Clement E. Blanchet

Versatile sample environments and automation for biological solution X-ray scattering experiments at the P12 beamline (PETRA III, DESY)

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.

Small-Angle X-Ray Scattering on Biological Macromolecules and Nanocomposites in Solution

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.

BioSAXS Sample Changer: a robotic sample changer for rapid and reliable high-throughput X-ray solution scattering experiments

A robotic sample changer for solution X-ray scattering experiments optimized for speed and to use the minimum amount of material has been developed. This system is now in routine use at three high-brilliance European synchrotron sites, each capable of several hundred measurements per day.

Instrumental setup for high-throughput small- and wide-angle solution scattering at the X33 beamline of EMBL Hamburg

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.

Limiting radiation damage for high‐brilliance biological solution scattering: practical experience at the EMBL P12 beamline PETRAIII

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 × 10(12) 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.

Automated Pipeline for Purification, Biophysical and X-Ray Analysis of Biomacromolecular Solutions

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).

A small and robust active beamstop for scattering experiments on high-brilliance undulator beamlines

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.

Quaternary structure of human, Drosophila melanogaster and Caenorhabditis elegans MFE‐2 in solution from synchrotron small‐angle X‐ray scattering

DmMFE‐2 and DmMFE‐2 bind by x ray scattering (View interaction)

Multi-channel in situ dynamic light scattering instrumentation enhancing biological small-angle X-ray scattering experiments at the PETRA III beamline P12

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.

Study and mitigation of radiation damage on the P12 BioSAXS beamline

With the progress in X-ray sources and increasing X-ray beam flux, radiation damage is a major challenge for biological SAXS measurements on modern synchrotron beamlines. In macromolecular solutions, radiation damage occurs mostly indirectly. Water molecules are dissociated into hydrogenand hydroxyl-radicals by the X-ray beam. In turn, these free radicals oxidise the macromolecules, which eventually start to aggregate. Since the SAXS intensity scales with the square of the particle size, a few large aggregates may quickly spoil the scattering pattern of the intact molecules. The high brilliance P12 BioSAXS beamline at Petra-3 storage ring (DESY, Hamburg) [1], dedicated and optimised for solution scattering, delivers 5*10^12 photons/s in a 120*200 μm^2 beam (full width half maximum) at the sample position. With this flux, aggregates may be rapidly formed and the data become unusable after exposure of a few tens of ms. Different approaches have been explored to reduce radiation damage [2]: attenuation of the beam, “in-flow” sample measurement, addition of free radicals scavengers or additives that stabilizes proteins and prevent their aggregation. All these methods help to reduce the damage, but all have their limits, and their action can vary depending on the nature of the sample. The “in-flow” measurement, where fresh sample is continuously flowed in the beam path during the exposure, is routinely used at many beamlines and may be the most versatile approach using no additives. On the downside, more sample is required compared to the measurement on a fixed sample. To improve the efficiency of in-flow measurements, radiation sensitive samples were measured in capillaries of different diameter (Schreoer et al., 2017, in preparation). SAXS cells are typically dimensioned such that the signal of aqueous sample is maximised, while the limited lifetime of the sample in the beam and the sample consumption are sometimes ignored. Using smaller capillaries (e.g. 1 mm instead of 2 mm diameter), the scattering signal is still decent and can be detected on low background beamline, but the sample volume required drops significantly (with the square of the diameter). In practice, for a given sample volume, in-flow measurement in small capillaries leads to less noisy data compared to the standard capillaries. This exploration of radiation damage is particularly important in the context of high flux operations on the P12 beamline (Blanchet et al., 2017, in preparation) using the recently commissioned double multilayer monochromator. With the intense flux of 4*10^14 photons/s in 85 x 285 μm^2 at the sample position delivered in this setup, proteins aggregate in a couple of ms and macroscopic effects such as bubble formation and very large aggregates are present in less than a second of exposure. Proper characterization and mitigation of radiation damage are required to make optimal use of this powerful beam.