scholarly journals Automated sample-changing robot for solution scattering experiments at the EMBL Hamburg SAXS station X33

2008 ◽  
Vol 41 (5) ◽  
pp. 913-917 ◽  
Author(s):  
A. R. Round ◽  
D. Franke ◽  
S. Moritz ◽  
R. Huchler ◽  
M. Fritsche ◽  
...  
Keyword(s):  
Large Scale ◽  
Ease Of Use ◽  
Protein Solutions ◽  
Biological Macromolecules ◽  
Control Software ◽  
Automated Data Collection ◽  
Client Server ◽  
Neutron Sources ◽  
X Ray ◽  
X Ray Scattering

There is a rapidly increasing interest in the use of synchrotron small-angle X-ray scattering (SAXS) for large-scale studies of biological macromolecules in solution, and this requires an adequate means of automating the experiment. A prototype has been developed of an automated sample changer for solution SAXS, where the solutions are kept in thermostatically controlled well plates allowing for operation with up to 192 samples. The measuring protocol involves controlled loading of protein solutions and matching buffers, followed by cleaning and drying of the cell between measurements. The system was installed and tested at the X33 beamline of the EMBL, at the storage ring DORIS-III (DESY, Hamburg), where it was used by over 50 external groups during 2007. At X33, a throughput of approximately 12 samples per hour, with a failure rate of sample loading of less than 0.5%, was observed. The feedback from users indicates that the ease of use and reliability of the user operation at the beamline were greatly improved compared with the manual filling mode. The changer is controlled by a client–server-based network protocol, locally and remotely. During the testing phase, the changer was operated in an attended mode to assess its reliability and convenience. Full integration with the beamline control software, allowing for automated data collection of all samples loaded into the machine with remote control from the user, is presently being implemented. The approach reported is not limited to synchrotron-based SAXS but can also be used on laboratory and neutron sources.

scholarly journals Smaller capillaries improve the small-angle X-ray scattering signal and sample consumption for biomacromolecular solutions

2018 ◽  
Vol 25 (4) ◽  
pp. 1113-1122 ◽  
Author(s):  
Martin A. Schroer ◽  
Clement E. Blanchet ◽  
Andrey Yu. Gruzinov ◽  
Melissa A. Gräwert ◽  
Martha E. Brennich ◽  
...  
Keyword(s):  
Small Angle ◽  
A Priori ◽  
Protein Solutions ◽  
Biological Macromolecules ◽  
Saxs Data ◽  
X Ray ◽  
X Ray Scattering ◽  
A Cell ◽  
Flow Through ◽  
Ray Scattering

Radiation damage by intense X-ray beams at modern synchrotron facilities is one of the major complications for biological small-angle X-ray scattering (SAXS) investigations of macromolecules in solution. To limit the damage, samples are typically measured under a laminar flow through a cell (typically a capillary) such that fresh solution is continuously exposed to the beam during measurement. The diameter of the capillary that optimizes the scattering-to-absorption ratio at a given X-ray wavelength can be calculated a priori based on fundamental physical properties. However, these well established scattering and absorption principles do not take into account the radiation susceptibility of the sample or the often very limited amounts of precious biological material available for an experiment. Here it is shown that, for biological solution SAXS, capillaries with smaller diameters than those calculated from simple scattering/absorption criteria allow for a better utilization of the available volumes of radiation-sensitive samples. This is demonstrated by comparing two capillary diameters d i (d i = 1.7 mm, close to optimal for 10 keV; and d i = 0.9 mm, which is nominally sub-optimal) applied to study different protein solutions at various flow rates. The use of the smaller capillaries ultimately allows one to collect higher-quality SAXS data from the limited amounts of purified biological macromolecules.

scholarly journals ASAXS measurements on ferritin and apoferritin at the bioSAXS beamline P12 (PETRA III, DESY)

2021 ◽  
Vol 54 (3) ◽  
Author(s):  
D. C. F. Wieland ◽  
M. A. Schroer ◽  
A. Yu. Gruzinov ◽  
C. E. Blanchet ◽  
C. M. Jeffries ◽  
...  
Keyword(s):  
Small Angle ◽  
Protein Solutions ◽  
Anomalous Scattering ◽  
Biological Macromolecules ◽  
X Ray ◽  
Protein Cages ◽  
Additional Information ◽  
X Ray Scattering ◽  
Structural Details ◽  
Ray Scattering

