SgcC5 was crystallized using the sitting-drop vapor-diffusion technique in 96-well CrystalQuick plates (Greiner Bio-one) prepared by a Mosquito liquid dispenser (TTP Labtech). For each screening condition, 0.4 μL of protein and 0.4 μL of crystallization formulation were mixed. The mixture was equilibrated against 140 μL of the reservoir in the well. The plates were incubated at 4 °C. The ligand-free SgcC5 (SgcC5apo) was crystallized by the crystallization formulation containing 1 M sodium citrate and 0.1 M sodium cacodylate/HCl, pH 6.5. To prepare SgcC5 in complex with an acceptor substrate, the protein solution was mixed with 4 mM (R)-1-phenyl-1,2-ethanediol (in DMSO) or (R)-1-(2-naphthyl)-1,2-ethanediol (in DMSO) and incubated at 4°C. While no crystal of SgcC5 in complex with (R)-1-phenyl-1,2-ethanediol was obtained, SgcC5 in complex with (R)-1-(2-naphthyl)-1,2-ethanediol (SgcC5NE) was crystallized by the crystallization formulation containing 0.2 M Li2SO4, 2 M (NH4)2SO4, and 0.1 M 3-(cyclohexylamino)-1-propanesulfonic acid/NaOH, pH 10.5.
Crystalquick plates
Manufactured by Greiner
Sourced in United States, United Kingdom
CrystalQuick plates are a type of lab equipment designed for protein crystallization experiments. They provide a controlled environment for the growth and study of protein crystals.
Automatically generated - may contain errors
Lab products found in correlation
Mosquito liquid dispenser, by SPT Labtech (13 mentions)
Jbscreen classic 1 10, by Jena Biosciences (3 mentions)
Jbscreen membrane 1 3, by Jena Biosciences (3 mentions)
Pi peg screen hts, by Jena Biosciences (2 mentions)
Easyxtal plates, by Qiagen (2 mentions)
Wizard 1 2, by Jena Biosciences (2 mentions)
Mosquito nanoliter liquid handler, by SPT Labtech (2 mentions)
Mosquito robot, by SPT Labtech (1 mentions)
Mcsg1 4, by Anatrace (1 mentions)
Centricon ym 10, by Merck Group (1 mentions)
Pilatus3x 6m detector, by Dectris (1 mentions)
20 protocols using crystalquick plates
1
Structural Determination of SgcC5 Complexes
2
Crystallization Screening of Protein EipA
3
Crystallization of Membrane Proteins
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Initial crystallization screens were performed by the sitting-drop vapour-diffusion technique (96-well Crystal Quick plates, Greiner Bio-One) using a nanodispenser robot (Gryphon, Art Robbins). Screening over a broad variety of crystallization conditions (Crystallization Basic Kit for Membrane Proteins, Crystallization Low Ionic Strength Kit for Proteins and Crystallization Basic Kit for Proteins from Sigma–Aldrich; Pi-PEG Screen HTS, JBScreen Classic 1–10 and JBScreen Membrane 1–3 from Jena Bioscience) gave the first hits (microcrystalline precipitant) for crystallization.
The first microcrystals were obtained by the hanging-drop vapour-diffusion technique (15-well EasyXtal 10 × 15 plates, Qiagen) using 10% PEG 4000, 15 mM MgCl2, 25 mM Tris–HCl pH 7.5 at 291 K as the crystallization condition (Fig. 2 ▶ a). The final crystallization condition for the growth of single crystals suitable for diffraction measurements was 10% PEG 4000, 1 mM Na6[TeW6O24]·22H2O, 25 mM Tris–HCl pH 7.5 at 291 K using 1 µl protein solution (10 mg ml−1) and 0.5 µl reservoir solution in the hanging drop and 500 µl solution in the reservoir (Fig. 2 ▶ b). The first crystals appeared after 1–2 d and crystal growth came to a stop after approximately 5 d.
