Ternary 9°N crystals were prepared similar to the KOD crystals. The protein was mixed with the annealed p/t duplex (same sequence as for KOD crystals), ddCTP and dATP in a ratio of 1:1.2:3:10. MgCl2 and MnCl2 were added to a final concentration of 10mM each. After addition of ddCTP and dATP the mixture was incubated for 30min at 30°C each. The final protein concentration was 8.3mg/ml. After filtration through a 0.1 μm sterile filter crystallization was performed. Well diffracting crystals grew in condition E10 of the PEG RX screen (Hampton Research) containing 6% Isopropanol, 0.1M sodium acetate trihydrate pH 4.5 and 26% PEG 550 MME. Before freezing crystals were cryoprotected in the reservoir solution containing 20% Glycerol. The crystals containing two or three divalent metal ions in the active site were harvested from the same crystallization drop.
Pegrx screen
Manufactured by Hampton Research
Sourced in United Kingdom
The PEGRx screen is a laboratory equipment product designed for screening and optimization of protein crystallization conditions. It provides a comprehensive set of pre-formulated solutions containing varying concentrations of polyethylene glycol (PEG) and other commonly used crystallization reagents. The core function of the PEGRx screen is to facilitate the systematic exploration of PEG-based crystallization conditions for a wide range of proteins and macromolecules.
Automatically generated - may contain errors
Lab products found in correlation
Morpheus screen, by Molecular Dimensions (1 mentions)
Crystalquick 96 well x plates, by Greiner (1 mentions)
Swissci mrc plates, by Molecular Dimensions (1 mentions)
Structure 1 2, by Molecular Dimensions (1 mentions)
Saltrx, by Hampton Research (1 mentions)
Ampicillin sodium salt, by Merck Group (1 mentions)
Potassium phosphate dibasic, by Merck Group (1 mentions)
Chloramphenicol, by Merck Group (1 mentions)
Yeast extract, by Merck Group (1 mentions)
Hispur ni nta resin, by Thermo Fisher Scientific (1 mentions)
Deoxyribonuclease 1, by Merck Group (1 mentions)
L histidine monohydrochloride, by Merck Group (1 mentions)
Terrific broth, by Merck Group (1 mentions)
Ribonuclease a, by Merck Group (1 mentions)
Glycerol, by Merck Group (1 mentions)
Ethylenediaminetetraacetic acid, by Merck Group (1 mentions)
Phenylmethylsulfonyl fluoride, by Merck Group (1 mentions)
Potassium phosphate monobasic, by Merck Group (1 mentions)
7 protocols using pegrx screen
1
Ternary 9°N Crystals with Divalent Ions
2
Eukaryotic Translation Initiation Factor 4E Crystallization
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The murine eukaryotic translation initiation factor 4E (eIF4E, residues 28–217) was expressed and purified as described previously [35 (link)]. After the final purification step on a gel filtration column, eIF4E was stored in a buffer containing 20 mM HEPES pH 7.2, 100 mM KCl, 0.5 mM EDTA, and 2 mM DTT. For crystallization, aliquots of the protein concentrated to 4.75 mg/mL (Amicon 10 kDa cut-off Ultra Centrifugal Filter Device) were incubated with 1 mM of the cap analogs 1a , 1c and 1h at rt for approximately 15 min. Crystallization trials were performed at 18 °C using the sitting-drop vapor diffusion method. Initial crystallization hits were identified in PEGRx Screen (Hampton Research). After the optimization of crystal growth conditions using the hanging-drop vapor diffusion method, the best diffracting crystals were obtained in: 0.1 M Bis-Tris propane pH 9.0, 0.1 M NaCl, 25% w/v PEG 1500 for complex eIF4E–1a (Bn7GpppG); 0.1 M sodium citrate tribasic pH 5.5, 20% PEG 3350 for complex eIF4E–1c (3-MeBn7GpppG); 0.1 M Bicine pH 8.5, 0.2 M sodium formate, 19% PEGMME 5000 for complex eIF4E–1h (4-Cl-Bn7GpppG). Crystals were cryoprotected in 30% glycerol and flash frozen in liquid nitrogen.
3
Protein Crystallization and Radiolabeled Assays
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Reagents. For crystallization, we used the PEGRx screen (Hampton Research). For the biochemical experiments, we used α-33P-dATP 3000 Ci/mmol, 10 mCi/ml, 250 uCi, Hartman Analytics.
Biological resources. The present study used the following E. coli strains: BL21 Star (DE3) (F−ompT hsdSB [rB− mB−] gal dcm+rne131 [DE3]), BL21 Gold (DE3) (B F−ompT hsdS [rB− mB−] dcm+ Tetrgal alt hsdSendA Hte), Top10 (F−mcrA crAmrr-hsdRMS-mcrBC] rr-lacZΔa15 ΔlacX74 recA1 araD139 acara-leu]7697 galU galK rpsL [Strr] endA1 nupG) and BW25113 K-12 strain (F−, Δ(araD-araB)567, ΔlacZ4787(::rrnB-3), λ−, rph-1, Δ(rhaD-rhaB)568, hsdR514).
Biological resources. The present study used the following E. coli strains: BL21 Star (DE3) (F−ompT hsdSB [rB− mB−] gal dcm+rne131 [DE3]), BL21 Gold (DE3) (B F−ompT hsdS [rB− mB−] dcm+ Tetrgal alt hsdSendA Hte), Top10 (F−mcrA crAmrr-hsdRMS-mcrBC] rr-lacZΔa15 ΔlacX74 recA1 araD139 acara-leu]7697 galU galK rpsL [Strr] endA1 nupG) and BW25113 K-12 strain (F−, Δ(araD-araB)567, ΔlacZ4787(::rrnB-3), λ−, rph-1, Δ(rhaD-rhaB)568, hsdR514).
