established detection methods in this time frame, this screening approach has the potential to overcome one of the bottlenecks of intracellular crystal detection. Moreover, the association of the Bragg peak positions in the scattering curves with the unit-cell composition of the protein crystals raises the possibility of investigating the impact of environmental conditions on the crystal structure of the intracellular protein crystals. This information provides valuable insights helping to further understand the in cellulo crystallization process.

Coronaviral nsp7+8 complexes follow species- and stoichiometry-specific assembly pathways determined by few amino acid changes., Coronaviruses infect many different species including humans. The last two decades have seen three zoonotic coronaviruses, with SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) causing a pandemic in 2020. Coronaviral non-structural proteins (nsps) form the replication-transcription complex (RTC). Nsp7 and nsp8 interact with and regulate the RNA-dependent RNA-polymerase and other enzymes in the RTC. However, the structural plasticity of nsp7+8 complexes has been under debate. Here, we present the framework of nsp7+8 complex stoichiometry and topology based on native mass spectrometry and complementary biophysical techniques of nsp7+8 complexes from seven coronaviruses in the genera Alpha- and Betacoronavirus including SARS-CoV-2. Their complexes cluster into three groups, which systematically form either heterotrimers or heterotetramers or both, exhibiting distinct topologies. Moreover, even at high protein concentrations, SARS-CoV-2 nsp7+8 consists primarily of heterotetramers. From these results, the different assembly paths can be pinpointed to specific residues and an assembly model proposed.

Messenger ribonucleic acid (mRNA)-based nanomedicines have shown to be a promising new lead in a broad field of potential applications such as tumor immunotherapy. Of these nanomedicines, lipid-based mRNA nanoparticles comprising ionizable lipids are gaining increasing attention as versatile technologies for fine-tuning toward a given application, with proven potential for successful development up to clinical practice. Still, several hurdles have to be overcome to obtain a drug product that shows adequate mRNA delivery and clinical efficacy. In this study, pH-induced changes in internal molecular organization and overall physicochemical characteristics of lipoplexes comprising ionizable lipids were investigated using small-angle X-ray scattering and supplementary techniques. These changes were determined for different types of ionizable lipids, present at various molar fractions and N/P ratios inside the phospholipid membranes. The investigated systems showed a lamellar organization, allowing an accurate determination of pH-dependent structural changes. The differences in the pH responsiveness of the systems comprising different ionizable lipids and mRNA fractions could be clearly revealed from their structural evolution. Measurements of the degree of ionization and pH-dependent mRNA loading into the systems by fluorescence assays supported the findings from the structural investigation. Our approach allows for direct in situ determination of the structural response of the lipoplex systems to changes of the environmental pH similar to that observed for endosomal uptake. These data therefore provide valuable complementary information for understanding and fine-tuning of tailored mRNA delivery systems toward improved cellular uptake and endosomal processing.

Journal of applied crystallography 54(3), 830-838 (2021). doi:10.1107/S1600576721003034, Small-angle X-ray scattering is widely utilized to study biological macromol��ecules in solution. For samples containing specific (e.g. metal) atoms, additional information can be obtained using anomalous scattering. Here, measuring samples at different energies close to the absorption edges of relevant elements provides specific structural details. However, anomalous small-angle X-ray scattering (ASAXS) applications to dilute macromolecular solutions are challenging owing to the overall low anomalous scattering effect. Here, pilot ASAXS experiments from dilute solutions of ferritin and cobalt-loaded apoferritin are reported. These samples were investigated near the resonance X-ray K edges of Fe and Co, respectively, at the EMBL P12 bioSAXS beamline at PETRA III, DESY. Thanks to the high brilliance of the P12 beamline, ASAXS experiments are feasible on dilute protein solutions, allowing one to extract the Fe- or Co-specific anomalous dispersion terms from the ASAXS data. The data were subsequently used to determine the spatial distribution of either iron or cobalt atoms incorporated into the ferritin/apoferritin protein cages., Published by Wiley-Blackwell, [S.l.]

