FMX - the Frontier Microfocusing Macromolecular Crystallography Beamline at the National Synchrotron Light Source II

Abstract

Two new macromolecular crystallography (MX) beamlines at the National Synchrotron Light Source II, FMX and AMX, opened for general user operation in February 2017 [Schneider et al. (2013). J. Phys. Conf. Ser. 425, 012003; Fuchs et al. (2014). J. Phys. Conf. Ser. 493, 012021; Fuchs et al. (2016). AIP Conf. Proc. SRI2015, 1741, 030006]. FMX, the micro-focusing Frontier MX beamline in sector 17-ID-2 at NSLS-II, covers a 5-30 keV photon energy range and delivers a flux of 4.0 × 10¹² photons s^-1 at 1 Å into a 1 µm × 1.5 µm to 10 µm × 10 µm (V × H) variable focus, expected to reach 5 × 10¹² photons s^-1 at final storage-ring current. This flux density surpasses most MX beamlines by nearly two orders of magnitude. The high brightness and microbeam capability of FMX are focused on solving difficult crystallographic challenges. The beamline's flexible design supports a wide range of structure determination methods - serial crystallography on micrometre-sized crystals, raster optimization of diffraction from inhomogeneous crystals, high-resolution data collection from large-unit-cell crystals, room-temperature data collection for crystals that are difficult to freeze and for studying conformational dynamics, and fully automated data collection for sample-screening and ligand-binding studies. FMX's high dose rate reduces data collection times for applications like serial crystallography to minutes rather than hours. With associated sample lifetimes as short as a few milliseconds, new rapid sample-delivery methods have been implemented, such as an ultra-high-speed high-precision piezo scanner goniometer [Gao et al. (2018). J. Synchrotron Rad. 25, 1362-1370], new microcrystal-optimized micromesh well sample holders [Guo et al. (2018). IUCrJ, 5, 238-246] and highly viscous media injectors [Weierstall et al. (2014). Nat. Commun. 5, 3309]. The new beamline pushes the frontier of synchrotron crystallography and enables users to determine structures from difficult-to-crystallize targets like membrane proteins, using previously intractable crystals of a few micrometres in size, and to obtain quality structures from irregular larger crystals.

Keywords: beamlines; endstations; macromolecular crystallography; microfocus; serial crystallography.

Figure 1

(a) The FMX and AMX beamline layout (not to scale). AMX is served by the upstream (17-ID-1) IVU21 undulator, FMX by the downstream (17-ID-2) one. Beam and labels of AMX are red, those of FMX in blue. All distances are given with respect to the FMX source position. (b) A top-down plan view of the beamline layout on the NSLS-II experimental floor. The white-beam path to the monochromators is contained in the 17-ID-A lead hutches, followed by the AMX experimental hutch 17-ID-B and the FMX experimental hutch 17-ID-C.

Figure 2

The FMX beam profile, showing the FMX beam spot at focus as detected by a Cr nanowire scan. (a) Horizontal profile with a 1.49 µm FWHM. (b) Vertical profile with a 1.04 µm FWHM.

Figure 3

The FMX photon flux at the sample position across the operational photon energy range. At lower energies, the flux is diminished due to partial absorption of the horizontally polarized synchrotron radiation in the horizontal-bounce DCM, as well as by absorption in the Be exit window and the remaining air path to the sample.

Figure 4

Flux density and beam size of FMX and selected bright microfocus MX beamlines worldwide (Allan et al., 2015; Burkhardt et al., 2016; Cianci et al., 2017; Fischetti et al., 2013; Hasegawa et al., 2013; Hirata et al., 2013; Mueller-Dieckmann et al., 2015; Riekel et al., 2010; Schulze-Briese et al., 2002; Smith et al., 2012; Soltis et al., 2008; von Stetten et al., 2020; Ursby et al., 2020 ▸). Spot size encodes maximum flux, see legend.

Figure 5

The FMX experimental station. (1) Main goniometer. (2) Secondary goniometer. (3) Eiger X 16M detector. (4) CryoStream CS800. (5) Sample-mounting robot. (6) Cooling-gas exhaust tube. (7) Microscope deflection mirror. (8) Sample back-illumination. (9) Beam stop. (10) Questar QM100 microscope objective. (11) Beam-conditioning unit. Panels (b) to (d) show three experiment states of the Governor state controller (see Section 5.2) – (b) sample exchange, (c) sample alignment and (d) data acquisition.

Figure 6

The controls architecture of the FMX and AMX beamlines. Communication between the different databases, servers and controllers is indicated by white arrows. The LSDC GUI and server communicate through EPICS Channel Access, indicated by green arrows.

Figure 7

The LSDC GUI for AMX and FMX. The sample requests view is displayed in the left-hand column, data acquisition parameters are set in the center, and the sample video view is displayed in the right-hand column.

Figure 8

The benefits of microfocus crystallography, or why a micrometre-sized beam is also beneficial for data collection on larger crystals.

Figure 9

(a) X-shaped crystals of the ppGpp riboswitch. A red arrow shows the tip of the crystal used for collecting data on FMX. (b) The crystal structure of the ppGpp-bound riboswitch (Peselis & Serganov, 2018 ▸). The RNA is in cartoon representation, with the sugar–phosphate backbone depicted as a cylindrical ribbon, colored according to its structural elements. ppGpp is shown as a stick model, surrounded by Mg²⁺ ions.

Figure 10

A structure model of Ric 8A. Anomalous difference electron-density map showing the 40 sulfur sites comprising the sulfur atom substructure in the asymmetric unit (see text), contoured at 4.5σ.

Figure 11

Serial crystallography with an LCP jet on FMX. (Right) The high-viscosity extrusion injector mounted on the secondary goniometer on the FMX beamline. The beam (yellow arrow) hits the LCP stream ejected downwards from the injector nozzle. (Bottom left) A view of the injector nozzle tip in the sample microscope. The nozzle is aligned so the beam (red cross) hits the viscous jet just underneath the nozzle. (Top left) Still diffraction images are recorded on the Eiger X 16M detector.