Small-angle X-ray scattering is widely utilized to study biological macromolecules 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.

scholarly journals REGALS: a general method to deconvolve X-ray scattering data from evolving mixtures

2020 ◽  
Author(s):  
Steve P. Meisburger ◽  
Da Xu ◽  
Nozomi Ando
Keyword(s):  
A Priori ◽  
Scattering Data ◽  
Biological Macromolecules ◽  
Time Resolved ◽  
X Ray ◽  
X Ray Scattering ◽  
Open Source Software Package ◽  
Saxs Analysis ◽  
Structural Methods ◽  
Ray Scattering

AbstractMixtures of biological macromolecules are inherently difficult to study using structural methods, as increasing complexity presents new challenges for data analysis. Recently, there has been growing interest in studying evolving mixtures using small-angle X-ray scattering (SAXS) in conjunction with time-resolved, high-throughput, or chromatography-coupled setups. Deconvolution and interpretation of the resulting datasets, however, are nontrivial when neither the scattering components nor the way in which they evolve are known a priori. To address this issue, we introduce the REGALS method (REGularized Alternating Least Squares), which incorporates simple expectations about the data as prior knowledge and utilizes parameterization and regularization to provide robust deconvolution solutions. The restraints used by REGALS are general properties such as smoothness of profiles and maximum dimensions of species, which makes it well-suited for exploring datasets with unknown species. Here we apply REGALS to analyze experimental data from four types of SAXS experiment: anion-exchange (AEX) coupled SAXS, ligand titration, time-resolved mixing, and time-resolved temperature jump. Based on its performance with these challenging datasets, we anticipate that REGALS will be a valuable addition to the SAXS analysis toolkit and enable new experiments. The software is implemented in both MATLAB and python and is available freely as an open-source software package.

scholarly journals The new NCPSS BL19U2 beamline at the SSRF for small-angle X-ray scattering from biological macromolecules in solution

2016 ◽  
Vol 49 (5) ◽  
pp. 1428-1432 ◽  
Author(s):  
Na Li ◽  
Xiuhong Li ◽  
Yuzhu Wang ◽  
Guangfeng Liu ◽  
Ping Zhou ◽  
...  
Keyword(s):  
Data Processing ◽  
Small Angle ◽  
Dynamic Range ◽  
Silicon Crystal ◽  
High Dynamic Range ◽  
Biological Macromolecules ◽  
Shanghai Synchrotron Radiation Facility ◽  
X Ray ◽  
X Ray Scattering ◽  
Ray Scattering

The beamline BL19U2 is located in the Shanghai Synchrotron Radiation Facility (SSRF) and is its first beamline dedicated to biological material small-angle X-ray scattering (BioSAXS). The electrons come from an undulator which can provide high brilliance for the BL19U2 end stations. A double flat silicon crystal (111) monochromator is used in BL19U2, with a tunable monochromatic photon energy ranging from 7 to 15 keV. To meet the rapidly growing demands of crystallographers, biochemists and structural biologists, the BioSAXS beamline allows manual and automatic sample loading/unloading. A Pilatus 1M detector (Dectris) is employed for data collection, characterized by a high dynamic range and a short readout time. The highly automated data processing pipeline SASFLOW was integrated into BL19U2, with help from the BioSAXS group of the European Molecular Biology Laboratory (EMBL, Hamburg), which provides a user-friendly interface for data processing. The BL19U2 beamline was officially opened to users in March 2015. To date, feedback from users has been positive and the number of experimental proposals at BL19U2 is increasing. A description of the new BioSAXS beamline and the setup characteristics is given, together with examples of data obtained.

