1. Volume 25, Issue 4, Pages 663-670.e3 (April 2017)
A Fast and Effective Microfluidic Spraying-Plunging Method for High-Resolution Single- Particle Cryo-EM 
Xiangsong Feng, Ziao Fu, Sandip Kaledhonkar, Yuan Jia, Binita Shah, Amy Jin, Zheng Liu, Ming Sun, Bo Chen, Robert A. Grassucci, Yukun Ren, Hongyuan Jiang, Joachim Frank, Qiao Lin 
Structure 
Volume 25, Issue 4, Pages e3 (April 2017)
DOI: /j.str
Copyright © Terms and Conditions

2. Structure 2017 25, 663-670.e3DOI: (10.1016/j.str.2017.02.005)
Copyright © Terms and Conditions

3. Figure 1
Design of the Microsprayer Chip and Schematic of the Experimental Setup
(A) Design of the microsprayer chip. The sprayer is composed of the liquid injector and gas nozzle with orifice diameters of 75 and 360 μm, respectively. The space between these two orifices is 500 μm long and forms the mixing chamber. The liquid is introduced into the microsprayer chip, issuing from the liquid injector as a cylindrical liquid jet. Simultaneously, the nitrogen gas is fed into the chip, regulated by the gas nozzle as a co-flowing gas stream surrounding the liquid jet in the mixing chamber, which facilitates the atomization.
(B) Experimental setup. The position of the tweezers shown is only one transient point of its trajectory (see Figure S1), with the cryo-EM grid in the process of intersecting the spray plume. For adjustment of the precise position of the microsprayer nozzle, the chip holder can be slid back and forth in the guide groove and the microsprayer chip can be slid up and down in the chip holder. Hence the solution in the form of droplets is sprayed onto the EM grid, which is then quickly plunged into the cryogen (see also Figure S1). The resulting grid bears thin blobs of vitreous ice where the droplets have impinged on the grid surface.
See also Figure S1 and Movies S1–S4.
Structure  , e3DOI: ( /j.str )
Copyright © Terms and Conditions

4. Figure 2
Measurements of Ice Thickness of Droplets Sprayed on the EM Grid with the Following Settings: Liquid Flow Rate 6 μL/s, Gas Pressure 16 psi, and Sprayer-Grid Distance 5 mm
(A) Half of a grid showing the droplet distribution and the droplet size.
(B) Area marked cyan in (A), at 120× magnification, was used to find a set of squares (red boxes) with particle-collectible droplets for further imaging.
(C–F) Four squares targeted in (B) were imaged at 550×. Droplets with vitreous ice (marked with orange) allowing collection of particles are visible in these squares. Positions of holes covered with ice were randomly chosen for drilling tunnels (red crosses in D).
(G) A series of tunnels were drilled into the ice at the tilt angle of −30°. The grid was then tilted to +30°, and images of projections of the tunnels were acquired.
See also Figure S2.
Structure  , e3DOI: ( /j.str )
Copyright © Terms and Conditions

5. Figure 3 Ice Distribution in the Holes
(A and B) The ice thickness is different from the leading to the trailing side of each hole (blue arrows), which is different from grids obtained by the blotting method.
(C and D) The ice is thinner on one side than on the other side as indicated by the different lengths of the tunnels drilled on the two sides. The thicker ice region on each hole is marked in yellow, the thinner ones in blue.
See also Figure S3.
Structure  , e3DOI: ( /j.str )
Copyright © Terms and Conditions

6. Figure 4
3.0-Å Resolution Structure of Apoferritin Obtained by Spraying with the Microsprayer
(A) High-quality electron micrograph of apoferritin from equine spleen (Sigma A3641; 7 mg/mL in PBS buffer, 0.2-μm filtered) sprayed onto cryo-EM holy carbon grid and collected using the FEI Polara microscope with a Gatan K2 Summit direct electron detection camera. Scale bars for the micrograph and magnified region represent 1,000 and 100 Å, respectively.
(B) Cryo-EM structure of apoferritin at 3.0-Å resolution with each subunit color coded.
(C) The Fourier shell correlation curve for the final cryo-EM 3D reconstruction, generated with RELION 1.4.
(D and E) Representative cryo-EM densities (gray mesh), superimposed on our atomic model (main chain in yellow) for different apoferritin domains.
See also Figure S6.
Structure  , e3DOI: ( /j.str )
Copyright © Terms and Conditions

7. Figure 5 Three α-Helical Segments from One Subunit
(A) The cryo-EM density of long α-helical segment is shown in blue mesh. The model was docked with rigid-body fitting using Chimera first, then manually optimized by fitting in Coot.
(B) Short α-helical segment near the N terminus. Long-base amino acid arginine is well preserved (R5).
(C) The additional density accounting for the hydroxyl group of the tyrosine side chain (Y164) is clearly seen compared with the side chain for phenylalanine (F166).
See also Figure S6.
Structure  , e3DOI: ( /j.str )
Copyright © Terms and Conditions