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Ultraufoil r1.2 1 3 300 mesh grid

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The UltrAuFoil R1.2/1.3 300-mesh grids are a type of specimen support structure used in electron microscopy. They consist of a gold (Au) thin film with a grid pattern supported on a copper (Cu) mesh. The grids provide a stable and electrically conductive platform for mounting samples for imaging and analysis in transmission electron microscopes (TEMs) and scanning transmission electron microscopes (STEMs).

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Lab products found in correlation

Vitrobot mark 4, by Thermo Fisher Scientific (13 mentions) Whatman no 1 filter paper, by Cytiva (6 mentions) Em gp automatic plunge freezer, by Leica (4 mentions) K3 direct electron detector, by Ametek (3 mentions) Solarus plasma cleaner, by Ametek (3 mentions) Superdex 200 increase column, by GE Healthcare (3 mentions) Pelco easiglow, by Ted Pella (3 mentions) Superose 6 10 300 gl column, by GE Healthcare (2 mentions) Atpγs, by Merck Group (2 mentions) Titan krios microscope, by Thermo Fisher Scientific (2 mentions) Pelco easiglow device, by Ted Pella (2 mentions) Lauryl maltose neopentyl glycol, by Anatrace (2 mentions) Em gp2 plunge freezer, by Leica (2 mentions) Mpp iodide, by Merck Group (2 mentions) Epu software, by Thermo Fisher Scientific (2 mentions) Em gp2, by Leica (2 mentions) Superdex200 10 30, by GE Healthcare (1 mentions) Em gp2 automatic plunge freezer, by Leica (1 mentions) Pelco easiglow glow discharge cleaning system, by Ted Pella (1 mentions) 595 blotting paper, by Cytiva (1 mentions)

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UltrAuFoil (17 citations) UltrAuFoil grids (10 citations) UltrAuFoil 2/2 grids (5 citations) Quantifoil 2/2 grids (4 citations)

27 protocols using ultraufoil r1.2 1 3 300 mesh grid

1

Structural Analysis of CtLas1-Grc3 Complex

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Purified CtLas1-Grc3 complex (10 μM) was cross-linked with 50 μM bis(sulfosuccinimidyl)suberate (BS3; Sigma) in 20 mM Hepes pH 7.7, 200 mM NaCl, 5 mM MgCl2, 5% glycerol at room temperature for 5 minutes before quenching with 30 mM Tris pH 7.5 for 15 minutes at 4°C. Cross-linked reactions were resolved over a Superose 6 10/300 GL column (GE Healthcare) in 10 mM Tris pH 8.0, 200 mM NaCl, 5 mM MgCl2. CtLas1-Grc3 (0.15 mg/ml) was incubated in the absence (apo state) and presence (ATP-γS bound state) of 2 mM ATP-γS (Sigma) and 10 μM CT-ITS2-RNA (5′-UGUGUUGGGGdeoxyACCCGCGGCUGCUCG CGGGCCCUGAAAAGCA-3′) for 1 hour at 4°C. The CT-ITS2-RNA substrate represents part of the C. thermophilum ITS2 pre-rRNA sequence. Protein mixtures (3 μL) were applied to glow-discharged UltrAuFoil R1.2/1.3 300 mesh grids (Quantifoil) and vitrified after 5 sec blotting using the Automatic Plunge Freezer EM GP (Leica).
Pillon M.C., Hsu A.L., Krahn J.M., Williams J.G., Goslen K.H., Sobhany M., Borgnia M.J, & Stanley R.E. (2019). Cryo-EM Reveals Active Site Coordination Within a Multienzyme pre-rRNA Processing Complex. Nature structural & molecular biology, 26(9), 830-839.
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2

