Cryo-EM Sample Preparation for Ribosomes

Sample quality was initially examined using negative stain cryo-EM using in-house microscopes (TF20 and T12) and standard protocols (30 (link)). Once sample quality was ensured, cryo-EM samples were prepared on Quantifoil R1.2/1.3 grids coated with graphene oxide (GO) prepared in-house. Ribosome samples (3.5 μl) at a concentration of 100 pM were frozen using an FEI Vitrobot with a 30-s wait time, followed by a blot of 10 s with a force of +5. Once the sample was frozen, sample quality was ensured using an in-house TF20 microscope and shipped for high-resolution data collection.

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Morgan C.E., Huang W., Rudin S.D., Taylor D.J., Kirby J.E., Bonomo R.A, & Yu E.W. (2020). Cryo-electron Microscopy Structure of the Acinetobacter baumannii 70S Ribosome and Implications for New Antibiotic Development. mBio, 11(1), e03117-19.

Publication 2020

R1.2/1.3 grids

Frozen Graphene oxide Microscope Pm 100 Ribosome Stain

Corresponding Organization :

Other organizations : Case Western Reserve University, University School, Louis Stokes Cleveland VA Medical Center, Beth Israel Deaconess Medical Center, Harvard University

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Variable analysis

independent variables

Sample preparation method (negative stain cryo-EM vs. cryo-EM on Quantifoil R1.2/1.3 grids coated with graphene oxide)
Ribosome sample concentration (100 pM)

dependent variables

Sample quality

control variables

Microscopes used (TF20 and T12 for negative stain cryo-EM, in-house TF20 for cryo-EM sample quality check)
Cryo-EM sample preparation protocol (3.5 μl sample volume, 30-s wait time, 10-s blot with +5 force)

positive controls

Not explicitly mentioned

negative controls

Not explicitly mentioned

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