High-Dose Endstation

The first XFP endstation, termed the High-Dose Endstation, is centered at 28m from the source, at the 1:1 focus of the beamline’s front-end toroidal focusing mirror. At this location, beam sizes as small as 100 μm x 400 μm (vert x horz., FWHM) can be achieved, permitting very high flux densities and beam powers to be delivered to the sample (photon flux ~1016 photons/sec, maximum power of 500 W/mm2 at 500mA NSLS-II ring current). This endstation is well-suited for X-ray footprinting of highly scavenging systems requiring an intense hydroxyl radical pulse, such as membrane proteins, large multi-component protein complexes, and investigations under in vivo conditions. Most experiments are conducted by flowing samples through Kapton-coated glass capillaries (200 μm and 530 μm IDs standard), with the cross-section of the beam and the flow rate defining X-ray exposure times.

The centerpiece of this endstation is a flexible three-axis motorized table that can support a wide variety of apparatus (Figure 1). Standard configurations available for user experiments include a continuous flow pumping scheme with incubator and fraction collector for in vivo footprinting (Figure 2), and a capillary flow setup using a syringe pump for sample delivery (Figure 3) for equilibrium measurements at high X-ray/radical doses. Non-standard endstation setups provided by users can also be accommodated at this location, following discussion with beamline staff.

For details of the performance of XFP at the high-dose endstation, see our recent beamline paper (Asuru et al., J. Synch. Rad. 2019, 26, 1388-1399. DOI: 10.1107/S1600577519003576). Protocols for performing in vivo footprinting experiments can be found in a recent article from Sarah Woodson’s group at Johns Hopkins University (Hao et al., Current Protocols in Nucleic Acid Chemistry, 2018, e52. DOI: 10.1002/cpnc.52).

View of the blue 3-axis table on XFP, with a large breadboard on top for holding equipment and samples.

Figure 1. Three axis motorized experimental table for the High-Dose Endstation. This flexible table is equipped with a 36 in. square breadboard and can handle several hundred lbs of equipment.  It features a changeable sample platform, 4 lift jacks, a Z translator, and an X translator.

the in vivo footprinting setup is mounted on top of the sample table, just downstream of the exit window.

Figure 2. In vivo footprinting setup. The VICI M50 continuous flow pump moves sample from the heated sample incubator through a glass capillary intersecting X-ray beam in the water-cooled capillary holder, and then to a fraction collector (hidden). The capillary can be precisely aligned into the beam using a motorized XY stage and a diamond screen based beam alignment camera.

the standard capillary flow setup has a capillary centered in the beam for exposing flowing samples at short exposure times.

Figure 3. Conventional capillary flow setup with water-cooled capillary holder using a Harvard syringe pump for sample delivery and a small sample collection stage for exposed samples. Several capillary diameters are available; most experiments are performed using a 200 μm ID capillary permitting exposure times of 75 μs to 1 ms.

the water cooled capillary holder consists of a cooper block attached to an aluminum plate that holds two slits that fix the capillary in place.

Figure 4. Close-up view of the water-cooled capillary holder. The capillary is held between 2 stainless steel blades in thermal contact with a water-cooled copper block. The assembly is mounted to a translation stage that allows different regions of the capillary to be exposed, reducing damage caused by the X-ray beam.