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

Abstract 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).

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

Journal of Synchrotron Radiation ◽

10.1107/s1600577515000375 ◽

2015 ◽

Vol 22 (2) ◽

pp. 273-279 ◽

Cited By ~ 68

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.

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Further application of size-exclusion chromatography combined with small-angle X-ray scattering optics for characterization of biological macromolecules

Analytical and Bioanalytical Chemistry ◽

10.1007/s00216-010-4140-7 ◽

2010 ◽

Vol 399 (4) ◽

pp. 1449-1453 ◽

Cited By ~ 9

Author(s):

Yasushi Watanabe ◽

Yoji Inoko

Keyword(s):

Size Exclusion Chromatography ◽

Small Angle ◽

Size Exclusion ◽

Biological Macromolecules ◽

X Ray ◽

X Ray Scattering ◽

Exclusion Chromatography ◽

Ray Scattering

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A microfabricated fixed path length silicon sample holder improves background subtraction for cryoSAXS

Journal of Applied Crystallography ◽

10.1107/s1600576714027782 ◽

2015 ◽

Vol 48 (1) ◽

pp. 227-237 ◽

Cited By ~ 3

Author(s):

Jesse B. Hopkins ◽

Andrea M. Katz ◽

Steve P. Meisburger ◽

Matthew A. Warkentin ◽

Robert E. Thorne ◽

...

Keyword(s):

Radiation Damage ◽

Background Subtraction ◽

Path Length ◽

Sample Volume ◽

Biological Macromolecules ◽

Silicon Sample ◽

X Ray ◽

X Ray Scattering ◽

Fixed Path

The application of small-angle X-ray scattering (SAXS) for high-throughput characterization of biological macromolecules in solution is limited by radiation damage. By cryocooling samples, radiation damage and required sample volumes can be reduced by orders of magnitude. However, the challenges of reproducibly creating the identically sized vitrified samples necessary for conventional background subtraction limit the widespread adoption of this method. Fixed path length silicon sample holders for cryoSAXS have been microfabricated to address these challenges. They have low background scattering and X-ray absorption, require only 640 nl of sample, and allow reproducible sample cooling. Data collected in the sample holders from a nominal illuminated sample volume of 2.5 nl are reproducible down toq≃ 0.02 Å−1, agree with previous cryoSAXS work and are of sufficient quality for reconstructions that match measured crystal structures. These sample holders thus allow faster, more routine cryoSAXS data collection. Additional development is required to reduce sample fracturing and improve data quality at lowq.

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Upgrade of MacCHESS facility for X-ray scattering of biological macromolecules in solution

Journal of Synchrotron Radiation ◽

10.1107/s1600577514020360 ◽

2015 ◽

Vol 22 (1) ◽

pp. 180-186 ◽

Cited By ~ 40

Author(s):

Alvin Samuel Acerbo ◽

Michael J. Cook ◽

Richard Edward Gillilan

Keyword(s):

Scattering Data ◽

Biological Macromolecules ◽

Wide Angle ◽

X Ray ◽

Pristine Sample ◽

X Ray Scattering ◽

Angle Scattering ◽

Order Of Magnitude ◽

Ray Scattering

X-ray scattering of biological macromolecules in solution is an increasingly popular tool for structural biology and benefits greatly from modern high-brightness synchrotron sources. The upgraded MacCHESS BioSAXS station is now located at the 49-pole wiggler beamline G1. The 20-fold improved flux over the previous beamline F2 provides higher sample throughput and autonomous X-ray scattering data collection using a unique SAXS/WAXS dual detectors configuration. This setup achieves a combinedq-range from 0.007 to 0.7 Å−1, enabling better characterization of smaller molecules, while opening opportunities for emerging wide-angle scattering methods. In addition, a facility upgrade of the positron storage ring to continuous top-up mode has improved beam stability and eliminated beam drift over the course of typical BioSAXS experiments. Single exposure times have been reduced to 2 s for 3.560 mg ml−1lysozyme with an average quality factorI/σ of 20 in the Guinier region. A novel disposable plastic sample cell design that incorporates lower background X-ray window material provides users with a more pristine sample environment than previously available. Systematic comparisons of common X-ray window materials bonded to the cell have also been extended to the wide-angle regime, offering new insight into best choices for variousq-space ranges. In addition, a quantitative assessment of signal-to-noise levels has been performed on the station to allow users to estimate necessary exposure times for obtaining usable signals in the Guinier regime. Users also have access to a new BioSAXS sample preparation laboratory which houses essential wet-chemistry equipment and biophysical instrumentation. User experiments at the upgraded BioSAXS station have been on-going since commissioning of the beamline in Summer 2013. A planned upgrade of the G1 insertion device to an undulator for the Winter 2014 cycle is expected to further improve flux by an order of magnitude.

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Analysis Strategies for MHz XPCS at the European XFEL

Applied Sciences ◽

10.3390/app11178037 ◽

2021 ◽

Vol 11 (17) ◽

pp. 8037

Author(s):

Francesco Dallari ◽

Mario Reiser ◽

Irina Lokteva ◽

Avni Jain ◽

Johannes Möller ◽

...

Keyword(s):

New Technologies ◽

Soft Condensed Matter ◽

Pixel Detector ◽

Analysis Pipeline ◽

X Ray ◽

X Ray Scattering ◽

Analysis Strategies ◽

Scientific Questions ◽

Sophisticated Analysis ◽

Data Analysis Pipeline

The nanometer length-scale holds precious information on several dynamical processes that develop from picoseconds to seconds. In the past decades, X-ray scattering techniques have been developed to probe the dynamics at such length-scales on either ultrafast (sub-nanosecond) or slow ((milli-)second) time scales. With the start of operation of the European XFEL, thanks to the MHz repetition rate of its X-ray pulses, even the intermediate μs range have become accessible. Measuring dynamics on such fast timescales requires the development of new technologies such as the Adaptive Gain Integrating Pixel Detector (AGIPD). μs-XPCS is a promising technique to answer many scientific questions regarding microscopic structural dynamics, especially for soft condensed matter systems. However, obtaining reliable results with complex detectors at free-electron laser facilities is challenging and requires more sophisticated analysis methods compared to experiments at storage rings. Here, we discuss challenges and possible solutions to perform XPCS experiments with the AGIPD at European XFEL; in particular, at the Materials Imaging and Dynamics (MID) instrument. We present our data analysis pipeline and benchmark the results obtained at the MID instrument with a well-known sample composed by silica nanoparticles dispersed in water.

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Small angle X-ray scattering study of porosity variation in a styrene-divinylbenzene copolymer

Collection of Czechoslovak Chemical Communications ◽

10.1135/cccc19811675 ◽

1981 ◽

Vol 46 (7) ◽

pp. 1675-1681 ◽

Cited By ~ 13

Author(s):

Josef Baldrian ◽

Božena N. Kolarz ◽

Henrik Galina

Keyword(s):

Small Angle ◽

Structural Parameters ◽

Two Phase ◽

X Ray ◽

Porosity Variation ◽

Swelling Agent ◽

X Ray Scattering ◽

Styrene Divinylbenzene ◽

Ray Scattering

Porosity variations induced by swelling agent exchange were studied in a styrene-divinylbenzene copolymer. Standard methods were used in the characterization of copolymer porosity in the dry state and the results were compared with related structural parameters derived from small angle X-ray scattering (SAXS) measurements as developed for the characterization of two-phase systems. The SAXS method was also used for porosity determination in swollen samples. The differences in the porosity of dry samples were found to be an effect of the drying process, while in the swollen state the sample swells and deswells isotropically.

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