Development of an X-ray fluorescence holographic measurement system for protein crystals

Abstract

Experimental procedure and setup for obtaining X-ray fluorescence hologram of crystalline metalloprotein samples are described. Human hemoglobin, an α2β2 tetrameric metalloprotein containing the Fe(II) heme active-site in each chain, was chosen for this study because of its wealth of crystallographic data. A cold gas flow system was introduced to reduce X-ray radiation damage of protein crystals that are usually fragile and susceptible to damage. A χ-stage was installed to rotate the sample while avoiding intersection between the X-ray beam and the sample loop or holder, which is needed for supporting fragile protein crystals. Huge hemoglobin crystals (with a maximum size of 8 × 6 × 3 mm(3)) were prepared and used to keep the footprint of the incident X-ray beam smaller than the sample size during the entire course of the measurement with the incident angle of 0°-70°. Under these experimental and data acquisition conditions, we achieved the first observation of the X-ray fluorescence hologram pattern from the protein crystals with minimal radiation damage, opening up a new and potential method for investigating the stereochemistry of the metal active-sites in biomacromolecules.

Size and Shape Controlled Crystallization of Hemoglobin for Advanced Crystallography

“…For the XFH measurement, the large COHbA crystal was briefly soaked in CO-saturated 2.30 M phosphate buffer containing 0.958 M NaH2PO4, 1.342 M K2HPO4, and 15% (v/v) glycerol, and then flash cooled to 100 K to reduce X-ray radiation damage (Figure 8a). The first XFH data-set using a COHbA crystal was collected at the beamline BL6C of Photon Factory [13]. A step-scan data collection was performed with an interval of 0.25° and 1.0° in pitch and yaw, respectively.…”

“…We checked the crystal quality by measuring the Bragg diffraction pattern for X-rays incident on the crystal (Figure 8c). More details of the XFH measurement are described in elsewhere [13]. Note that the crystals grown at the 3 ) was mounted on a customized loop made by a polyimide film (0.125 mm thickness) with a copper wire (1.2 mm diameter), and cooled to 100 K by nitrogen gas flow; (b) Typical hologram pattern of the COHbA crystal (excited by 7.25 keV X-ray photons), represented as a stereographic projection of an interference pattern in the reciprocal space (a*, b*, c*) after applying a Gaussian low path spatial filter [13]; (c) An X-ray diffraction pattern of the COHbA crystal used for the XFH measurement.…”

“…The first XFH data-set using a COHbA crystal was collected at the beamline BL6C of Photon Factory [13]. A step-scan data collection was performed with an interval of 0.25 • and 1.0 • in pitch and yaw, respectively.…”

“…We checked the crystal quality by measuring the Bragg diffraction pattern for X-rays incident on the crystal (Figure 8c). More details of the XFH measurement are described in elsewhere [13]. Note that the crystals grown at the oil-solution interface tend to have shapes with well-developed crystal faces (Figures 7b,c and 8a).…”

“…However, this is not always the case, since protein crystallography experiments are becoming more diverse and challenging, and thereby requirements for crystals are also becoming more demanding in terms of size and shape. The purpose of this article is to illustrate our approach to the growth of Hb crystals to dimensions that meet the needs of advanced crystallography, including XRC combined with photoexcitation (e.g., photolysis of CO bound to Hb [7,8]) and/or spectrophotometry [9,10], neutron crystallography (NC) [11,12], and recently developed X-ray fluorescent holography (XFH) [13]. The first one requires optically thin but well-diffracting Hb crystals, while the latter two, especially XFH, require large-volume, well-ordered Hb crystals, due to feeble signals.…”