Peg ion screen

Manufactured by Hampton Research

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The PEG/Ion Screen is a laboratory tool designed to facilitate the screening of protein crystallization conditions. It consists of a set of solutions containing various polyethylene glycol (PEG) and salt concentrations, which can be used to explore the optimal conditions for protein crystal growth.

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Lab products found in correlation

Pegrx, by Hampton Research (1 mentions) Crystal screen, by Hampton Research (1 mentions) Intelli plate, by Art Robbins Instruments (1 mentions) Jcsg plus, by Molecular Dimensions (1 mentions) Mosquito crystallization robot, by SPT Labtech (1 mentions) Index screen, by Hampton Research (1 mentions) Cryoloop, by Hampton Research (1 mentions) Pact suite, by Qiagen (1 mentions) Phoenix crystallization robot, by Art Robbins Instruments (1 mentions) Jcsg core suite, by Qiagen (1 mentions) Pilatus3 6 m detector, by Dectris (1 mentions) Saltrx, by Hampton Research (1 mentions)

10 protocols using peg ion screen

Crystallization of BpsR Protein and Complex

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Crystallization of BpsR was carried out using the sitting drop vapor diffusion method with equal volumes of protein and crystallization solution in the drop reservoir. Initial crystals were identified in the PEG-ION screen (Hampton Research) and a PEG-infactorial screen [24 (link)]. Optimized crystallization conditions contained 18% PEG 2250, 0.2 M potassium formate, and 15% butanediol in the reservoir solution. Crystals were transferred to a drop containing 50% mineral oil, 50% parafin oil (Hampton Research) prior to cryocooling in a liquid nitrogen stream.
Co-crystallization of the 6HNA-BpsR complex was performed with 10 mg/ml BpsR. Crystals were obtained by mixing 2.6 μL of protein solution with 3 μL of the reservoir solution containing 0.1 M HEPES (pH 7.5), 2 mM 6-HNA, 0.2 M MgCl₂, 18.5% PEG 3350. The crystals were obtained by incubation at 12 ºC.

Booth W.T., Davis R.R., Deora R, & Hollis T. (2019). Structural mechanism for regulation of DNA binding of BpsR, a Bordetella regulator of biofilm formation, by 6-hydroxynicotinic acid. PLoS ONE, 14(11), e0223387.

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Structural Determination of AtWRI1-DNA Complex

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AtWRI1 (7.5 mg/ml) was mixed with equimolar AtWRI1-binding dsDNA BCCP2(-34/-11) (5′-TACTTCCTCGGTTTCATCGTCCAC) and incubated at 4°C for 1 hour. Crystallization screening was performed with Crystal Screen, Index, PEG Rx, and PEG/Ion Screen (Hampton Research) using the sitting drop vapor diffusion method at 20°C on an Intelli-Plate 96-3 LVR (Art Robbins Instruments). Crystals appeared during Crystal Screening under the A9 condition [0.2 M ammonium acetate, 0.1 M sodium citrate tribasic dihydrate (pH 5.6), and 30% w/v polyethylene glycol 4000]. The final optimized condition for crystal growth was 0.2 M ammonium acetate, 0.1 M MES (pH 6.5), and 28% w/v PEG 4000.
The native dataset of AtWRI1-DNA crystals was collected from the Swiss Light Source (SLS). SAD datasets for Se-Met–labeled AtWRI1-DNA crystals were collected at an inflection wavelength of 0.9795 Å with an Australian light source and SLS. All the datasets were processed using XDS (x-ray detector software) (48 (link)). The Phenix AutoSol program was used for phasing, and four Se atoms were found in the substructure solution (figure of merit, 0.23). The structure served as a template to resolve native AtWRI1-DNA structures through molecular replacement. The models were built and refined using Phenix and Coot (41 (link), 49 (link)). All the structure-related figures were generated with PyMOL (50 ).

Qiao Z., Kong Q., Tee W.T., Lim A.R., Teo M.X., Olieric V., Low P.M., Yang Y., Qian G., Ma W, & Gao Y.G. (2022). Molecular basis of the key regulator WRINKLED1 in plant oil biosynthesis. Science Advances, 8(34), eabq1211.

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Crystallization of Virus-Like Particles

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VLPs were crystallized at 293 K in 96-well plates using the sitting-drop vapour-diffusion method. Prior to crystallization, the buffer solution of purified VLPs was replaced with 20 mM MES pH 6.5 and they were concentrated to 4 mg ml⁻¹. Initial screening was performed using PEG/Ion Screen (Hampton Research), which was diluted fivefold and tenfold because the crystallization-condition information in VIPERdb (Carrillo-Tripp et al., 2009 ▸ ) shows that many viruses tend to crystallize in a combination of low concentrations of PEG and salt. Crystals of ChV VLPs were obtained under several conditions, and the largest crystals were obtained in the presence of dibasic phosphate ions. However, these crystals did not have sharp edges and appeared to be clusters of several crystals. Using a microseeding technique and optimizing the concentrations of PEG and (NH₄)₂HPO₄ and the molecular weight of PEG resulted in the growth of large single crystals (Fig. 2 ▸). Higher concentrations of (NH₄)₂HPO₄ or PEG tended to cause the formation of aggregates. The final crystallization condition is shown in Table 2 ▸. The speed of crystal growth was quite slow; it took one to three months to obtain crystals of 100 µm in size.

