Serial X-ray Crystallography

3.3.1 Dd Myosin2 Micro-Crystallization

At the beginning of this work, no protocol for the micro-crystallization of myosin motor constructs had been established. To establish a batch crystallization protocol, suitable seeds were targeted. Therefore, crystals of Dd myosin2 were grown in classic vapor diffusion plates and setup (Figure 22a). The crystals were collected and crushed using a 10 µL pipette tip upon transfer into a 200 µL PCR reaction tube. Any

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55 remaining crystal fragments were collected and transferred by washing the well twice with 2 µL of reservoir solution. This combined solution served as seed solution. Micro-crystallization was performed in batch by mixing 50 µL of freshly thawed protein trapped in pre-powerstroke state with 50 µL of reservoir solution (as described in Chapter 2.2.4.1). Then, 5 µL seed solution was added and mixed by short vortexing for 5 seconds. This was followed by incubation at 18°C over night. Obtained crystals ranged from 10 to 50 µm. However, loading microcrystals obtained in an initial batch crystallization step on the chip proved to be precarious procedure, because pores were frequently clocked by crystal clumps or precipitation when excess solution was removed by the soak-through method (Figure 22b). On-chip crystallization proved to be more successful. It was performed similar to vapor diffusion experiments, but on a larger scale. Up to 200 µL of each crystallization mix were applied to the chip surface (32.4 x 12.8 mm² silicone chip, Suna precision) for these setups (Figure 22c). Excess solution was also removed by the soak-through method.

Figure 22: crystallization of Dd myosin2. a) Dd myosin2 crystal seeds grown with vapor-diffusion technique. b) Micro-crystals grown in batch. C) Micro-Micro-crystals grown on silicon Chip.

3.3.2 Human NM2B-2R Micro-Crystallization

The initially developed technique of Dd myosin2 micro-crystallization was subsequently adopted and optimized for the NM2B-2R construct. The established vapor diffusion crystallization conditions were supplemented with 5% DMSO. NM2B-2R (5 mg/mL) was mixed 1:1 (v/v) with the precipitation solution to a final volume of 200 µL. The mixture was applied to the chip surface (36.5 x 13.2 mm² silicone chip, Suna precision) (Figure 23). After 16 hours of incubation at 18°C, the volume decreased to a thin liquid film and crystals formed. For co-crystallization experiments the precipitation solution was supplemented with 625 µM EMD 57033.

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Figure 23: Micro-crystallization of NM2B-2R. a) Human NM2B-2R crystals grown on silicon chip (Suna precision) with vapor-diffusion technique. b) Micro-crystals grown in batch for seed generation and/or soaking load procedure.

3.3.3 Initial T-Jump Experiments

3.3.3.1 Dd Myosin2 pink beam crystallography

So far, the yield of Dd myosin2 micro-crystals diffracting to high resolution is limited. The maximum diffraction reaches 3.5 Å, with most micro-crystals diffracting to 5 Å or less. As the overall data quality was insufficient for our targeted T-jump crystallography experiments, further processing was not pursued.

Instead, we focused on the generation of better diffracting samples.

3.3.3.2 Lysozyme as Initial Test Target

Initial T-Jump test experiments were carried out at the ESRF beamline ID09 with the Roadrunner III setup from December 5^th to 9^th, 2018. Crystallization of lysozyme was conducted by Dr. Julia Lieske and Dr.

Sebastian Guenther. Data Processing was performed by Dr. Oleksandr Yefanov and Aleksandra Tolstikova.

Processed MTZ-files were used by me as paradigm for the analysis of Jump experiment datasets. For T-jump induction, a 1490 nm infrared laser was used. The focus size was 60 µm² and single nanosecond laser pulses triggered a local 30°C temperature jump. In addition to the initial ground state, post-T-Jump diffraction data sets were collected after 5, 10, 15, 30, 100, 200, and 1,000 µs. Each time point corresponds to a single chip collection with 4000 to 6000 single crystal hits. Each dataset was processed in an identical manner. B-factors were scaled using Equation 7. Based on data completeness the resolution cut off was determined. For most datasets the resolution was cut at 1.8 Å. Data recorded 30 µs or more after the IR-pulse exhibit a gap of completeness around 2.8 Å and have lower maximum diffraction. In these cases, the resolution was cut at 2.3 Å. The overall Wilson-B was slightly higher, compared to earlier time points. In Figure 24 the ribbon representations of all states of lysozyme are depicted, using color and thickness as B-factor indicators. In nearly all states the C-terminus exhibits the highest B-B-factors. Additionally, two loop regions at G 71 to S72 and D101 to G102 show high B-factors. The C-terminus exhibits a B-factor maximum

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57 at 5 µs delay, the two prior mentioned loops (G71 - S72 and D101 - G102) show a maximum at 15 µs delay.

Secondary structures located in the enzyme core, exhibit the lowest B-factors. Regions that are in direct contact with the enzyme core exhibit only small changes with the maximum increase occurring after 15 µs.

Figure 24: T-jump crystallography model system lysozyme. a-h) In addition to the ground state, seven time points were recorded after the T-Jump. Scaled B-factor is visualized with color (1 = blue, 2 = white, 3 = red) and ribbon thickness (standard PyMol scaling, 1 = thin, 3 = thick). i) The scaled B-factor distribution as function of the peptide chain. Color-code is shown in box on the right. The 15 µs delay graph is shown in bold and orange. Area between graphs is marked in light orange, when the 15 µs delay graph exhibits highest B-factors.

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58 3.3.3.3 Human NM2B-2R XFEL serial crystallography

Serial diffraction data obtained at the European XFEL were preprocessed from stream files by Dr.

Oleksandr Yefanov. These stream files were indexed and merged by me, with the help of Dr. Oleksandr Yefanov. The dataset of the native NM2B-2R crystals corresponded to a single chip measurement with 9,984 unique crystals. The unit cell parameters (a = 65.9 Å, b = 159.0 Å, c = 143.5 Å, α = 90.0°, β = 93.9°, γ = 90.0) were comparable to those of NM2B-2R structures obtained by conventional single crystal X-ray crystallography. The resolution cut-off was applied at 3.2 Å, based on Rsplit analysis as described by White and colleagues (2012). The structure was refined to a final Rfree of 0.3072. The small detector size of 0.5 megapixel resulted in a greatly limited availability of low-resolution diffraction data. Thus, the resolution range used in refinement was 3.2 – 15 Å, whereas maximum protein diffraction was observable to 2.24 Å.

On chip crystallization in the presence of EMD 57033 produced more but smaller crystals.

To increase completeness at low resolution, the detector distance to the chip was increased. For this measurement, diffraction data were collected from 6,748 crystals on a single chip. For refinement, diffraction spots in the resolution range 3.4 – 18.8 Å were used. The defined unit cell parameters were: a

= 66.17 Å, b = 160.87 Å, c = 143.78 Å, α = 90.0°, β = 93.99°, γ = 90.0°. Surprisingly, the unit cell was larger.

Therefore, it was not possible to calculate an isomorphic difference map calculation (Figure 25). The final Rfree in model refinement was 0.3347. Conformational changes or electron density related to the presence of EMD 57033 were not observed.

Figure 25: Comparison of XFEL derived structures of NM2B-2R with and without EMD 57033. A cartoon representation of the NM2B-2R structure is shown in green. The structure of NM2B-2R, co-crystallized with EMD 57033 is shown in red.

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