Polysarcosine (pSar) is a polypeptoid based on the endogenous amino acid sarcosine (N-methylated glycine), which has previously shown potent stealth properties. Here, lipid nanoparticles (LNPs) for therapeutic application of messenger RNA were assembled using pSarcosinylated lipids as a tool for particle engineering. Using pSar lipids with different polymeric chain lengths and molar fractions enabled the control of the physicochemical characteristics of the LNPs, such as particle size, morphology, and internal structure. In combination with a suited ionizable lipid, LNPs were assembled, which displayed high RNA transfection potency with an improved safety profile after intravenous injection. Notably, a higher protein secretion with a reduced immunostimulatory response was observed when compared to systems based on polyethylene glycol (PEG) lipids. pSarcosinylated nanocarriers showed a lower proinflammatory cytokine secretion and reduced complement activation compared to PEGylated LNPs. In summary, the described pSar-based LNPs enable safe and potent delivery of mRNA, thus signifying an excellent basis for the development of PEG-free RNA therapeutics.

Excitation frequencies in the terahertz (THz) range are expected to lead to functionally relevant domain movements within the biological macromolecules such as proteins. The possibility of examining such movements in an aqueous environment is particularly valuable since here proteins are not deprived of any motional degrees of freedom. Small angle x-ray scattering (SAXS) is a powerful method to study the structure and domain movements of proteins in solution. Here, we present a microfluidic cell for SAXS experiments, which is also transparent for THz radiation. Specifically, cell dimensions and material were optimized for both radiation sources. In addition, the polystyrene cell can be 3D printed and easily assembled. We demonstrate the practicality of our design for SAXS measurements on several proteins in solution.

Cells 9(9), 2034 (1-19) (2020). doi:10.3390/cells9092034, Hybrid nanoparticles from lipidic and polymeric components were assembled to serve as vehicles for the transfection of messenger RNA (mRNA) using different portions of the cationic lipid DOTAP (1,2-Dioleoyl-3-trimethylammonium-propane) and the cationic biopolymer protamine as model systems. Two different sequential assembly approaches in comparison with a direct single-step protocol were applied, and molecular organization in correlation with biological activity of the resulting nanoparticle systems was investigated. Differences in the structure of the nanoparticles were revealed by thorough physicochemical characterization including small angle neutron scattering (SANS), small angle X-ray scattering (SAXS), and cryogenic transmission electron microscopy (cryo-TEM). All hybrid systems, combining lipid and polymer, displayed significantly increased transfection in comparison to lipid/mRNA and polymer/mRNA particles alone. For the hybrid nanoparticles, characteristic differences regarding the internal organization, release characteristics, and activity were determined depending on the assembly route. The systems with the highest transfection efficacy were characterized by a heterogenous internal organization, accompanied by facilitated release. Such a system could be best obtained by the single step protocol, starting with a lipid and polymer mixture for nanoparticle formation., Published by MDPI, Basel

Crystals 10(11), 975 (1-18) (2020). doi:10.3390/cryst10110975, We describe the updated size-exclusion chromatography small angle X-ray scattering (SEC-SAXS) set-up used at the P12 bioSAXS beam line of the European Molecular Biology Laboratory (EMBL) at the PETRAIII synchrotron, DESY Hamburg (Germany). The addition of size exclusion chromatography (SEC) directly on-line to the SAXS capillary has become a well-established approach to reduce the effects of the sample heterogeneity on the SAXS measurements. The additional use of multi-angle laser light scattering (MALLS), UV absorption spectroscopy, refractive index (RI), and quasi-elastic light scattering (QELS) in parallel to the SAXS measurements enables independent molecular weight validation and hydrodynamic radius estimates. This allows one to address sample monodispersity as well as conformational heterogeneity. The benefits of the current SEC-SAXS set-up are demonstrated on a set of selected standard proteins. The processed SEC-SAXS data and models are provided in the Small Angle Scattering Biological Data Bank (SASBDB) and are labeled as “bench-marked” datasets that include the unsubtracted data frames spanning the respective SEC elution profiles and corresponding MALLS-UV-RI-QELS data. These entries provide method developers with datasets suitable for testing purposes, in addition to an educational resource for SAS data analysis and modeling., Published by MDPI, Basel