Hasegawa K., Someya Y., Shigematsu H., Kimura-Someya T., Nuemket N, & Kumasaka T. (2017). Crystallization and X-ray analysis of 23 nm virus-like particles from Norovirus Chiba strain. Acta Crystallographica. Section F, Structural Biology Communications, 73(Pt 10), 568-573.

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Crystallization of NADPH-Vv2KGR Complex

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Crystals were grown in sitting drops by vapor diffusion in 96-well INTELLI-PLATE (Art Robbins Instruments) at 16 °C. The protein sample used for crystallization was concentrated to ∼10 mg/ml in the above-mentioned dialysis buffer. Each well consisted of a 1-μl/1-μl ratio of well solution to protein solution and the well solution reservoir (75 μl). The screening plates were set up using a Phoenix robot system (Art Robbins Instruments). In addition to the ligand-free sample, an enzyme solution containing 2 mm NADPH was also tested to obtain NADPH-Vv2KGR co-crystals. Crystals were obtained in a number of solutions in the Wizzard Classic 1 and 2 block (Rigaku) and PEG/ion screen (Hampton Research). The protein crystals used for diffraction were obtained in solution containing 1.26 m ammonium sulfate, 100 mm sodium acetate/acetic acid (pH 4.5), and 200 mm sodium chloride.

Jia Y., Burbidge C.A., Sweetman C., Schutz E., Soole K., Jenkins C., Hancock R.D., Bruning J.B, & Ford C.M. (2019). An aldo-keto reductase with 2-keto-l-gulonate reductase activity functions in l-tartaric acid biosynthesis from vitamin C in Vitis vinifera. The Journal of Biological Chemistry, 294(44), 15932-15946.

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Crystallization of Nxph1-LNS2 Complexes

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For crystallization of Nxph1‐LNS2 complexes, glycosylation of Nxph1 was prevented by mutating its N‐linked glycosylation sites to aspartic acid (Nxph1^3ND). Nxph1^3ND‐LNS2 complexes crystallized at 2–3 mg/ml in the 16% PEG 3350 with 2% Tacsimate pH 5.0 and 0.1 M sodium citrate tribasic condition from the PEG/Ion screen (Hampton Research). Nxph1^3ND‐LNS2 crystals were cryoprotected in mother liquor containing 10% glycerol and 20% PEG 3350 and frozen in liquid N₂. For the Nxph1^3ND‐LNS2^SS2− crystals, data were collected at the Advanced Photon Source (APS) using the microfocus NE‐CAT beamline 24‐ID‐C (Table 1). For the Nxph1^3ND‐LNS2^SS2A+ crystals, data were collected at the Stanford Synchrotron Radiation Light Source (SSRL) using the microfocus beamline 12‐2 (Table 1). For simplicity, we refer to Nxph1^3ND simply as Nxph1 when discussing the crystal structures. We note, however, that all biochemical studies were performed with Nxph1 without the 3ND mutations, as we were unable to prepare well‐behaved Nxph1^3ND alone.

Wilson S.C., White K.I., Zhou Q., Pfuetzner R.A., Choi U.B., Südhof T.C, & Brunger A.T. (2019). Structures of neurexophilin–neurexin complexes reveal a regulatory mechanism of alternative splicing. The EMBO Journal, 38(22), e101603.

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Structural Determination of Pf PNP-MMV000848 Complex

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The PfPNP-MMV000848 complex structure was obtained by incubating 37 mg/ml of PfPNP with 3 mM of MMV000848 on ice for 15 min followed by screening for crystallization conditions of co-crystals with commercial screening kits (JCSG-plus and Morpheus from Molecular Dimensions, PEG/Ion Screen from Hampton Research). Co-crystal screens were set up with a mosquito crystallization robot (TTP Labtech) and sitting-drop vapor diffusion method at 20 °C. Multiple crystal hits were observed after 5 days and left to grow up to 14 days. Crystals were fished in nylon loops and flash-frozen in liquid nitrogen for data collection. The complex structure of PfPNP and MMV000848 was obtained from co-crystals grown in buffer condition of JCSG+ C12 (10% (w/v) PEG 1000).
X-ray diffraction intensities were collected at the ANSTO MXI beamline, integrated, and scaled using XDS (52 (link)). Structures were solved with molecular replacement as implemented in the program Phaser (53 (link)) using the P. falciparum ligand-free structure (PDB access code: 5ZNI (31 )) as a search probe. Refinement was done using the Phenix (54 (link)) package and interspersed with manual model correction using Coot (55 (link)).

Chung Z., Lin J., Wirjanata G., Dziekan J.M., El Sahili A., Preiser P.R., Bozdech Z, & Lescar J. (2023). Identification and structural validation of purine nucleoside phosphorylase from Plasmodium falciparum as a target of MMV000848. The Journal of Biological Chemistry, 300(1), 105586.

