Microcrystallography, high-pressure cryocooling and BioSAXS at MacCHESS

Abstract

The Macromolecular Diffraction Facility at the Cornell High Energy Synchrotron Source (MacCHESS) is a national research resource supported by the National Center for Research Resources of the US National Institutes of Health. MacCHESS is pursuing several research initiatives designed to benefit both CHESS users and the wider structural biology community. Three initiatives are presented in further detail: microcrystallography, which aims to improve the collection of diffraction data from crystals a few micrometers across, or small well diffracting regions of inhomogeneous crystals, so as to obtain high-resolution structures; pressure cryocooling, which can stabilize transient structures and reduce lattice damage during the cooling process; and BioSAXS (small-angle X-ray scattering on biological solutions), which can extract molecular shape and other structural information from macromolecules in solution.

Figure 1

(a) Diagram of a cross section of a microfocusing glass capillary; the capillary is drawn to an elliptical internal profile. A divergent X-ray source located in the left focus of the ellipse is reflected on the inside of the glass capillary (shown here in green) towards the second focus of the ellipse, where the sample resides (L is the distance from the source, L _c the actual length of the capillary, F the focal length; the angle θ_div describes the beam divergence at the sample). A beamstop centered upstream of the capillary blocks the direct beam. (Diagram courtesy of S. Cornaby, PhD thesis 2008, Cornell University, USA.) (b) A glass capillary (red) mounted in a precision-bored and reamed brass barrel. The capillary is guided in the front and back in two brass bores while being supported in the center by two Viton O-rings (blue) at 1/4 and 3/4 of its length. A brass cap in the back pushes onto a Viton cushion that holds the capillary in place. A clean-up aperture made of lead is added onto the front of the brass barrel.

Figure 2

(Top) A Keyence laser device measures the runout of the air-bearing rotation stage as it reflects from a calibrated polished steel sphere (Bal-tek, Los Angeles, CA; http://www.precisionballs.com/). (Bottom) Radial runout of the stepper stage with encoders. The runout determined by this set-up over two full revolutions is ±1 m.

Figure 3

Steps in manipulation of a crystal for pressure cryocooling. (a) The crystal is mounted in a standard cryoloop on a steel pin and a piano wire is attached. (b) The pin with crystal is placed into a steel pressure tube. A magnet on the outside holds the pin in place. The pressure tubes are connected to the manifold and lowered into the liquid-nitrogen bath [panel (c) shows tubes and bath, with manifold removed for clarity]. (d) After pressure cooling the samples are removed from the pressure tubes under liquid nitrogen.

Figure 4

A large mobile steel cabinet (188 cm × 112 cm × 74 cm) houses the pressure cryocooling apparatus. A safety enclosure on the upper shelf contains the liquid-nitrogen bath and manifold for the steel tubes with the samples. The lower shelf carries the pump that generates a maximum pressure of 200 MPa.

Figure 5

The BioSAXS set-up, shown here at CHESS G1 line, includes a quartz capillary flow cell housing (block at center) with a port at the top through which the robotic pipetting system (top) loads a sample. The X-ray beam travels entirely through vacuum from left to right in this figure. The video camera (front) provides a transverse in vacuo view of the flow cell for sample alignment.

Figure 6

BioSAXS robot control software. The Python-based graphical user interface controls the pipetting action of the robot, the pumping which positions the sample in the beam, and the X-ray exposure. By using disposable pipette tips to load the flow cell, sample cross contamination and loss are minimized. The sample can be oscillated in the beam or flowed at a constant rate to reduce radiation damage.