The first microcrystals were obtained by the hanging-drop vapour-diffusion technique (15-well EasyXtal 10 × 15 plates, Qiagen) using 10% PEG 4000, 15 mM MgCl2, 25 mM Tris–HCl pH 7.5 at 291 K as the crystallization condition (Fig. 2
4
Protein Crystallization Optimization for SgcJ and NCS-Orf16
5
Crystallization Screening for Wildtype MdPPO1
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Initial screening for suitable crystallization conditions for wild-type MdPPO1 was performed by the sitting-drop vapour-diffusion method using a nanodispenser robot (Gryphon, Art Robbins) and 96-well Crystal Quick plates (Greiner Bio-One). A wide range of crystallization conditions were screened by using a variety of commercially available screening kits (JBScreen Classic 1–10, JBScreen Membrane 1–3 and Pi-PEG Screen HTS from Jena Bioscience). The screening procedure yielded some promising hits, which were further optimized manually by applying the hanging-drop vapour-diffusion technique using 15-well EasyXtal plates (Qiagen). Single crystals of wild-type MdPPO1 and of the mutants MdPPO1-Ala239Thr and MdPPO1-Phe259Ala (Fig. 3 ▸ ) were grown at 20°C by mixing 1 µl protein solution (5–10 mg ml−1) with 2 µl reservoir solution (50 mM Tris–HCl pH 7.0, 19–21% PEG 3350). Crystals usually appeared after 10–15 d. Crystallization information is summarized in Table 2 ▸ . Crystallization trials of the mutants MdPPO1-Leu243Arg and MdPPO1-Glu234Ala applying the above-described methods yielded only low-quality crystals which were not suitable for adequate data collection (Fig. 3 ▸ ).
6
Crystallization of AtmS13 Protein
7
Crystallization of SARS-CoV-2 Protease Complexes
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Crystallizations were performed with the protein-to-matrix ratio of 1:1 using the sitting drop vapor-diffusion method with the help of the Mosquito liquid dispenser (TTP LabTech) in 96-well CrystalQuick plates (Greiner Bio-One). MCSG1, MCSG2, MCSG3, and MCSG4 (Anatrace), Index (Hampton Research) and Wizard 1&2 (Jena Bioscience) screens were used at 16 °C. The PLproCoV-2-C111S:hISG15 complex (13 mg/ml) crystallized in Index E11 (0.02 M MgCl2, 0.1 M HEPES pH 7.5, 22% (w/v) polyacrylic acid sodium salt 5,100). For the PLproCoV-2-C111S:Ub2-K48R,D77 complex (11 mg/ml), crystals appeared in MCSG-2 F11 and were improved in hanging drops with a protein-to-matrix ratio of 2:1 in 0.2 M disodium tartrate, 15 % (w/v) PEG3350 after seeding with 1/10 volume of PLproCoV-2-C111S:Ub2-K48R, D77 microcrystals. Crystals of the PLproCoV-2C111S,D286N:Ub2-K48R complex were obtained in 0.2 M disodium tartrate, 15 % (w/v) PEG3350 (as above). The Ub2 protein crystallized in Pi-PEG D1 (50 mM acetate buffer pH 4.8, 8.6% PEG 2000 MME, 17.1% PEG 400). The hISG15 protein crystallized in MCSG1 G2 (40 mM potassium phoshate, 16% PEG 8000, 20% glycerol). Crystals selected for data collection were washed in the crystallization buffer supplemented with 25% glycerol and flash-cooled in liquid nitrogen.
8
Crystallization of SARS-CoV-2 Nsp15 Protein
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Crystallization experiments also were conducted as described previously, with slight modifications.29 The sitting‐drop vapor‐diffusion method was used with the help of the Mosquito liquid dispenser (TTP LabTech) in 96‐well CrystalQuick plates (Greiner Bio‐One). Crystallizations were performed with the protein‐to‐matrix ratio of 1:1. MCSG4 (Anatrace), SaltRX (Hampton), and INDEX (Hampton) screens were used for protein crystallization at 16°C. The crystallization conditions were MCSG4 H11 (0.2 M calcium acetate, 0.1 M HEPES/NaOH pH 7.5, 10% PEG 8000). These crystals belong to hexagonal space group P63 diffract X‐rays to 2.20 Å resolution. Diffraction‐quality crystals of Nsp15 suitable for data collection appeared after 12 hr. The second hexagonal crystal form, also in P63 space group, was obtained from 0.1 M trisodium citrate pH 5.6, 10%(w/v) PEG4000, and 10%(v/v) isopropanol. These crystals diffracted X‐rays to 1.90 Å.