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Crystallization of BcsX Protein

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Initial screening for crystallization conditions was performed at 18 °C using Index Screen, SaltRx1 Screen and PEG/Ion Screen (Hampton Research) and the hanging-drop vapor diffusion method with drops containing 1 µL of protein solution and 1 µL of reservoir solution. The initial crystals of BcsX were obtained using the following solutions: (1) 2.1 M DL-Malic acid pH 7.0; (2) 0.2 M sodium chloride, 0.1 M Tris pH 8.5, 25% PEG 3350; (3) 0.2 M ammonium acetate, 0.1 M HEPES pH 7.0, 25% PEG 3350. Crystals obtained using solutions 2 and 3 were cryoprotected by transfer into mother liquor containing 22.5 % (v/v) PEG 400 and flash-cooled in liquid nitrogen. X-ray diffraction measurements were carried out on beamline 14.1 of the BESSY II storage ring (Berlin, Germany; [57 (link)]).

Szymczak I., Pietrzyk-Brzezińska A.J., Duszyński K, & Ryngajłło M. (2022). Characterization of the Putative Acylated Cellulose Synthase Operon in Komagataeibacter xylinus E25. International Journal of Molecular Sciences, 23(14), 7851.

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Crystallization of CD1d-glycolipid-TCR Complexes

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Crystals were grown at 22.3 °C by sitting drop vapor diffusion, while mixing 0.5–1 µL of ternary complex solution with 0.5–1 µL of precipitate. All CD1d–glycolipid–TCR complexes were crystallized in conditions contained within the PEG/Ion-screen (Hampton Research) and needed no further optimization. 15a and 15o were crystallized in 0.2 m ammonium tartrate dibasic, 20% PEG 4000 (PEG/Ion 1, D2); 15m, 15p, 15q, 15s, 15w and 15x were crystallized in 0.1 m sodium malonate pH 4.0, 12% PEG 3350 (PEG/Ion 1, E1). Single crystals were harvested using a CryoLoop (Hampton Research) and flash cooled with liquid nitrogen (−196 °C) in the same precipitate mixture containing 20 v/v% glycerol. Samples were stored and shipped in liquid nitrogen.

Janssens J., Bitra A., Wang J., Decruy T., Venken K., Van der Eycken J., Elewaut D., Zajonc D.M, & Van Calenbergh S. (2018). 4”-O-Alkylated α-Galactosylceramide Analogues as iNKT-Cell Antigens: Synthetic, Biological and Structural Studies. ChemMedChem, 14(1), 147-168.

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Crystallization of ccGFP Variants

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Seven ccGFP variants, 5, E6, 7, 8, 9, were crystallized using the sitting drop vapor diffusion method. Protein and reservoir solutions of 0.1 μL each were mixed and equilibrated against 30 μL reservoir at 298 K using a PHOENIX crystallization robot (Art Robbins Instruments). A set of crystallization reagents consisting of Crystal Screens, PEG/Ion screens (Hampton Research), PACT suite (Qiagen), and JCSG core suites (Qiagen) was used to screen for the propensity of crystallization. Subsequent grid‐screens to optimize buffer pH, concentrations of salt and precipitants, and additives screens were employed as needed until diffraction‐quality crystals were obtained. The final crystallization conditions for the ccGFP variants reported herein are listed in Table S3.

Hung L., Terwilliger T.C., Waldo G.S, & Nguyen H.B. (2024). Engineering highly stable variants of Corynactis californica green fluorescent proteins. Protein Science : A Publication of the Protein Society, 33(2), e4886.

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Optimized Crystallization Protocol for Protein

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Initial crystallization screening was performed in sitting drops by mixing 1.0 ml protein solution with 1.0 ml reservoir solution of commercial screening kits such as SaltRx, Index, and PEG/ION screens (Hampton Research, USA) on 96-well Swissci MRC plates (SWISSCI, Switzerland) at 295 K. Several hits were obtained, and crystal optimization was performed by varying the concentration of the components. The final optimized composition was 12% (w/v) polyethylene glycol (PEG) 3350 and 0.1 M sodium acetate trihydrate. The size of the crystals reached the maximum dimensions after a month, with typical dimensions of 0.1 mm × 0.1 mm × 0.3 mm. For cryoprotection, the concentrations of glycerol and PEG 3350 were continuously increased to 20% (w/v) PEG 3350 and 20% (v/v) glycerol. The crystal was taken from the drop and directly flash-frozen in liquid nitrogen before data collection. Data were collected at the 11C beamline of the Pohang Accelerator Laboratory (Korea) using a PILATUS 3 6M detector (DECTRIS, Switzerland) at a wavelength of 0.9794 Å and a crystal-to-detector distance of 400.0 mm. A total of 360 images were collected with an exposure time of 1 s and 1° oscillation. The data-collection statistics are listed in Supplementary Table S1.

Kim S., Koh S., Kang W, & Yang J.K. (2022). The Crystal Structure of L-Leucine Dehydrogenase from Pseudomonas aeruginosa. Molecules and Cells, 45(7), 495-501.

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