Small-angle X-ray scattering (SAXS) analysis of biomolecules is increasingly common with a constantly high demand for comprehensive and efficient sample quality control prior to SAXS experiments. As monodisperse sample suspensions are desirable for SAXS experiments, latest dynamic light scattering (DLS) techniques are most suited to obtain non-invasive and rapid information about the particle size distribution of molecules in solution. A multi-receiver four-channel DLS system was designed and adapted at the BioSAXS endstation of the EMBL beamline P12 at PETRA III (DESY, Hamburg, Germany). The system allows the collection of DLS data within round-shaped sample capillaries used at beamline P12. Data obtained provide information about the hydrodynamic radius of biological particles in solution and dispersity of the solution. DLS data can be collected directly prior to and during an X-ray exposure. To match the short X-ray exposure times of around 1 s for 20 exposures at P12, the DLS data collection periods that have been used up to now of 20 s or commonly more were substantially reduced, using a novel multi-channel approach collecting DLS data sets in the SAXS sample capillary at four different neighbouring sample volume positions in parallel. The setup allows online scoring of sample solutions applied for SAXS experiments, supports SAXS data evaluation and for example indicates local inhomogeneities in a sample solution in a time-efficient manner. Biological macromolecules with different molecular weights were applied to test the system and obtain information about the performance. All measured hydrodynamic radii are in good agreement with DLS results obtained by employing a standard cuvette instrument. Moreover, applying the new multi-channel DLS setup, a reliable radius determination of sample solutions in flow, at flow rates normally used for size-exclusion chromatography–SAXS experiments, and at higher flow rates, was verified as well. This study also shows and confirms that the newly designed sample compartment with attached DLS instrumentation does not disturb SAXS measurements.

Structure 28(3), 348 - 354.e3 (2020). doi:10.1016/j.str.2019.12.001, Recent structures of full-length ATP-binding cassette (ABC) transporter MsbA in different states indicate large conformational changes during the reaction cycle that involve transient dimerization of its nucleotide-binding domains (NBDs). However, a detailed molecular understanding of the structural changes and associated kinetics of MsbA upon ATP binding and hydrolysis is still missing. Here, we employed time-resolved small-angle X-ray scattering, initiated by stopped-flow mixing, to investigate the kinetics and accompanying structural changes of NBD dimerization (upon ATP binding) and subsequent dissociation (upon ATP hydrolysis) in the context of isolated NBDs as well as full-length MsbA in lipid nanodiscs. Our data allowed us to structurally characterize the major states involved in the process and determine time constants for NBD dimerization and dissociation. In the full-length protein, these structural transitions occur on much faster time scales, indicating close-proximity effects and structural coupling of the transmembrane domains with the NBDs., Published by Cell Press, Cambridge, Mass.

Biomaterials 192, 612 - 620 (2019). doi:10.1016/j.biomaterials.2018.10.020, mRNA pharmaceuticals represent a new class of therapeutics, with applications, in cancer vaccination, tumour therapy and protein substitution. Formulations are required to deliver messenger RNA (mRNA) to the target sites where induction of genetic transfection following receptor mediated cell uptake & translation is required.In the current study, the cationic polysaccharide diethylaminoethylen (DEAE) – Dextran was selected as a model system carrier for the investigation of polyplex nanoparticle formation together with mRNA as a function of the molar ratio of the components. The structure of the mRNA/Dextran colloids was investigated as a function of the polymer-to-mRNA ratio and correlated with the biological activity determined by cellular transfection with luciferase coding mRNA. Dynamic light scattering (DLS), small angle x-ray scattering (SAXS), and small angle neutron scattering (SANS) with deuterium contrast variation were used to achieve structural insight into the systems. Similarly to previously investigated lipid based systems, colloidally stable particles with confined size were obtained with either excess of positive or negative charge. Highest activity was obtained with positive charge excess. From the scattering experiments information on the internal organization inside the polymer/mRNA systems was derived. Indication for the presence of structural elements in the length scale of ten to 20 nm were found in the excess of dextran, which could be due to either excess or particulate polymer. Information on the molecular organization of the mRNA nanoparticle products may provide a valuable basis for defining critical quality attributes of drug products for pharmaceutical application., Published by Elsevier Science, Amsterdam [u.a.]

