Time resolution in cryo-EM using a novel PDMS-based microfluidic chip assembly and its application to the study of HflX-mediated ribosome recycling

Abstract

The rapid kinetics of biological processes and associated short-lived conformational changes pose a significant challenge in attempts to structurally visualize biomolecules during a reaction in real time. Conventionally, on-pathway intermediates have been trapped using chemical modifications or reduced temperature, giving limited insights. Here we introduce a novel time-resolved cryo-EM method using a reusable PDMS-based microfluidic chip assembly with high reactant mixing efficiency. Coating of PDMS walls with SiO₂ virtually eliminates non-specific sample adsorption and ensures maintenance of the stoichiometry of the reaction, rendering it highly reproducible. In an operating range from 10 to 1000 ms, the device allows us to follow in vitro reactions of biological molecules at resolution levels in the range of 3 Å. By employing this method, we show for the first time the mechanism of progressive HlfX-mediated splitting of the 70S E. coli ribosome in the presence of the GTP, via capture of three high-resolution reaction intermediates within 140 ms.

Figure 1.. The modular PDMS-based microfluidic chip assembly for TRCEM sample preparation.

(A) Schematic showing the setup for TRCEM using the mixing-spraying-plunging method. (B) The microfluidic chip assembly comprises three parts: micro-mixer, micro-reactor and micro-sprayer. (C) The splitting-and-recombination (SAR) based micro-mixer is fabricated by soft-lithography. (D) Fluorescence distribution along the micromixer with five mixing units under different inlet flow rate conditions. The mixing efficiency is characterized by the evenness in the distribution of fluorescent fluid. (E) Mixing efficiency for the micro-mixer at the exit under different flow rate conditions. The high mixing performance of this micro-mixer was validated both numerically and experimentally. (F) Micro-sprayer, with inner and outer tubing aligned and centered, used for depositing the reaction product onto the grid. The micro-spray (illuminated by red laser) is generated under the conditions of liquid flow rate 6 μL/s and gas pressure 8 psi, (G) A set of microfluidic chips was employed to achieve required reaction time points of 10, 25, 140, and 900 ms for the HflX study. (H) Compared with PDMS surface without any coating layer, and with DDM coating, the SiO₂ coating shows effective mitigation of protein adsorption (E. coli 70S is used as sample). (I) The SiO₂ coating functions well even after one month (here both E. coli 70S and HflX are used as samples).

Figure 2.. Data collection strategies on droplet-based cryo-grid prepared by mixing-spraying TRCEM method.

(A) Collect as many micrographs as possible on each droplet. The number of particles gradually decreases when the target moves away from the grid bar, in the direction of areas marked in blue, cyan, yellow and red. (B) Collect only along two or three lines of holes which are near and parallel to the grid bar.

Figure 3.. Molecular details of subunit interface during the progressive opening of the 70S

(A) Superimposition of reconstructions to show the opening of the 30S subunit from apo-70S (yellow) to i70S_HflX-I (red) to accommodate HflX. The green line represents the initial axis of 30S rotation, Axis I. (B) and (C), reconstructions of second and third intermediates overlapped with first intermediate, showing the stepwise splitting of the 70S by HflX by rotation of the 30S subunit around Axis II (green line). The corresponding rotation angles and direction of 30S rotation are shown in cartoon book representations. (D), reconstruction of the 50S-HflX complex after the departure of the 30S subunit, overlapped with the first 70S intermediate. In (A) through (D), all reconstructions are aligned on the 50S subunit. (E) to (H) are the high-resolution densities of control 70S at 900 ms and three intermediates obtained within 140 ms, and showing the stepwise rotation of 30S during recycling with respect to 50S (gray). (I) and (J) are the zoomed views of Coulomb densities in yellow for apo-70S and red for i70S_HflX-I, respectively, and corresponding atomic models (gray) showing the rearrangement of the protein uL2 of the 50S and bS6 of 30S to accommodate HflX. (K) and (L), Coulomb densities, and corresponding ribbon models of H69 from apo-70S (yellow), and i70S_HflX-I (red), respectively, showing the very first movement of helix H69. HflX is shown in magenta. (M) Kinetics of the splitting reaction in terms of the number of particles per class as a function of time, obtained by 3D classification.

