Computational protocol: Expression, purification, and contaminant detection for structural studies of Ralstonia metallidurance ClC protein rm1

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Protocol publication

[…] We used SEC to assess the stability and quality of purified ClC-rm1 in detergent. SEC fractions of dimeric ClC-rm1 in DDM, DM, or OG were kept at 4°C for three days and then run again on a SEC column to determine long-term stability.Electron microscope micrographs of negatively-stained ClC-rm1 were used to assess the quality and purity of the sample. Grids were prepared by applying 3 μl of ClC-rm1 (~ 40 ng/μl) onto a carbon-coated copper grid. To avoid interference with the staining of the protein, the detergent-containing buffer was removed, after ClC-rm1 was adsorbed to the carbon film, by washing the grid with eight drops of deionized water prior to staining with 1.5% (w/v) uranyl formate. The grids were then screened under low-dose conditions using a Technai T12 microscope (FEI) operating at 120 kV, and a Technai F20 microscope (FEI) operating at 200 kV. Micrographs taken on the T12 were recorded at nominal magnifications of 49,000x and 68,000x on a 4k x 4k TVIPS CMOS camera, resulting in 2.1 and 1.5 Å/pixel, respectively. Micrographs taken on the F20 were recorded at nominal magnifications of 62,000x on a 4k x 4k TVIPS CMOS camera, resulting in 1.33 Å/pixel. Single-particle image processing was carried out using IMAGIC software []. Micrographs with excessive drift, low contrast, and poor power spectra were removed based on visual inspection with TIGRIS (http://tigris.sourceforge.net/). 50 particles were picked manually using TIGRIS, summed and rotationally averaged to serve as a reference for correlation-based particle picking in IMAGIC. These particle images were band-pass filtered to suppress very low and high spatial frequencies, and a circular mask was applied to remove unwanted background. The particle images were then normalized to zero average density and variance of 1. Those pre-processed images underwent cycles of classification and reference-free alignment, as implemented in IMAGIC, without imposing any symmetry.We then tested whether the purified ClC-rm1_eGFP sample was suitable for high-resolution data collection. The following specimen preparation procedure was used to make cryo-grids of ClC-rm1_eGFP. C-flat Cu grids (Protochips, Protochips, Inc) were glow discharged for 60 s at 25 mA. 3 μl of sample with a concentration of ~2 mg/ml was applied to the grid and plunged into liquid ethane using FEI Vitrobot Mark 2 (FEI Company, Hillsboro, OR) after blotting for 3 s at 4°C and ~85% relative humidity. Cryo-EM data were collected in movie mode on an FEI Krios microscope (FEI Company, Hillsboro, OR) operating in super-resolution mode with pixel size of 0.82 Å per super-resolution pixel. Each movie consisted of 75 frames collected over 23 s with an exposure per frame of 1.4 e-/Å2 as shown by Digital Micrograph (Gatan Inc., Pleasanton, CA), giving a total exposure of 100 e-/Å2. The image defocus range between ~0.8 to ~2.5 μm underfocus. The gain-corrected super-resolution movie frames were downsampled by Fourier cropping to a pixel size of 1.64 Å. The down-sampled frames were then motion-corrected and exposure filtered using Unblur []. The micrograph defocus was determined using CTFFIND4 [] on non-exposure-filtered micrographs and micrographs with excessive motion, low contrast, ice contamination or poor power spectra were removed based on visual inspection using TIGRIS. 50 particles were picked manually using TIGRIS, summed and rotationally averaged to serve as a reference for correlation-based particle picking in IMAGIC. 2D classification was performed as above using IMAGIC.The purified ClC-rm1 samples were loaded on NUPAGE 4–12% Bis-Tris Gel (Invitrogen) and visualized by SimplyBlue SafeStain (Invitrogen) or Silver stain to detect the presence of contaminants. Visible protein bands were cut out and sent to a facility for mass spectrometry analyses to confirm identity (Taplin Biological Mass Spectrometry Facility at Harvard Medical School, Boston, MA 02115). […]

Pipeline specifications

Software tools IMAGIC, Unblur, CTFFIND
Application cryo-EM
Organisms Cupriavidus metallidurans, Escherichia coli, Escherichia coli BL21(DE3)
Chemicals Acriflavine