4
Optimizing AbiA Protein Crystallization
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A purified AbiA protein without SUMO tag was initially used for crystallization. It did not, however, produce any crystals. On the contrary, AbiA with an uncleaved SUMO tag crystallized in a wide range of concentrations and conditions. Because of this, we continued the crystallization experiments with this protein variant. Protein crystals were grown in the PEGRx screen (Hampton Research) and further optimized manually. The following crystallization robots were used: Phoenix (Art Robbins Instruments, Sunnyvale, CA, USA) and Oryx (Douglas Instruments, Hungerford, UK). Crystals that diffracted X-rays to the highest resolution were obtained at a protein concentration of 5 mg/ml under the following conditions: 0.2 M sodium potassium tartrate tetrahydrate, 0.1 M Bis–Tris (pH 6.0), and 9% PEG 10 000. For crystallization, both the hanging-drop and sitting-drop vapor diffusion methods were used at 18°C. For data collection, the crystals were flash frozen in liquid nitrogen.
5
SARS-CoV-2 RBD Fab Complex Crystallization
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FD-5D and FI-3A Fabs were mixed with RBD separately with a final concentration of 13 mg/mL. FI-3A and FD-11A Fabs were mixed with RBD in a 1:1:1 molar ratio with a final concentration of 7 mg/mL. After incubation at room temperature for 30 min, the samples were used for initial screening of crystals in Crystalquick 96-well X plates (Greiner Bio-One) with a Cartesian Robot using the nanoliter sitting-drop vapor-diffusion method with 100 nL of protein and 100 nL of reservoir in each drop, as previously described 18 (link). Crystals of RBD/FD-5D Fab complex were obtained from Molecular Dimensions Morpheus screen, condition C9 containing 10% (w/v) PEG 20 000, 20% (v/v) PEG MME 550, 0.03 M of each NPS (NaNO3; Na2HPO4; (NH4)2SO4) and 0.1 M bicine/Trizma base pH 8.5. Tiny Crystals of RBD/FI-3A Fab complex were first formed in Hampton Research PEGRx screen, condition A10, containing 0.1 M sodium citrate tribasic and 30% (w/v) PEG 550 and further optimized in 0.08 M sodium citrate tribasic and 24% (w/v) PEG 550. Crystals of RBD/FI-3A/FD-11A Fabs complex were formed in Hampton Research Index screen, condition 25, containing 3.5 M sodium formate pH 7.0 and further optimized in 2.9 M sodium formate pH 7.0.
6
Optimizing Protein Crystallization Conditions
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Initial crystallization screening was performed at two protein concentrations: 4.2 and 10.9 mg ml−1. The vapor-diffusion method was performed in sitting drops by mixing 0.6 µl protein solution with 0.6 µl reservoir solution using 96-well Swissci MRC plates (Molecular Dimensions, Suffolk, UK) at 291 K. The commercial Structure 1 + 2 (Molecular Dimensions), Index, SaltRx and PEGRx screens (Hampton Research) were assessed. Several hits were obtained and crystal optimization was performed in Swissci 48-Well MRC Maxi Optimization Plates. The best diffraction crystals derived from these optimizations were obtained by mixing 1.5 µl protein solution at 10.9 mg ml−1 with 1.5 µl reservoir solution consisting of 0.1 M sodium acetate pH 4.5, 32% PEG 300 (Table 2 ▸ ). The crystals were cryocooled in liquid nitrogen without any cryoprotection prior to data collection.
7
Heterologous Protein Expression and Purification
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Tryptone enzymatic digest, yeast extract, glycerol, potassium phosphate dibasic, potassium phosphate monobasic, L-histidine monohydrochloride, ampicillin sodium salt, phenylmethylsulfonyl fluoride (PMSF), sodium chloride, ethylenediaminetetraacetic acid (EDTA), chloramphenicol, Terrific Broth, deoxyribonuclease I and ribonuclease A were purchased from Sigma-Aldrich (St. Louis, MO, USA). Difco Luria-Bertani broth lennox from Becton Dickinson (Franklin Lakes, NJ, USA). Isopropyl β-F-1-thiogalactopyranoside (IPTG) Dioxane-Free and 5-aminolevulinic acid hydrochloride were obtained from BOC Sciences (Upton, NY, USA). CHAPS(2-2-Cholamidopropyldimethylammonio)-1-propanesulfonate and Tris(2-carboxyethyl)phosphine hydrochlorate (TCEP) were obtained from Soltec venture (Beverly, MA, USA). HisPur Ni-NTA resin was from ThermoFisher Scientific (Waltham, MA, USA). CM Macroprep was from Bio-Rad (Hercules, CA, USA). 5-Cyclohexyl-1-hexylpentyl-β-D-maltoside (CYMAL-5) was obtained from Anatrace (Maumee, OH, USA). The 3α-Hydroxyl-7α,12α-di-(((2-(trimethylamino)ethyl)phosphoryl)ethyloxy)-cholane (FA-7/234-chol) is a facial amphiphile used as described previously [31 (link)]. PEGRx screens were obtained from Hampton Research. Human cytochrome P450 reductase was obtained from OriGene (Rockville, MD, USA).
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