Structural Analysis of Mfd-DNA Complex

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Size-exclusion chromatography (Superdex 200 10/30, GE Healthcare) was performed prior to complex reconstitution in a buffer containing 20 mm Tris-HCl pH 8, 100 mm NaCl, 20 mm MgCl2, 2 mm TCEP. For complex formation, Mfd was incubated with 21mer dsDNA in a 1:1.5 molar excess in the presence of 2 mm ADP, 10 mm NaF, and 1 mm AlCl3. 2.5 µl Mfd-DNA complex at 1.0 mg/mL was applied to UltrAuFoil R1.2/1.3 300 mesh grids (Quantifoil) that were previously plasma-cleaned using a Gatan Solarus (75% argon/25% oxygen atmosphere, 15 W for 7 s), then manually blotted with a Whatman No. 1 filter paper in a cold room with >80% humidity, and plunged into liquid ethane using a manual plunger. The dsDNA used for reconstitution had the following sequence: 5′-ATAGGATACTTACAGCCATCG-3′.
Brugger C., Zhang C., Suhanovsky M.M., Kim D.D., Sinclair A.N., Lyumkis D, & Deaconescu A.M. (2020). Molecular determinants for dsDNA translocation by the transcription-repair coupling and evolvability factor Mfd. Nature Communications, 11, 3740.
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3

Cryo-EM Analysis of NPR1–TGA3–LS7-DNA Complex

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Cryo-EM grids containing the NPR1–TGA3–LS7-DNA complex were prepared using the Leica EM GP2 Automatic Plunge Freezer at 4 °C and 85% humidity. The UltrAuFoil R1.2/1.3 300-mesh grids (Quantifoil) were glow-discharged using the PELCO easiGlow Glow Discharge Cleaning System (Ted Pella). A 3 μl sample of NPR1–TGA3–LS7-DNA (~1.3 mg ml−1) was applied to the grid, incubated for 60 s in the chamber and blotted for 2.4 s with Whatman No. 1 filter paper (Whatman International) to remove excess sample, and then plunge-frozen into liquid ethane cooled by liquid nitrogen. A total of 9,442 micrograph stacks were collected with SerialEM35 (link) on a Titan Krios microscope (Thermo Fisher Scientific) at 300 kV equipped with a K3 direct electron detector (Gatan), at a nominal magnification of 81,000× and defocus values from −2.0 μm to −1.0 μm, yielding a resolution of 1.066 Å px−1. Each stack was exposed for 8.3 s with an exposing time of 0.138 s per frame, resulting in 60 frames per stack. The total dose was approximately 60 e− Å−2 per stack.
Kumar S., Zavaliev R., Wu Q., Zhou Y., Cheng J., Dillard L., Powers J., Withers J., Zhao J., Guan Z., Borgnia M.J., Bartesaghi A., Dong X, & Zhou P. (2022). Structural basis of NPR1 in activating plant immunity. Nature, 605(7910), 561-566.
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4

Cryogenic Protein Sample Preparation

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4-μL volumes of the respective samples were applied to UltrAuFoil R1.2/1.3 300-mesh grids (Quantifoil Micro Tools GmbH; Germany) that were prior glow discharged in a Pelco easiGlow device (Ted Pella; CA) at 10 mA for 90 s (IM172N22 complex) or at 15 mA for 30 s (IM459 complex). Grids were blotted for 3 s at −3 blot-force setting in a Vitrobot mark IV (ThermoFisher Scientific; operated at 4 °C and 100% humidity) before being plunge frozen in liquid ethane.
Kirk N.S., Chen Q., Wu Y.G., Asante A.L., Hu H., Espinosa J.F., Martínez-Olid F., Margetts M.B., Mohammed F.A., Kiselyov V.V., Barrett D.G, & Lawrence M.C. (2022). Activation of the human insulin receptor by non-insulin-related peptides. Nature Communications, 13, 5695.
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5

