SPring-8 New Apparatus, Upgrades & Methodology Research Frontiers 2019 Research Frontiers 2019 88 New highly efficient and fully automatic MX at SPring-8 BL45XU beamline Recently, high flux and microfocus X-rays have enabled structural analysis from tiny (less than 20 μ m) protein crystals, such as LCP crystals of membrane proteins, at macromolecular crystallography (MX) beamlines. However, there still remains demand for highly efficient data collection from relatively large crystals (crystal size: 50 μ m or more) obtained by soaking chemical compounds aimed at, for example, drug discovery. Thus, SPring-8 BL45XU , operated a s t h e S A X S b e a m l i n e , w a s r e d e s i g n e d a n d reconstructed as a high-throughput automatic MX beamline with high flux and microfocus X-rays. The new BL45XU was designed on the basis the undulator beamlines BL41XU and BL32XU [1], at each of which an in-vacuum undulator and double crystal monochromator with Si (111) were installed. Available wavelengths range from 0.775 Å to 1.9 Å (Fig. 1(a)). The size of the focused X-rays at the sample position can be changed from 5 (H) × 5 (V) to 50 (H) × 50 (V) μ m 2 by adjusting the aperture of the virtual source slit and the glancing angles of vertical and horizontal focusing mirrors with a photon flux of 5.2 × 10 12 – 1.8 × 10 13 photons/s at 1.0 Å (Fig. 1(b)). The diffractometer in the experimental hutch was designed to enable high-speed and fully automatic measurement. The pixel array detector Pilatus 3 6M was installed to enable high-frame-rate measurement. The automatic sample changer with twin arms, SPACE-II [2], enables high-speed sample exchange (Fig. 1(c)). All the motorized axes are controlled by the beamline control software BSS [3]. The automated data collection system ZOO [4] has achieved automation for all goniometer-based data collection schemes in protein crystallography via communication with BSS. ZOO can estimate the absorbed dose in implemented schemes to mitigate severe radiation damage that may interfere with precise structure analyses (Table 1). The system has enabled ‘unattended’ automatic measurements. Users can obtain good datasets simply by sending their crystals without the need to visit the SPring-8 site. The automatic and unattended measurements, the so-called “mail-in” service, started at the end of May 2019. Fig. 1. Overview of BL45XU. (a) Layout of the optics. (b) Photon flux and beam profiles at the sample position. (c) Photograph of BL45XU experimental hutch showing diffractometer (sample changer and pixel array detector (PAD)). (a) (b) (c) DCM Horizontal Virtual source slit Guard slit focusing direction Horizontal Focal Point focusing direction Vertical focusing direction 36.5 m Wavelength (Å) Photon Flux (photons/sec) 0.75 1.0 × 10 11 4.1 × 10 12 8.1 × 10 12 1.2 × 10 13 1.6 × 10 13 2.0 × 10 13 0.95 1.15 1.35 1.55 1.75 1.95 40.4 m Distance from the source Beamsize PAD PILATUS3 6M SPACE-II High speed sample changer 20(H) × 16(V) μ m 2 10(H) × 10(V) μ m 2 5(H) × 5(V) μ m 2 48.0 m 56.5 m 57.0 m 58.0 m Research Frontiers 2019 Research Frontiers 2019 89 S. Baba a, *, K. Hirata b , N. Mizuno a and T. Kumasaka a a Japan Synchrotron Radiation Research Institute (JASRI) b RIKEN SPring-8 Center *Email: baba@spring8.or.jp References [1] K. Hirata et al. : J. Phys. Conf. Ser. 425 (2013) 012002. [2] H. Murakami et al. : Acta Crystallogr. D 76 (2020) 155. [3] G. Ueno et al. : J. Synchrotron Rad. 12 (2005) 380. [4] K. Hirata, K. Yamashita, G. Ueno, Y. Kawano, K. Hasegawa, T. Kumasaka and M. Yamamoto: Acta Cryst. D 75 (2019)138. Table 1. Experimental results of automatic measurements using ZOO system in BL45XU Type Single crystal Data collection by ZOO Data processing by KAMO (XDS) Multiple crystals 73 pins/6 pucks 11 h19 min 8 min 23 s 20 (H) × 16 (V) μ m 2 (9.75 × 10 12 photons/sec) 5 - 20 μ m (Membrane protein - LCP) 128 pins/8 pucks 8 h13 min 3 min 51 s 20 (H) × 16 (V) μ m 2 (1.73 × 10 13 photons/sec) 50 – 150 μ m Number of Sample Experimental time Ave. exp. Time Beam size (Photon flux) Crystal size Data Collect Processed 121 pins: Single point (102 pins) 121 data (Indexing failed: 5set) Completeness > 94% : 116 data Beyond 2.0 Å 33 data 3.0 – 2.0 Å 25 data Below 3.0 Å 58 data (9.17–3.03 Å) Helical (9 pins) Total Φ = 360º, (Dose = 10 MGy) 64 pins: Multiple small wedge 64 pins, 940 data Resolution, Completeness, I o / σ [outer shell] 1.92 Å , 99.6%, 1.56 2.04 Å , 99.8%, 1.42 4.78 Å , 99.8%, 1.94 Failed to a small number of data Sample A: (30 pins, 287 data) Sample B: (23 pins, 446 data) Sample C (9 pins 190 data) Sample D (2 pins, 17 data) Total Φ = 10º (Dose = 10 MGy)
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