Journal: bioRxiv

Article Title: MISO: Microfluidic protein isolation enables single particle cryo-EM structure determination from a single cell colony

doi: 10.1101/2025.01.10.632437

Figure Lengend Snippet: | Preparation and structure determination of bt TMEM206 using MISO and conventional purification approach . a , Schematics of the protein construct and MISO purification on a single 3 μl microfluidics streptavidin column. b , Complete MISO purification chromatogram of btTMEM206-YFP from cleared detergent-solubilized HEK cells. Fluorescent signal was detected at WL 525 nm and originates from YFP. c , Elution peak was fractionated into 1 μl fractions used for protein characterization. d , SDS-PAGE from bt TMEM206-YFP purified on MISO (left) and bt TMEM206 purified by conventional approach (right). The conventionally purified TMEM206 was gel-filtered (panel h) and the peak fraction was used for SDS-PAGE. e , Negative stain micrograph from MISO- purified sample shows homogeneous protein particles. f and i , Cryo-EM micrographs of MISO-purified bt TMEM206-YFP and conventionally purified TMEM206 on holey EM grids. h , SEC from bt TMEM206 purified by the conventional method. g, j-n , Comparison of 2D class averages and 3D reconstructions from bt TMEM206-YFP (g, k, m) and bt TMEM206 (j, l, n). Additional diffuse density from YFP in 2D classes is marked with white asterisk (panel g). o , Structural alignment between bt TMEM206 (grey) and bt TMEM206-YFP (coloured) models shows that YFP does not modify the structure.

Article Snippet: 1 μl affinity column was loaded with Pierce High-capacity streptavidin agarose resin and the downstream 10 μl column was loaded with Superose 6 Increase resin (GE Healthcare).

Techniques: Purification, Construct, SDS Page, Staining, Cryo-EM Sample Prep, Comparison

Journal: bioRxiv

Article Title: MISO: Microfluidic protein isolation enables single particle cryo-EM structure determination from a single cell colony

doi: 10.1101/2025.01.10.632437

Figure Lengend Snippet: | De termination of m TMEM16F structure using half a 10 cm dish of HEK cells. a , Schematics of the protein construct and MISO purification on a single 3 μl streptavidin column. b , Complete MISO purification chromatogram from a cleared lysate of HEK cells. Fluorescent signal from YFP was detected at WL 525 nm. c , Elution peak was fractionated into 1 μl fractions used for protein characterization. d , SDS-PAGE from m TMEM16F-YFP purified on MISO. e , Negative stain micrograph from MISO-purified sample shows homogeneous protein particles. f, Cryo-EM micrographs of MISO-purified m TMEM16F-YFP on holey EM grids. g, 3D reconstruction of TMEM16F-YFP to a resolution of 3.5 Å . High-resolution map is overlayed with a low-pass filtered map to show the detergent micelle. h , Close-up view on the Ca 2+ binding site with bound Ca 2+ ion. i , Details of density map for a trans-membrane helix and for a β-hairpin. j , Structural alignment between MISO purified m TMEM16F-YFP and m TMEM16F prepared by conventional approach (PDB 6P46).

Article Snippet: 1 μl affinity column was loaded with Pierce High-capacity streptavidin agarose resin and the downstream 10 μl column was loaded with Superose 6 Increase resin (GE Healthcare).

Techniques: Construct, Purification, SDS Page, Staining, Cryo-EM Sample Prep, Binding Assay, Membrane

Journal: bioRxiv

Article Title: MISO: Microfluidic protein isolation enables single particle cryo-EM structure determination from a single cell colony

doi: 10.1101/2025.01.10.632437

Figure Lengend Snippet: a , Protein constructs and schematics of the experiment. 80 μl of extract was purified on a 3 μl streptavidin column. b, Elution peak detected at WL of 525 nm was fractionated into 1 μl fraction used for negative stain EM (grey in the chromatogram) while subsequent 80 nl fractions were spotted on holey gold grids (blue) and cryo-plunged. c , Negative stain micrograph and an example of cryo-EM micrograph from MISO device. Panel below shows 2D classes from the cryo-EM dataset. d, 3D cryo-EM maps of the of the complete membrane protein complex solved to resolution of 3.5 Å. e, 3D reconstruction of intracellular domain (ICD) only resolved to 3.4 Å. f, Density for two distinct Ca 2+ ions bound to calcium binding sites (CBS) with fitted structure of human TRPC6 (PDB 6UZ8). g, Experimental scheme for purification of TRPC6 on a 2-column MISO chip. h, Protein purified on 1 μl streptavidin column was further polished on a 10 μl Superose 6. i, SDS-PAGE imaged using fluorescence at WL 488 nm. j, Negative stain EM micrographs from the selected fractions.

Article Snippet: 1 μl affinity column was loaded with Pierce High-capacity streptavidin agarose resin and the downstream 10 μl column was loaded with Superose 6 Increase resin (GE Healthcare).

Techniques: Construct, Purification, Staining, Cryo-EM Sample Prep, Membrane, Binding Assay, SDS Page, Fluorescence