Affinity Purified Ribosomes Vitrification

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Affinity purified ribosomes at a concentration of ∼ 65 nM (A260 of 3.2) were vitrified on UltrAuFoil R1.2/1.3 300-mesh grids (Quantifoil), coated with graphene oxide (GO). For GO coating, gold grids were washed with deionized water, dried and subsequently glow-discharged for 5 min with an Edwards glowdischarger at 0.1 torr and 30 mA. 3 μl of a 0.2 mg/ml GO suspension in deionized water (Sigma) was pipetted onto the glow-discharged grids and incubated for 1 min. Next the GO solution was blotted away, and the grids were washed 3x by dipping into 20 μl deionized water drops followed by blotting (washed twice the top-side and once the bottom-side). 3 μl sample was pipetted onto the grids, blotted for 5.5 sec, -15 blot force, 0 sec wait at 100% humidity, 4°C, Whatman 595 blotting paper, with a Vitrobot Mark IV and plunge frozen into liquid ethane. Grids were stored in liquid nitrogen until data-collection. The dataset was collected with a Gatan K3 camera on a Titan Krios4 microscope at eBIC (Diamond) in super-resolution counting mode and binning 2, using EPU software in faster acquisition mode (AFIS), yielding 20932 micrographs (105000x magnification, pixel size= 0.829 Å, total dose 44.7 e-/Å2, 44 frames, resulting in 1 e-/ Å2 dose/frame). Refer to Table S1 for data collection statistics.
Höpfler M., Absmeier E., Peak-Chew S.Y., Vartholomaiou E., Passmore L.A., Gasic I, & Hegde R.S. (2023). Mechanism of ribosome-associated mRNA degradation during tubulin autoregulation. Molecular Cell, 83(13), 2290-2302.e13.
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6

Structural Analysis of dGTPase by CryoEM

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About 1 mg of dGTPase was thawed on ice and then run over a 10/30 Superdex 200 Increase column (GE Healthcare) in buffer D (20 mM Tris [pH 7.5], 100 mM NaCl). The peak fraction (approximately 0.2 mg/ml hexamer) was used directly to set grids. The protein was mixed with appropriate substrates and MgCl2 (5 mM free concentration) at room temperature and used to set grids within 30 min (WT apo: 5 mM MgCl2; WT + dATP: 10 mM dATP and 15 mM MgCl2; H125A + dGTP: 10 mM dGTP and 15 mM MgCl2; H125A + dGTP + dATP: 10 mM dATP, 10 mM dGTP, and 25 mM MgCl2). Grids were prepared by applying 3 μl of the mixtures to glow-discharged UltrAuFoil R 1.2/1.3300 mesh grids (Quantifoil Micro Tools GmbH) followed by 2.5 to 3.5 s of blotting and vitrification in liquid ethane using an Automatic Plunge Freezer EM GP (Leica).
Klemm B.P., Sikkema A.P., Hsu A.L., Horng J.C., Hall T.M., Borgnia M.J, & Schaaper R.M. (2022). High-resolution structures of the SAMHD1 dGTPase homolog from Leeuwenhoekiella blandensis reveal a novel mechanism of allosteric activation by dATP. The Journal of Biological Chemistry, 298(7), 102073.
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7

Cryo-EM Sample Preparation for mSerRS-mtRNA Complex

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The human mSerRS-mtRNASer(UGA) complex was diluted to a concentration of 0.75 mg/ml and samples were mixed with 0.05% v/v Lauryl Maltose Neopentyl Glycol (Anatrace) immediately prior to plunge freezing. UltrAuFoil R1.2/1.3 300-mesh grids (Quantifoil) were plasma cleaned in a Solarus plasma cleaner (Gatan, Inc.) with a 75% nitrogen, 25% oxygen atmosphere at 15 W for 7 s. Cryo-EM grids were prepared by application of 4 μL protein sample at 4 °C in 95% humidity. The grids were manually blotted for 4-5 s using Whatman No. 1 filter paper, followed by plunge freezing in liquid ethane.
Kuhle B., Hirschi M., Doerfel L.K., Lander G.C, & Schimmel P. (2023). Structural basis for a degenerate tRNA identity code and the evolution of bimodal specificity in human mitochondrial tRNA recognition. Nature Communications, 14, 4794.
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8

