High-speed raster-scanning synchrotron serial microcrystallography with a high-precision piezo-scanner

Abstract

The Frontier Microfocus Macromolecular Crystallography (FMX) beamline at the National Synchrotron Light Source II with its 1 µm beam size and photon flux of 3 × 10¹² photons s^-1 at a photon energy of 12.66 keV has reached unprecedented dose rates for a structural biology beamline. The high dose rate presents a great advantage for serial microcrystallography in cutting measurement time from hours to minutes. To provide the instrumentation basis for such measurements at the full flux of the FMX beamline, a high-speed, high-precision goniometer based on a unique XYZ piezo positioner has been designed and constructed. The piezo-based goniometer is able to achieve sub-100 nm raster-scanning precision at over 10 grid-linepairs s^-1 frequency for fly scans of a 200 µm-wide raster. The performance of the scanner in both laboratory and serial crystallography measurements up to the maximum frame rate of 750 Hz of the Eiger 16M's 4M region-of-interest mode has been verified in this work. This unprecedented experimental speed significantly reduces serial-crystallography data collection time at synchrotrons, allowing utilization of the full brightness of the emerging synchrotron radiation facilities.

Keywords: high-precision goniometer; macromolecular crystallography; nano-positioning; raster-scanning; serial crystallography.

Figure 1

Piezo scanner in the FMX endstation. (a) Schematic of the system we developed. Xf, Yf and Zf are the fine-scanning directions with a 200 µm travel range in each direction. Yc and Zc are the coarse motion directions. Ω is the rotation around the X-axis. GMX, GMY and GMZ are the motion directions of the XYZ stage assembly. (b) Schematic of the piezo scanner. P1, P2 and P3 are the motion directions of three shear piezo actuators of the NanoCube scanner. Yc and Zc are the motions of the NEXLINE coarse stages. Both fine and coarse stages are mounted on top of an air-bearing rotational stage, Ω. (c) Photograph of the system we developed installed at the FMX endstation, key components are enumerated: (1) cryogenic sample loaded on a SPINE cap, (2) piezo scanner, (3) microscope, (4) collimator, (5) beam stopper, (6) Nelson Air rotary stage. (d) Resonance frequencies of the piezo scanning system we developed.

Figure 2

Laboratory characterization of the piezo scanner. (a) Resolution measurements, 10 nm steps in the X-, Y- and Z-directions directly measured using interferometry. (b) Repeatability measurements, 1 µm steps in the X-direction performed with the fine piezo stage. (c) Radial runout error measured using capacitive displacement sensors. (d) Scanning trajectory measured in the x-direction at a 40 Hz scanning frequency (blue solid line). The red dashed line demonstrates the selected trajectory.

Figure 3

Crystallography data collection and processing at FMX. Raster scans of (a) a bovine trypsin rod-shaped crystal and (b) 5–10 µm-sized proteinase K crystals loaded on MiTeGen loops. The raster-scan areas are shown as red-dashed rectangles; the corresponding heat maps acquired after the raster scans are shown in the insets. The grid coloring is the diffraction pattern spot count. (c) Clustering of the serial crystallography partial datasets based on unit-cell size. The data shown were acquired from raster scans of proteinase K microcrystals with a 200 Hz detector frame rate. Of these 279 partial datasets, 234 (blue dots) were indexed to c = 106.58 ± 0.41 Å, with 99.6% completeness (Table 1 ▸). The remaining 45 partial datasets (orange dots) with c = 110.06 ± 0.84 Å did not yield a complete dataset and were excluded from refinement. The unit-cell size was converted to Cartesian coordinates for better visualization. The data were clustered into two groups using a K-mean clustering algorithm. (d) Hierarchical cluster-analysis dendrogram for the proteinase K partial datasets acquired at a 750 Hz detector frame rate. The cutoff limit was set to 0.6 (dashed line).