Lab on a chip 16(7), 1161 - 1170(2016). doi:10.1039/C5LC01580D, We present a centrifugal microfluidic LabDisk for protein structure analysis via small-angle X-ray scattering (SAXS) on synchrotron beamlines. One LabDisk prepares 120 different measurement conditions, grouped into six dilution matrices. Each dilution matrix: (1) features automatic generation of 20 different measurement conditions from three input liquids and (2) requires only 2.5 μl of protein solution, which corresponds to a tenfold reduction in sample volume in comparison to the state of the art. Total hands on time for preparation of 120 different measurement conditions is less than 5 min. Read-out is performed on disk within the synchrotron beamline P12 at EMBL Hamburg (PETRA III, DESY). We demonstrate: (1) aliquoting of 40 nl aliquots for five different liquids typically used in SAXS and (2) confirm fluidic performance of aliquoting, merging, mixing and read-out from SAXS experiments (2.7–4.4% CV of protein concentration). We apply the LabDisk for SAXS for basic analysis methods, such as measurement of the radius of gyration, and advanced analysis methods, such as the ab initio calculation of 3D models. The suitability of the LabDisk for SAXS for protein structure analysis under different environmental conditions is demonstrated for glucose isomerase under varying protein and NaCl concentrations. We show that the apparent radius of gyration of the negatively charged glucose isomerase decreases with increasing protein concentration at low salt concentration. At high salt concentration the radius of gyration (Rg) does not change with protein concentrations. Such experiments can be performed by a non-expert, since the LabDisk for SAXS does not require attachment of tubings or pumps and can be filled with regular pipettes. The new platform has the potential to introduce routine high-throughput SAXS screening of protein structures with minimal input volumes to the regular operation of synchrotron beamlines., Published by RSC, Cambridge

An integrated environment for biological small-angle X-ray scattering (BioSAXS) at the high-brilliance P12 synchrotron beamline of the EMBL (DESY, Hamburg) allows for a broad range of solution scattering experiments. Automated hardware and software systems have been designed to ensure that data collection and processing are efficient, streamlined and user friendly., A high-brilliance synchrotron P12 beamline of the EMBL located at the PETRA III storage ring (DESY, Hamburg) is dedicated to biological small-angle X-ray scattering (SAXS) and has been designed and optimized for scattering experiments on macromolecular solutions. Scatterless slits reduce the parasitic scattering, a custom-designed miniature active beamstop ensures accurate data normalization and the photon-counting PILATUS 2M detector enables the background-free detection of weak scattering signals. The high flux and small beam size allow for rapid experiments with exposure time down to 30–50 ms covering the resolution range from about 300 to 0.5 nm. P12 possesses a versatile and flexible sample environment system that caters for the diverse experimental needs required to study macromolecular solutions. These include an in-vacuum capillary mode for standard batch sample analyses with robotic sample delivery and for continuous-flow in-line sample purification and characterization, as well as an in-air capillary time-resolved stopped-flow setup. A novel microfluidic centrifugal mixing device (SAXS disc) is developed for a high-throughput screening mode using sub-microlitre sample volumes. Automation is a key feature of P12; it is controlled by a beamline meta server, which coordinates and schedules experiments from either standard or nonstandard operational setups. The integrated SASFLOW pipeline automatically checks for consistency, and processes and analyses the data, providing near real-time assessments of overall parameters and the generation of low-resolution models within minutes of data collection. These advances, combined with a remote access option, allow for rapid high-throughput analysis, as well as time-resolved and screening experiments for novice and expert biological SAXS users.

Using an indirect detection scheme, a small-size beamstop was developed to accurately measure X-ray beam flux in a wide energy range with reduced radiation level on the electronics and significantly increased in-beam lifetime., A small active in-vacuum beamstop has been developed to monitor the flux of intense third-generation synchrotron X-ray beams protecting the downstream detector from the direct beam. Standard active beamstops, where a built-in diode directly absorbs the beam, have limitations in size and lifetime. In the present design, a silicon PIN diode detects the photons back-scattered from a cavity in the beamstop. This approach drastically reduces the radiation dose on the diode and thus increases its lifetime. The beamstop with a diameter of 2 mm has been fabricated to meet the requirements for the P12 bioSAXS beamline of EMBL Hamburg at PETRA III (DESY). The beamstop is in regular user operation at the beamline and displays a good response over the range of energies tested (6–20 keV). Further miniaturization of the diode is easily possible as its size is not limited by the PIN diode used.

