Cryschem plate

Manufactured by Hampton Research

Sourced in United States

Cryschem plates are laboratory equipment used for protein crystallization experiments. They provide a controlled environment for the growth and observation of protein crystals. The plates feature multiple wells or compartments to hold and separate individual crystallization conditions.

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Lab products found in correlation

Microloop, by MiTeGen (2 mentions) Cryoloop, by Hampton Research (2 mentions) E coli polar extract, by Avanti Polar Lipids (2 mentions) Insulin from porcine pancreas, by Merck Group (1 mentions) Intelli plate, by Hampton Research (1 mentions) Jcsg core suite, by Qiagen (1 mentions) Pilatus3 s 6m, by Dectris (1 mentions) Thaumatin from thaumatococcus danielii, by Merck Group (1 mentions) Bradford reagent, by Bio-Rad (1 mentions) Amicon ultra, by Merck Group (1 mentions) Crystal screen, by Hampton Research (1 mentions) Peg ion, by Hampton Research (1 mentions) Natrix, by Hampton Research (1 mentions)

15 protocols using cryschem plate

Crystallization of lmDegU Protein Domain

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The purified lmDegU_DBD protein was concentrated to 11.6 mg/ml for crystallization. lmDegU_DBD crystals were obtained by performing a sitting-drop vapor-diffusion method using a 24-well Cryschem plate (Hampton Research). For crystallization, 0.5 μl of the lmDegU_DBD protein was mixed with 0.5 μl of a well solution containing 22% PEG 3350 and 0.1 M Tris, pH 8.0, and was equilibrated via vapor diffusion against 500 μl of the well solution at 18 °C. A lmDegU_DBD crystal was briefly soaked in 25% glycerol, 24% PEG 3350, and 0.1 M Tris, pH 8.0, for cryoprotection and flash-cooled at − 173 °C under a nitrogen gas stream. X-ray diffraction data from a single lmDegU_DBD crystal were collected at beamline 7A, Pohang Accelerator Laboratory. The X-ray diffraction data were processed and scaled using the HKL2000 program^{38 (link)}. The data collection statistics are listed in Supplementary Table S1.

Oh H.B., Lee S.J, & Yoon S.I. (2022). Structural and biochemical analyses of the flagellar expression regulator DegU from Listeria monocytogenes. Scientific Reports, 12, 10856.

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Optimized Crystallization and Structural Determination

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cjRecO crystallization was performed at 18 °C via a sitting-drop vapor-diffusion method by equilibrating an equivolume mixture of protein and a crystallization solution against a reservoir solution in a Cryschem plate (Hampton Research, Aliso Viejo, CA, USA). The native cjRecO protein was crystallized in a solution containing 20% PEG 3350 and 0.2 M sodium formate, and the resulting crystal was cryoprotected in 22% PEG 3350, 0.2 M sodium formate, and 25% ethylene glycol. SeMet-cjRecO crystals were obtained using 22% PEG 3350 and 0.1 M Tris, pH 8.5, and were subjected to cryoprotection in 25% PEG 3350, 0.1 M Tris, pH 8.5, and 25% glycerol. The cryoprotected crystal was flash-cooled under a nitrogen gas cryostream. The X-ray diffraction of the cjRecO crystal was carried out at the Pohang Accelerator Laboratory, beamline 7A. The diffraction data were processed using the HKL2000 program [27 ].

Lee S.J., Oh H.B, & Yoon S.I. (2022). Crystal Structure of the Recombination Mediator Protein RecO from Campylobacter jejuni and Its Interaction with DNA and a Zinc Ion. International Journal of Molecular Sciences, 23(17), 9667.