9
Crystallization of Hpy GS Protein
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Purified Hpy GS was concentrated to 41 mg mL−1 using Centricon YM-10 (Millipore, USA) for crystallization trials. Initial screening was performed by the sitting-drop vapour diffusion method using 96-well CrystalQuick plates (Greiner Bio-One, Germany) with various commercial screens (Hampton Research, USA; Qiagen, Germany; Axygen, USA; Emerald Biosystems, USA). Each sitting drop was prepared by mixing 0.75 µl protein solution and 0.75 µl reservoir solution and incubated at room temperature. The initial crystals of Hpy GS were grown after four weeks of incubation under several conditions with polyethylene glycol (PEG) 6,000. The crystals of Hpy GS were further optimized, and the best crystals were grown in 10% (v/v) PEG 6,000, 100 mM HEPES pH 7.0, and 50 mM choline. To obtain the cocrystals with substrates, Hpy GS was concentrated to 19 mg mL−1 and mixed with 5 mM ATP, 5 mM PPT, and 5 mM magnesium chloride before crystallization. It was stored at 277 K for 1 h. The crystals of substrate-bound Hpy GS were grown in 2.0 M sodium formate and 100 mM sodium citrate at pH 5.0.
10
SgcE6 Protein Crystallization Screening
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SgcE6 crystallization screens
were prepared with a Mosquito liquid dispenser (TTP Labtech) using
the sitting-drop vapor-diffusion technique in 96-well CrystalQuick
plates (Greiner Bio-one). For each condition, 0.4 μL of protein
and 0.4 μL of crystallization formulation were mixed; the mixture
was equilibrated against 140 μL of the reservoir in the well.
The protein–ligand complex was prepared by mixing protein with
27.7 mM FAD and 27.7 mM NADH at 4 °C for several hours before
setting up crystallizations. The following commercially available
crystallization screens were used: MCSG-1-3 (Microlytic Inc.) at 24
°C for the ligand-free protein and MCSG-1-4 (Microlytic Inc.)
at 24 °C for the protein–ligand complexes. The crystals
for the ligand-free protein were obtained under 25% PEG 3350, 0.1
M HEPES (pH 7.5), and 0.2 M ammonium sulfate. The best crystal of
the protein–ligand complex of SgcE6 was produced under 20%
PEG 8000, 0.1 M MES (pH 6.0), and 0.2 M calcium acetate.
were prepared with a Mosquito liquid dispenser (TTP Labtech) using
the sitting-drop vapor-diffusion technique in 96-well CrystalQuick
plates (Greiner Bio-one). For each condition, 0.4 μL of protein
and 0.4 μL of crystallization formulation were mixed; the mixture
was equilibrated against 140 μL of the reservoir in the well.
The protein–ligand complex was prepared by mixing protein with
27.7 mM FAD and 27.7 mM NADH at 4 °C for several hours before
setting up crystallizations. The following commercially available
crystallization screens were used: MCSG-1-3 (Microlytic Inc.) at 24
°C for the ligand-free protein and MCSG-1-4 (Microlytic Inc.)
at 24 °C for the protein–ligand complexes. The crystals
for the ligand-free protein were obtained under 25% PEG 3350, 0.1
M HEPES (pH 7.5), and 0.2 M ammonium sulfate. The best crystal of
the protein–ligand complex of SgcE6 was produced under 20%
PEG 8000, 0.1 M MES (pH 6.0), and 0.2 M calcium acetate.
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