Small-angle X-ray scattering (SAXS) is a powerful method to study the structural properties of materials at the nanoscale. Recent progress in instrumentation and analysis methods has led to rapidly growing applications of this technique for the characterization of biological macromolecules in solution. Ab initio and rigid-body modeling methods allow one to build three-dimensional, low-resolution models from SAXS data. With the new approaches, oligomeric states of proteins and macromolecular complexes can be assessed, chemical equilibria and kinetic reactions can be studied, and even flexible objects such as intrinsically unfolded proteins can be quantitatively characterized. This review describes the analysis methods of SAXS data from macromolecular solutions, ranging from the computation of overall structural parameters to advanced three-dimensional modeling. The efficiency of these methods is illustrated by recent applications to biological macromolecules and nanocomposite particles.

Faraday discussions 191, 465 - 479(2016). doi:10.1039/C6FD00018E, We have developed a new class of lanthanide nano-clusters that self-assemble using flexible Schiff base ligands. Cd–Ln and Ni–Ln clusters, [Ln$_8$Cd$_{24}$(L$^1$)$_{12}$(OAc)$_{39}$Cl$_7$(OH)$_2$] (Ln = Nd, Eu), [Eu8Cd24(L1)12(OAc)44], [Ln$_8$Cd$_{24}$(L$^2$)$_{12}$(OAc)$_{44}$] (Ln = Nd, Yb, Sm) and [Nd$_2$Ni$_4$(L$^3$)$_2$(acac)$_6$(NO$_3$)$_2$(OH)$_2$], were constructed using different types of flexible Schiff base ligands. These molecular nano-clusters exhibit anisotropic architectures that differ considerably depending upon the presence of Cd (nano-drum) or Ni (square-like nano-cluster). Structural characterization of the self-assembled particles has been undertaken using crystallography, transmission electron microscopy and small-angle X-ray scattering. Comparison of the metric dimensions of the nano-drums shows a consistency of size using these techniques, suggesting that these molecules may share similar structural features in both solid and solution states. Photophysical properties were studied by excitation of the ligand-centered absorption bands in the solid state and in solution, and using confocal microscopy of microspheres loaded with the compounds. The emissive properties of these compounds vary depending upon the combination of lanthanide and Cd or Ni present in these clusters. The results provide new insights into the construction of novel high-nuclearity nano-clusters and offer a promising foundation for the development of new functional nanomaterials., Published by Royal Society of Chemistry , Cambridge [u.a.]

Acta crystallographica / D 71(1), 67 - 75(2015). doi:10.1107/S1399004714026959, Small-angle X-ray scattering (SAXS) of macromolecules in solution is in increasing demand by an ever more diverse research community, both academic and industrial. To better serve user needs, and to allow automated and high-throughput operation, a sample changer (BioSAXS Sample Changer) that is able to perform unattended measurements of up to several hundred samples per day has been developed. The Sample Changer is able to handle and expose sample volumes of down to 5 µl with a measurement/cleaning cycle of under 1 min. The samples are stored in standard 96-well plates and the data are collected in a vacuum-mounted capillary with automated positioning of the solution in the X-ray beam. Fast and efficient capillary cleaning avoids cross-contamination and ensures reproducibility of the measurements. Independent temperature control for the well storage and for the measurement capillary allows the samples to be kept cool while still collecting data at physiological temperatures. The Sample Changer has been installed at three major third-generation synchrotrons: on the BM29 beamline at the European Synchrotron Radiation Facility (ESRF), the P12 beamline at the PETRA-III synchrotron (EMBL@PETRA-III) and the I22/B21 beamlines at Diamond Light Source, with the latter being the first commercial unit supplied by Bruker ASC., Published by Wiley-Blackwell, Oxford

Scientific reports 5(1), 10734(2015). doi:10.1038/srep10734, Small angle X-ray scattering (SAXS), an increasingly popular method for structural analysis of biological macromolecules in solution, is often hampered by inherent sample polydispersity. We developed an all-in-one system combining in-line sample component separation with parallel biophysical and SAXS characterization of the separated components. The system coupled to an automated data analysis pipeline provides a novel tool to study difficult samples at the P12 synchrotron beamline (PETRA-3, EMBL/DESY, Hamburg)., Published by Nature Publishing Group, London

