Refinement of the crystallization conditions of SCOC (78–159) crystals

First crystallization conditions were optimized withgrid screens in 24-well format. Usually, pH was varied along rows in increments of one pH unit for first optimization. Precipitant concentration was varied along columns.

3.1 Characterization & structure determination of the SCOC ccd 77

A B

Figure 3.17: Crystals in hanging drops grown in 24-well format (A) Crystals from a grid screen varying pH (MES 5.5 – MES 6.5) against NaAc concentration (B) Crystals from a grid screen varying pH (MES 5.5 – HEPES 7.5) against PEG 3350 concentration

Figure 3.17) shows crystals obtained by two grid screens (see Table 3.4 for crystallization conditions). In general, 24 well plates yielded larger crystals suitable for flash cooling and data collection. The range of protein concentra-tion tested remained the same for 24-well plates. In order to obtain single, slow growing crystals instead of multiple, fast growing crystals, I also tested crystallization at 4 °C. However, this did not yield any crystals. I further tried to control the vapor diffusion rate to obtain slower growing crystals. In 24-well sitting drop plates, 5 µL (2.5 µL protein plus 2.5 µL crystallization buffer) drops were covered with silicon oil. Only phase separation but no crystals were observed.

Table 3.4: Crystallization conditions in 24 well plate screens condition construct buffer composition

A SCOC (78–159) 0.1 M MES pH 6.5

1.4 M NaAc

B SCOC (78–159) 0.1 M MES pH 6.5

19 % (w/v) PEG 3350 0.01 M MgCl₂

When refining the crystallization conditions in 24 well plates, I applied streak seeding, providing nucleation sites with the small crushed crystal fragments added by the streak in order to avoid multiple crystals. Figure 3.16 demonstrates the effect of this method: Panel A shows star-shaped crystals grown in a 96-well plate. Panel B shows a crystal with almost the same buffer condition, grown from a streak seeded drop in a 24-well plate.

78 SCOC and its interaction partners Crystals from 24-well and 96-well plates were soaked stepwise in a variety of cryoprotectants, and flash cooled. Diffraction quality was tested at 100 K at the synchrotron (Beamline X10SA, Paul Scherrer Institute, Swiss Light Source Villigen, Switzerland) and eventually data were collected. Crystals from PEG conditions diffracted only up to 6 Å. Other samples from salt conditions diffracted to better resolution (4 Å), but the lattice was of insufficient quality with multiple, smeary spots and high mosaicity. The best crystal from initial screening was taken out of a drop from the Cryo screen condition D6 (see Figure 3.15). It diffracted up to 3.6 Å, but with split and smeary spots.

Next,Random and Grid Screens were designed and dispensed with the Tecan robot, taking the Cryo screen condition D6 yielding the best crystals so far as basis. Several Grid Screens were set up, varying NaCl concentration in small increments against the pH of MES buffer between 5.2–6.9 or HEPES buffer between 6.8–8.0. As the Cryo condition contained several components, including two different phosphate salts and glycerol, a Random screen was designed to evaluate their effects on crystallization. Crystals obtained from these screens are shown in Figure 3.18, the respective buffer compositions are shown in Table 3.5.

Table 3.5: Exemplary crystallization conditions of Random and Grid screens

condition construct buffer composition A SeMet SCOC (78–159) 0.085 M MES pH 6.77

0.085 M NaH₂PO₄ 0.085 M KH₂PO₄ 1.43 M NaCl

15 % (v/v) glycerol B SCOC (78–159) w/o Tag 0.085 M MES pH 6.45

0.0122 M NaH₂PO₄ 0.147 M KH₂PO₄ 1.77 M NaCl

C SCOC (78–151) 0.1 M MES pH 5.55 2.79 M NaCl

D SCOC (78–141) 0.1 M HEPES pH 7.31

2.68 M NaCl

The designed screens yielded many crystals, most of them multiple or single

3.1 Characterization & structure determination of the SCOC ccd 79

A B

C D

Figure 3.18: Crystals obtained from refinement with grid and random screens

(A) selenomethionine-labelled SCOC (78–159) crystals from a random Screen (B) SCOC (78–159) crystals from a random screen, crystal-lized without StrepTag (C) SCOC (78–159) crystals from a grid screen (D) SCOC (78–159) crystals from a grid screen

crystals in elongated cuboid shapes. In addition, the screens were utilized to test the crystallization behaviour of SCOC (78–159) variants without the StrepTag (cleaved with Thrombin with a protocol according to Section 2.2.2.5) (see Figure 3.18 B) or selenomethionine-labelled SCOC (see Figure 3.18 A) (see Section A.2 for a table summarizing crystallization conditions from random and grid screens). Large and single crystals were soaked in cryoprotectant (only if necessary, as many conditions already contained glycerol as cryoprotectant), and their diffraction was tested. However, neither diffraction resolution nor lattice quality was significantly improved.

Hence, although SCOC (78–159) crystallized easily from a large variety of conditions, the diffraction quality even after extensive screening was not suffi-cient to solve the structure.

80 SCOC and its interaction partners 3.1.4.3 Screening of other SCOC ccd constructs

In parallel, I cloned and purifiedC-terminally truncated constructs SCOC (78–141) and SCOC (78–151). Crystallization screens were set up at 20 °C with 200 nL sitting drops. SCOC (78–151) at 15 and 7.5 mg/mL protein con-centration yielded mostly crystal clusters (Figure 3.15 C). SCOC (78–141) at 12 and 6 mg/mL was easily crystallized, forming needles or needle clusters in a few conditions (Figure 3.15 D) (see Table 3.3 for crystallization condi-tions). The crystal needles were very fragile and required careful handling during soaking and flash cooling. The ccd fragment crystals diffracted to only 5–6 Å resolution.

Another attempt to achieve better diffracting crystals was made by cloning and purifying two overlapping halves of SCOC ccd, SCOC (78–132) and SCOC (112–159). 96-well plates sitting drops were dispensed at 20 °C at 40 and 20 mg/mL (SCOC 78–132) and 17 and 8.5 mg/mL (SCOC 112–

159). Both constructs yielded only spherulites, gel or cluster-like structures in 96-well screening (Figure 3.15 E and F).

Summing up, the purpose-built constructs—some of them missing presum-ably unstructured parts of the ccd, others designed to pack better in the crystal—did not result in better diffracting crystals compared to SCOC (78–

159).

3.1.4.4 Crystallization of selenomethionine-labelled SCOC crystals Selenomethionine labelled crystals (SeMet SCOC (78–159) in Figure 3.18 A) grew predominantly in conditions similar to the native protein. The crystals showed similar diffraction quality as native crystals, however, the anomalous signal dropped rapidly between 5 and 6 Å, making structure determination from single or multi- wavelength anomalous diffraction (SAD or MAD) very challenging. Hence, two mutant constructs, SCOC L96M (78–159) and SCOC L105M (78–159), were cloned, purified and subjected to crystallization screens.

Initial screening of SCOC L96M (78–159) did not yield promising conditions.

SCOC L105M (78–159) showed similar crystallization behavior as wt selenome-thionine crystals and was subjected to in situ proteolysis (see below).