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

Blanchet CE, Spilotros A, Schwemmer F, Graewert MA, Kikhney A, Jeffries CM, Franke D, Mark D, Zengerle R, Cipriani F, Fiedler S, Roessle M, Svergun DI - J Appl Crystallogr (2015)

Bottom Line: 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.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.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: European Molecular Biology Laboratory, Hamburg Outstation , Notkestrasse 85, Hamburg, 22603, Germany.

ABSTRACT

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.

No MeSH data available.


Related in: MedlinePlus

Schematic representation of the major elements of the P12 beamline. The distances of the main components from the undulator source are indicated in blue.
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fig1: Schematic representation of the major elements of the P12 beamline. The distances of the main components from the undulator source are indicated in blue.

Mentions: Every component in contact with the beam (the sample cell, windows, optical elements, slits and air) may scatter X-rays that will contribute to the instrument background. As biological macromolecular SAXS is contingent on the accurate subtraction of all background scattering contributions, reducing the effects of parasitic scattering is vital not only to improve signal-to-noise ratios in the data but also to remove sources of scattering that may perturb the extraction of structural parameters. The instrument background at P12 was originally reduced by a standard three-paired-slits collimator system (slits 2, 3 and 4 in Fig. 1 ▶), where (a) slits 2 define the beam and adjust the flux, (b) slits 3 cut out the parasitic scattering fan coming from the optics and passing through the first pair of slits, and (c) the last pair of slits (slits 4) remove the weaker secondary scattering produced by the second pair of slits. However, the last pair of guard slits can still produce strong parasitic scattering, especially at low angles (Fig. 2 ▶). To further reduce this background the last pair of guard slits has been replaced by hybrid scatterless slits. These are composed of heavy metal blades with the monocrystal lateral edges oriented far from any Bragg peak position with respect to the incident beam (Li et al., 2008 ▶). The scatterless slits installed at P12 (from Xenocs, Sassenage) can sustain a higher radiation load, compared with regular guard slits, without producing significant parasitic scattering and they significantly reduce the overall instrument background (Fig. 2 ▶).


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

Blanchet CE, Spilotros A, Schwemmer F, Graewert MA, Kikhney A, Jeffries CM, Franke D, Mark D, Zengerle R, Cipriani F, Fiedler S, Roessle M, Svergun DI - J Appl Crystallogr (2015)

Schematic representation of the major elements of the P12 beamline. The distances of the main components from the undulator source are indicated in blue.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4379436&req=5

fig1: Schematic representation of the major elements of the P12 beamline. The distances of the main components from the undulator source are indicated in blue.
Mentions: Every component in contact with the beam (the sample cell, windows, optical elements, slits and air) may scatter X-rays that will contribute to the instrument background. As biological macromolecular SAXS is contingent on the accurate subtraction of all background scattering contributions, reducing the effects of parasitic scattering is vital not only to improve signal-to-noise ratios in the data but also to remove sources of scattering that may perturb the extraction of structural parameters. The instrument background at P12 was originally reduced by a standard three-paired-slits collimator system (slits 2, 3 and 4 in Fig. 1 ▶), where (a) slits 2 define the beam and adjust the flux, (b) slits 3 cut out the parasitic scattering fan coming from the optics and passing through the first pair of slits, and (c) the last pair of slits (slits 4) remove the weaker secondary scattering produced by the second pair of slits. However, the last pair of guard slits can still produce strong parasitic scattering, especially at low angles (Fig. 2 ▶). To further reduce this background the last pair of guard slits has been replaced by hybrid scatterless slits. These are composed of heavy metal blades with the monocrystal lateral edges oriented far from any Bragg peak position with respect to the incident beam (Li et al., 2008 ▶). The scatterless slits installed at P12 (from Xenocs, Sassenage) can sustain a higher radiation load, compared with regular guard slits, without producing significant parasitic scattering and they significantly reduce the overall instrument background (Fig. 2 ▶).

Bottom Line: 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.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.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: European Molecular Biology Laboratory, Hamburg Outstation , Notkestrasse 85, Hamburg, 22603, Germany.

ABSTRACT

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.

No MeSH data available.


Related in: MedlinePlus