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Crystallization of HIV-1 Env Complexes

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Fab NC-Cow1 (25 mg/ml) and the BG505 SOSIP.664-Fab NC-Cow1-Fab 35022-Fab PGT128 quaternary complex (15 mg/ml) were both screened for crystallization on our robotic Rigaku CrystalMation system at Scripps against 384 different conditions at both 4° and 20°C. The NC-Cow1 crystal used for data collection was grown at 4°C with a precipitant of 0.1 M sodium cacodylate, 40% methyl-pentanediol, and 5% PEG-8000 (polyethylene glycol, molecular weight 800) (pH 6.5). SOSIP-NC-Cow1-35022-PGT128 crystallized in several crystallization conditions, all with low–molecular weight PEG in the precipitant mix, but most of these crystals diffracted x-rays very poorly. The crystal used for data collection was grown in a 24-well Cryschem plate (Hampton Research) at 20°C with precipitant of 0.2 M sodium citrate, 0.1 M tris, and 30% PEG-400 (pH 8.6).

Stanfield R.L., Berndsen Z.T., Huang R., Sok D., Warner G., Torres J.L., Burton D.R., Ward A.B., Wilson I.A, & Smider V.V. (2020). Structural basis of broad HIV neutralization by a vaccine-induced cow antibody. Science Advances, 6(22), eaba0468.

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EnpA^CD H109A Protein Crystallization

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EnpA_CD H109A crystals were grown using the sitting-drop vapor-diffusion method at 291 K. A crystallization robot (Phoenix, Art Robbins Instruments) was used to mix 0.2 µL of JCSG-plus screen buffers (Molecular Dimensions) with an equal volume of 15 mg/mL protein solution in 50 mM Tris-HCl buffer pH 7.5 and 200 mM NaCl on 96-well crystallization plates (Molecular Dimensions). The crystals appeared after 3 days in a buffer consisting of 0.8 M succinic acid, pH 7.0. Crystallization in this condition was repeated on a 24-well Cryschem Plate (Hampton Research) by mixing 4.0 µL of 9.8 g/L protein solution with 2.0 µL of well solution. Again, the crystals appeared after 3 days and were harvested on 10th day, when they reached the largest size (co-crystallization with pentaglycine was also attempted, but no crystals were obtained). The crystals were cryo-protected by quickly immersing in the well solution mixed in a 1:1 volume ratio with 50% (v/v) glycerol and flash cooled in liquid nitrogen.

Małecki P.H., Mitkowski P., Jagielska E., Trochimiak K., Mesnage S, & Sabała I. (2021). Structural Characterization of EnpA D,L-Endopeptidase from Enterococcus faecalis Prophage Provides Insights into Substrate Specificity of M23 Peptidases. International Journal of Molecular Sciences, 22(13), 7136.

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Insulin Crystallization Protocol

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Insulin from porcine pancreas (catalogue No. I5523) was purchased from Sigma–Aldrich GmbH, Buchs, Switzerland and dissolved in 50 mM Na₂HPO₄, 10 mM EDTA pH 10.5 to a final concentration of 25 mg ml⁻¹. Crystals were grown using two different methods for different parts of the investigation. The crystals used in Sections 3.1 and 3.4 were grown in batch by mixing 0.5 ml of the insulin solution in a 1:1 ratio with the crystallization buffer [25%(w/v) PEG 6000, 0.1 M bis-Tris propane pH 7.5, 0.2 M sodium bromide] in a centrifuge tube at 20°C. The centrifuge tube was briefly vortexed and left on a revolver/rotator at 20°C. The mean crystal size and concentration were 30 µm and 1.5 × 10⁶, respectively. The crystals used in Sections 3.2 and 3.3 were grown overnight at 20°C in 24-well sitting-drop vapor-diffusion CrysChem plates (Hampton Research, USA) against 500 µl reservoir. The drops contained 2 µl insulin solution and 2 µl crystallization buffer.

Martiel I., Beale J.H., Karpik A., Huang C.Y., Vera L., Olieric N., Wranik M., Tsai C.J., Mühle J., Aurelius O., John J., Högbom M., Wang M., Marsh M, & Padeste C. (2021). Versatile microporous polymer-based supports for serial macromolecular crystallography. Acta Crystallographica. Section D, Structural Biology, 77(Pt 9), 1153-1167.