Radiation damage is the general curse of structural biologists who use synchrotron small-angle X-ray scattering (SAXS) to investigate biological macromolecules in solution. The EMBL-P12 biological SAXS beamline located at the PETRAIII storage ring (DESY, Hamburg, Germany) caters to an extensive user community who integrate SAXS into their diverse structural biology programs. The high brilliance of the beamline [5.1 × 1012 photons s−1, 10 keV, 500 (H) µm × 250 (V) µm beam size at the sample position], combined with automated sample handling and data acquisition protocols, enable the high-throughput structural characterization of macromolecules in solution. However, considering the often-significant resources users invest to prepare samples, it is crucial that simple and effective protocols are in place to limit the effects of radiation damage once it has been detected. Here various practical approaches are evaluated that users can implement to limit radiation damage at the P12 beamline to maximize the chances of collecting quality data from radiation sensitive samples.

Multifunctional enzyme type 2 (MFE-2) forms part of the fatty acid β-oxidation pathway in peroxisomes. MFE-2s from various species reveal proteins with structurally homologous functional domains assembled in different compilations. Crystal structures of all domain types are known. SAXS data from human, fruit fly and Caenorhabditis elegans MFE-2s and their constituent domains were collected, and both ab initio and rigid body models constructed. Location of the putative substrate binding helper domain SCP-2L (sterol carrier protein 2-like), which is not part of MFE-2 protein in every species and not seen as part of any previous MFE-2 structures, was determined. The obtained models of human and C. elegans MFE-2 lend a direct structural support to the idea of the biological role of SCP-2L.Structured summary of protein interactionsHsMFE-2 and HsMFE-2 bind by x ray scattering (View interaction)CeMFE-2 and CeMFE-2 bind by x ray scattering (View interaction)DmMFE-2 and DmMFE-2 bind by x ray scattering (View interaction)

Acta crystallographica / A 72(a1), s14 - s14(2016). doi:10.1107/S2053273316099770, Published by Blackwell, Oxford [u.a.]

A setup is presented for automated high-throughput measurements of small-angle X-ray scattering (SAXS) from macromolecular solutions on the bending-magnet beamline X33 of EMBL at the storage ring DORIS-III (DESY, Hamburg). A new multi-cell compartment allows for rapid switching between in-vacuum and in-air operation, for digital camera assisted control of cell filling and for colour sample illumination. The beamline is equipped with a Pilatus 1 M-W pixel detector for SAXS and a Pilatus 300 k-W for wide-angle scattering (WAXS), and results from the use of the Pilatus detectors for scattering studies are reported. The setup provides a broad resolution range from 100 to 0.36 nm without the necessity of changing the sample-to-detector distance. A new optimized robotic sample changer is installed, permitting rapid and reliable automated sample loading and cell cleaning with a required sample volume of 40 µl. All the devices are fully integrated into the beamline control software system, ensuring fully automated and user-friendly operation (attended, unattended and remote) with a throughput of up to 15 measurements per hour.

Structure 21(8), 1350 - 1360 (2013). doi:10.1016/j.str.2013.05.015, Proteins carrying nuclear export signals cooperatively assemble with the export factor CRM1 and the effector protein RanGTP. In lower eukaryotes, this cooperativity is coupled to CRM1 conformational changes; however, it is unknown if mammalian CRM1 maintains its compact conformation or shows similar structural flexibility. Here, combinations of small-angle X-ray solution scattering and electron microscopy experiments with molecular dynamics simulations reveal pronounced conformational flexibility in mammalian CRM1 and demonstrate that RanGTP binding induces association of its N- and C-terminal regions to form a toroid structure. The CRM1 toroid is stabilized mainly by local interactions between the terminal regions, rather than by global strain. The CRM1 acidic loop is key in transmitting the effect of this RanGTP-induced global conformational change to the NES-binding cleft by shifting its population to the open state, which displays enhanced cargo affinity. Cooperative CRM1 export complex assembly thus constitutes a highly dynamic process, encompassing an intricate interplay of global and local structural changes., Published by Elsevier Science, London [u.a.]