The nature of the scattering tail in Cu–Ni–Fe and Invar alloys investigated by anomalous small-angle X-ray scattering

1991 ◽  
Vol 24 (6) ◽  
pp. 1027-1034 ◽  
Author(s):  
J. P. Simon ◽  
O. Lyon
Keyword(s):  
Small Angle ◽  
Large Scale ◽  
Concentration Fluctuations ◽  
X Ray ◽  
Invar Alloys ◽  
X Ray Scattering ◽  
Angle Measurements ◽  
Two Systems ◽  
Fe Alloys ◽  
Ray Scattering

A large rapidly decreasing intensity called the `scattering tail' is generally observed at the smallest recorded angles during small-angle measurements of metallic alloys. Since this tail was interpreted as caused by a bimodal phase separation in Cu–Ni–Fe alloys and by long-wavelength concentration fluctuations in Invar alloys, these two systems were re-examined with anomalous X-ray scattering. The variation of the alloying atomic contrasts allows a discrimination between the different types of particles or defects. In neither of the two systems can the tails be interpreted as caused by large-scale concentration fluctuations. In Cu–Ni–Fe alloys, the tail is due to some kind of superficial defect (surface roughness etc.). In Invar alloys, the tail is probably due to residual impurity particles.

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

2015 ◽  
Vol 22 (2) ◽  
pp. 273-279 ◽  
Author(s):  
Cy M. Jeffries ◽  
Melissa A. Graewert ◽  
Dmitri I. Svergun ◽  
Clément E. Blanchet
Keyword(s):  
Radiation Damage ◽  
Practical Experience ◽  
Quality Data ◽  
Biological Macromolecules ◽  
Sample Handling ◽  
X Ray ◽  
X Ray Scattering ◽  
Sample Position ◽  
Effects Of Radiation

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.

Quality control of protein standards for molecular mass determinations by small-angle X-ray scattering

2010 ◽  
Vol 43 (2) ◽  
pp. 237-243 ◽  
Author(s):  
Shuji Akiyama
Keyword(s):  
Quality Control ◽  
Molecular Mass ◽  
Small Angle ◽  
Biological Macromolecules ◽  
Average Deviation ◽  
Saxs Data ◽  
X Ray ◽  
X Ray Scattering ◽  
Standard Quality ◽  
Ray Scattering

Small-angle X-ray scattering (SAXS) is a powerful technique with which to evaluate the size and shape of biological macromolecules in solution. Forward scattering intensity normalized relative to the particle concentration,I(0)/c, is useful as a good measure of molecular mass. A general method for deducing the molecular mass from SAXS data is to determine the ratio ofI(0)/cof a target protein to that of a standard protein with known molecular mass. The accuracy of this interprotein calibration is affected considerably by the monodispersity of the prepared standard, as well as by the precision in estimating its concentration. In the present study, chromatographic fractionation followed by hydrodynamic characterization is proposed as an effective procedure by which to prepare a series of monodispersed protein standards. The estimation of molecular mass within an average deviation of 8% is demonstrated using monodispersed bovine serum albumin as a standard. The present results demonstrate the importance of protein standard quality control in order to take full advantage of interprotein calibration.

Recent developments in small-angle X-ray scattering and hybrid method approaches for biomacromolecular solutions

2018 ◽  
Vol 2 (1) ◽  
pp. 69-79 ◽  
Author(s):  
Martin A. Schroer ◽  
Dmitri I. Svergun
Keyword(s):  
Small Angle ◽  
Scattering Data ◽  
Quality Data ◽  
Biological Macromolecules ◽  
Ambient Conditions ◽  
X Ray ◽  
X Ray Scattering ◽  
Analysis Methods ◽  
Recent Developments ◽  
Ray Scattering

Small-angle X-ray scattering (SAXS) has become a streamline method to characterize biological macromolecules, from small peptides to supramolecular complexes, in near-native solutions. Modern SAXS requires limited amounts of purified material, without the need for labelling, crystallization, or freezing. Dedicated beamlines at modern synchrotron sources yield high-quality data within or below several milliseconds of exposure time and are highly automated, allowing for rapid structural screening under different solutions and ambient conditions but also for time-resolved studies of biological processes. The advanced data analysis methods allow one to meaningfully interpret the scattering data from monodisperse systems, from transient complexes as well as flexible and heterogeneous systems in terms of structural models. Especially powerful are hybrid approaches utilizing SAXS with high-resolution structural techniques, but also with biochemical, biophysical, and computational methods. Here, we review the recent developments in the experimental SAXS practice and in analysis methods with a specific focus on the joint use of SAXS with complementary methods.

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