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Inactivation and Crystallization of Human OAT

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Potassium pyrophosphate buffer (500 μL, 100 mM, pH 8.0), containing human OAT (2.2 mg, 49 nmol), α-ketoglutarate (10 mM), β-mercaptoethanol (10 mM), and FCP (50 mM), was protected from light and incubated at room temperature for 14 h until the human OAT was inactivated (activity of ≲0.1%). The inactivated OAT was then concentrated to 10 mg/mL using a 30000 MWCO Amicon Ultra centrifugal filter device, and the concentrated protein was exchanged into a crystallization buffer [50 mM Tricine HCl (pH 7.8) and 10 μM PLP]. Crystals were grown using previously published conditions.^{14 (link)} Crystallization was performed in 24-well Cryschem Plates (Hampton Research). Crystals with the best morphology appeared in the well solution containing NaCl (175 mM) and 8–10% (v/v) PEG 6000 after incubation for 1 week at room temperature. OAT crystals with the best morphology were transferred into a cryo-protecting solution [well solution supplemented with 25% (v/v) glycerol] before being flash-cooled in liquid nitrogen.

Mascarenhas R., Le H.V., Clevenger K.D., Lehrer H.J., Ringe D., Kelleher N.L., Silverman R.B, & Liu D. (2017). Selective Targeting by a Mechanism-Based Inactivator against Pyridoxal 5′-Phosphate-Dependent Enzymes: Mechanisms of Inactivation and Alternative Turnover. Biochemistry, 56(37), 4951-4961.

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Structural Determination of LbaCas13a-crRNA Complex

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Initial screening was carried out with JCSG Core Suites (Qiagen) in
96-well sitting drop vapor diffusion format (Intelli-plate, Hampton Research) at
293 ± 1 K. Diffraction-quality crystals of the LbaCas13a:crRNA complex
(20-nt and 24-nt containing spacer) (A₂₈₀=4.0) and
LbaCas13a:pre-crRNA complex (24-nt containing spacer)
(A₂₈₀=5.7) grew in 0.2 M ammonium iodide and 20%
(w/v) PEG 3,350. Crystals were cryoprotected in 10–15% (v/v)
glycerol, and high-resolution diffraction data were collected at the Advanced
Light Source (ALS) beam line 8.3.1 on a Pilatus3 S 6M (Dectris) detector under
cryogenic conditions. To produce isomorphous crystals for phasing by native
single-anomalous dispersion (SAD), sitting drop vapor diffusion experiments
containing 2 μL purified LbaCas13a:crRNA complex (24-nt containing
spacer) complex (A₂₈₀=4.0) and 2 μL reservoir
solution (0.2 M ammonium iodide, 20–25% (w/v) PEG 3,350) were
prepared in 24-well Cryschem plates (Hampton Research). A total of 52 datasets
were collected from 26 crystals (cryoprotected in 10% (v/v) glycerol)
under cryogenic conditions at 6.0 keV (ALS beam line 8.3.1, Pilatus3 S 6M). Each
dataset covered 360° of crystal rotation divided into 0.2°
images collected in 45° inverse-beam wedges at 5 Hz. To minimize pixel
readout error, the detector was moved by 10 mm before collecting a second
360°-pass from the same position of each crystal.

Knott G.J., East-Seletsky A., Cofsky J.C., Holton J.M., Charles E., O’Connell M.R, & Doudna J.A. (2017). Guide-bound structures of an RNA-targeting A-cleaving CRISPR-Cas13a enzyme. Nature structural & molecular biology, 24(10), 825-833.

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Thaumatin Protein Crystallization Protocol

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Thaumatin from Thaumatococcus danielii was purchased from Sigma–Aldrich GmbH (Buchs, Switzerland). Crystals were grown at 20°C using the vapour-diffusion method in sitting-drop CrysChem plates (Hampton Research, USA). Drops were composed of 2 µl of thaumatin solution (50 mg ml⁻¹ dissolved in water) and 2 µl of reservoir solution (24% potassium sodium tartrate tetrahydrate, 100 mM bis-Tris propane pH 6.5) and were set up over wells containing 500 µl of reservoir solution. Crystals appeared overnight and grew over three to five days to a final size of 800 × 400 µm. The crystals were crushed in order to produce a seed stock, which was used to nucleate the growth of microcrystals as described below.
Micrometre-sized crystals were obtained by gently mixing 400 µl of thaumatin dissolved in 100 mM Na HEPES pH 7.0 at 88 mg ml⁻¹ with 400 µl of the precipitant (1.6 M potassium sodium tartrate tetrahydrate, 100 mM Na HEPES pH 7.0) supplemented with 20 µl of the seed stock. Microcrystals grew overnight to a size of ∼20 µm in length and 10 µm in width. The microcrystalline slurry was then centrifuged at 8000g for two minutes and washed twice with the washing solution (0.8 M potassium sodium tartrate, 100 mM Na HEPES pH 6.8).