Cryo-EM Grid Preparation and Data Acquisition

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UltrAuFoil R1.2/1.3 300 mesh grids (Quantifoil) were glow discharged in a Pelco easiGlow (TedPella Inc) for 90 seconds at 25 mA current. Using a Vitrobot Mark IV (ThermoFisher) grids were prepared at 4 °C, 100 % RH, blot force of 5, 0 s wait and drain time, and with blot time ranging from 2 to 8 second. Sample was diluted from 1.8 mg/mL to 0.1 mg/mL prior to freezing. Movies were collected using SerialEM on a Titan Krios G3 cryoTEM (Thermo Fisher) operating at 300 kV, equipped with BioQuantum Energy Filter (20 eV slit width, Gatan) and a K3 direct electron detector (Gatan) using beam image-shift in a 3×3×3 pattern. A total of 3618 movies were collected at 105,000 magnification in super resolution mode with the corrected pixel size of 0.427 Å/pix, ~60 electros/Å2 total dose, and 40 frames per movie.
Lyu M., Malyutin A.G, & Stadtmueller B.M. (2023). The structure of the teleost Immunoglobulin M core provides insights on polymeric antibody evolution, assembly, and function. bioRxiv.
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9

Structural Analysis of CtLas1-Grc3 Complex

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Purified CtLas1-Grc3 complex (10 μM) was cross-linked with 50 μM bis(sulfosuccinimidyl)suberate (BS3; Sigma) in 20 mM Hepes pH 7.7, 200 mM NaCl, 5 mM MgCl2, 5% glycerol at room temperature for 5 minutes before quenching with 30 mM Tris pH 7.5 for 15 minutes at 4°C. Cross-linked reactions were resolved over a Superose 6 10/300 GL column (GE Healthcare) in 10 mM Tris pH 8.0, 200 mM NaCl, 5 mM MgCl2. CtLas1-Grc3 (0.15 mg/ml) was incubated in the absence (apo state) and presence (ATP-γS bound state) of 2 mM ATP-γS (Sigma) and 10 μM CT-ITS2-RNA (5′-UGUGUUGGGGdeoxyACCCGCGGCUGCUCG CGGGCCCUGAAAAGCA-3′) for 1 hour at 4°C. The CT-ITS2-RNA substrate represents part of the C. thermophilum ITS2 pre-rRNA sequence. Protein mixtures (3 μL) were applied to glow-discharged UltrAuFoil R1.2/1.3 300 mesh grids (Quantifoil) and vitrified after 5 sec blotting using the Automatic Plunge Freezer EM GP (Leica).
Pillon M.C., Hsu A.L., Krahn J.M., Williams J.G., Goslen K.H., Sobhany M., Borgnia M.J, & Stanley R.E. (2019). Cryo-EM Reveals Active Site Coordination Within a Multienzyme pre-rRNA Processing Complex. Nature structural & molecular biology, 26(9), 830-839.
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10

Cryo-EM Grid Preparation Protocol

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A 4 μL volume of the above stock solution was then applied to UltrAuFoil R1.2/1.3 300-mesh grids (Quantifoil Micro Tools GmbH; Großlöbichau, Germany) which were glow discharged prior to sample application in a Pelco easiGlow device (Ted Pella; Redding, California) at 15 mA for 30 s. Grids were blotted for 3 s at -3 blot-force setting in a Vitrobot mark IV (ThermoFisher Scientific; operated at 4°C and 100 % humidity) before being plunge frozen in liquid ethane.
Xu Y., Kirk N.S., Venugopal H., Margetts M.B., Croll T.I., Sandow J.J., Webb A.I., Delaine C.A., Forbes B.E, & Lawrence M.C. (2020). How IGF-II Binds to the Human Type 1 Insulin-like Growth Factor Receptor. Structure(London, England:1993), 28(7), 786-798.e6.
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