The XFP (17-BM) beamline for X-ray footprinting at NSLS-II

Abstract

Hydroxyl-radical mediated synchrotron X-ray footprinting (XF) is a powerful solution-state technique in structural biology for the study of macromolecular structure and dynamics of proteins and nucleic acids, with several synchrotron resources available to serve the XF community worldwide. The XFP (Biological X-ray Footprinting) beamline at the NSLS-II was constructed on a three-pole wiggler source at 17-BM to serve as the premier beamline for performing this technique, providing an unparalleled combination of high flux density broadband beam, flexibility in beam morphology, and sample handling capabilities specifically designed for XF experiments. The details of beamline design, beam measurements, and science commissioning results for a standard protein using the two distinct XFP endstations are presented here. XFP took first light in 2016 and is now available for general user operations through peer-reviewed proposals. Currently, beam sizes from 450 µm × 120 µm to 2.7 mm × 2.7 mm (FWHM) are available, with a flux of 1.6 × 1016 photons s−1 (measured at 325 mA ring current) in a broadband (∼5–16 keV) beam. This flux is expected to rise to 2.5 × 1016 photons s−1 at the full NSLS-II design current of 500 mA, providing an incident power density of >500 W mm−2 at full focus.

New high-throughput endstation to accelerate the experimental optimization pipeline for synchrotron X-ray footprinting

“…Indeed, Alexa decay rates could only be reliably determined for concentrations <5 mM under our conditions, a level at which buffering capacity is low and the system is increasingly susceptible to undesirable pH perturbation. For systems where non-preferred buffers such as HEPES must absolutely be used, a considerably higher flux density and thus radical dose is required, such as can be obtained at the High Dose Endstation of the XFP beamline using a capillary flow device (Asuru et al, 2019).…”

“…A thorough understanding of the effects and influence of the sample matrix as well as fully characterized, flexible, and reliable beamline instrumentation are essential for constructive results. To this end, we recently described the design and capabilities of the X-ray Footprinting of Biological Materials (XFP) beamline located at the National Synchrotron Light Source II (NSLS-II, Brookhaven National Laboratory, New York, USA), which was developed to provide access to the synchrotron XF technology for the structural biology research community (Asuru et al, 2019).…”

“…The XFP beamline can deliver ca. 10 16 photons s À1 ($70 W power) of pink-beam X-ray radiation over a 4.5-16 keV energy range to two experimental endstations that differ in their achievable flux density and thus X-ray dose (Asuru et al, 2019). The first endstation uses a capillary flow device to flow samples through the X-ray beam, with focused beam sizes as small as 120 mm  450 mm [full width at half-maximum (FWHM), V  H], capable of delivering up to 500 W mm À2 power densities to the sample (at 500 mA NSLS-II ring current).…”

A coded aperture microscope for X-ray fluorescence full-field imaging

“…We used the XFP beamline at NSLS-II (Asuru et al, 2019) to demonstrate that the image processing method would also work on extended and fluorescing samples. XFP can operate in a pink beam mode with a partially focused spot of roughly 2 mm  2 mm.…”

Hydroxyl radical mediated damage of proteins in low oxygen solution investigated using X-ray footprinting mass spectrometry

“…Another advantage of OH footprinting over methods such as DNase footprinting or chemical footprinting methods is the small size of the hydroxyl radical, which permits finer structural features to be resolved in nucleic acids or proteins. Two national XFMS synchrotron beamlines are now in operation (Asuru et al, 2019;Gupta et al, 2014), and XFMS has several experimental aspects that distinguish it from the more well known X-ray protein structural methods, such as macromolecular crystallography (MX) and small angle X-ray scattering (SAXS).…”