Nass K., Cheng R., Vera L., Mozzanica A., Redford S., Ozerov D., Basu S., James D., Knopp G., Cirelli C., Martiel I., Casadei C., Weinert T., Nogly P., Skopintsev P., Usov I., Leonarski F., Geng T., Rappas M., Doré A.S., Cooke R., Nasrollahi Shirazi S., Dworkowski F., Sharpe M., Olieric N., Bacellar C., Bohinc R., Steinmetz M.O., Schertler G., Abela R., Patthey L., Schmitt B., Hennig M., Standfuss J., Wang M, & Milne C.J. (2020). Advances in long-wavelength native phasing at X-ray free-electron lasers. IUCrJ, 7(Pt 6), 965-975.

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Crystallization of GLIC Receptor

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GLIC wt in 100 mM NaCl, 10 mM Tris-HCl (pH 7.4), and 0.5 mM DDM was concentrated to between 9–10 mg/ml with an Amicon Ultra 50 KDa cutoff concentrator (EMD Millipore, Billerica, MA). Prior to crystallization setup, the protein was supplemented with 50 μM DHA (from 300 mM DHA stock in ethanol) and 0.5 mg/ml E.coli polar extract (Avanti Polar Lipids) and incubated on ice for 1 hr. The protein was crystallized at 4°C by sitting drop vapor diffusion in Cryschem plates (Hampton Research, Aliso Viejo, CA) with a 1:1 mixture (1 μl each) of protein and reservoir solution (225 mM ammonium sulfate, 50 mM sodium acetate, pH 3.9–4.2 and 9–12% PEG4000). Crystals typically formed within one week and typically took 2–3 weeks to reach full size. The crystals were cryoprotected by adding 6 μL reservoir solution supplemented with 30% ethyleneglycol to the drop, and directly frozen in liquid nitrogen using appropriately sized microloops (MiTeGen, Ithaca, NY) or cryoloops (Hampton Research).

Basak S., Schmandt N., Gicheru Y, & Chakrapani S. (2017). Crystal structure and dynamics of a lipid-induced potential desensitized-state of a pentameric ligand-gated channel. eLife, 6, e23886.

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Crystallization of QTRT1 Enzyme

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QTRT1 was crystallized using sitting-drop or hanging drop vapor diffusion technique with protein concentration adjusted to 6 mg/mL as determined with Bradford reagent (Bio-Rad, Hercules, CA, USA). Equal volumes of protein containing solution and crystallization condition (100 mM Tris pH 7.8, 200 mM KBr, 200 mM KSCN, 3% (w/v) γ-polyglutamic acid-LM (PGA-LM), 5% PEG 4000) were mixed. Crystallization was performed in EasyXtal-15-well tool plates (Qiagen, Hilden, Germany) with the cap loosened at a ¼ turn to allow for evaporation or in loosely sealed 24 well Cryschem plates (Hampton Research, Aliso Viejo, CA, USA). Crystals were obtained after five to seven days at 20 °C from crystallization drops of 0.5–6 µL total volume. To obtain QTRT1 in complex with queuine, respective crystals were soaked in the crystallization condition containing the free queuine base at 50 µM concentration. Crystals were cryo-protected by a stepwise increase of glycerol and PEG400 concentration to 15% (v/v) each.

Johannsson S., Neumann P, & Ficner R. (2018). Crystal Structure of the Human tRNA Guanine Transglycosylase Catalytic Subunit QTRT1. Biomolecules, 8(3), 81.

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