Figure 4.. The shift of S12 towards HflX and Involvement of HflX in 70S splitting

The zoomed view of HflX and S12 of 30S interactions from i70SHflX-I and i70SHflX-III states are shown in (A) and (B), respectively. Due to the stepwise separation of the 30S during splitting, there occurs a steric clash (red star) at 140 ms due to the subsequent shift of the whole S12, which is not present at earlier states like10 ms, and this clash is the cause of the final separation of 30S from 50S by a power-stroke from HflX upon GTP hydrolysis. (C) The same clash at 140 ms from i70SHflX-III is shown in Coulomb density with a fitted model. (D) Pulling of H71 by the NTD of HflX causes the disruption of intersubunit bridge B3 between H71 and h44, and the zoomed view in (E) shows the interacting residues of both H71 and HflX. During pulling of H71 HflX has to shift its position and is shown in (F). (G) The same interaction is shown in Coulomb density with a fitted model.

Figure 5.. Analysis of nucleotide states, and spring-like nature of H34 and molecular events

(A) Refined density map of i70S_HflX-III (in mesh) with an atomic model fitted in with the zoomed view of i70S_HflX-III with the fitted model of GTP·Pi. The distance of 4.5 Å between GDP and Pi is compared with the distance of 2.8 Å between two P atoms of GDP. This distance between GDP and Pi is too large to form a P-O bond as in GTP. (B) Refined density map of 50S_HflX (in mesh) with atomic model fitted in along with the zoomed view of 50S_HflX with the fitted model of GTP. (C) The superimposition of atomic models of apo-70S and three intermediates with the zoomed view of H34 showing its spring-like behavior. Corresponding colors are indicated. (D) The molecular events involved during the 70S splitting by HflX are tabulated.

Figure 6.. Axes of rotation of 30S subunit during splitting/recycling of the 70S ribosome:

(A), (B), and (C), rotation of 30S subunit from Apo-70S (yellow) to i70SHflX-I (red), i70SHflX-I (red) to i70SHflX-II (green), and i70SHflX-I (red) to i70SHflX-III (blue), respectively. (D), (E), and (F) characterization of the 30S subunit rotation while the 50S subunits (cyan) are fixed in space. The models show the rotated state of the 30S subunit in each case. For clarity, 30S subunits are represented with only their principal axes of inertia. Rotation axes (Axis I, Axis II) are shown as green arrows indicating the direction (right-hand thumb rule) of the rotation (black curled arrows). (D) Rotation by 5.9° of 30S subunit around Axis I from Apo-70S (yellow) to i70SHflX-I (red). (E) and (F), Rotations by 7.9° and 16.1°, respectively, of 30S subunit around Axis II. (G), (H), and (I), same representation as (D), (E), and (F) omitting the 30S subunit to show the axes clearly. (J) and (K), Intersubunit bridges through which Axes I and II pass on the 50S and 30S subunit, respectively. (L) Formulation of rigid body motion along with location of the rotation axis. Initial and transformed positions are respectively denoted by A (position vector ${\overrightarrow{x}}_0$) and B (position vector ${\overrightarrow{x}}_1$). The shift from A to B is given by translation vector $\overrightarrow{t}$. Points with dotted circles are on the plane perpendicular to the rotation axis $\widehat{l}$. Points $\overrightarrow{A_p}$ and $\overrightarrow{B_p}$ are projections of points A and B, respectively. ${\overrightarrow{t}}_{\parallel }$ and ${\overrightarrow{t}}_{\perp }$ are respectively the parallel and perpendicular components of the translation vector $\overrightarrow{t}$. Axis $\widehat{l}$ and angle θ describes the rotation of the rigid body from point A to B. The rotation axis $\widehat{l}$ passes through the point given by the position vector ${\overrightarrow{C}}_{\perp }$. The solution for the position vector ${\overrightarrow{C}}_{\perp }$ is obtained by using the remaining vectors, as indicated in the derivation in Methods Section in SI “Determining the position vector of the unique point through which the rotation axis passes”.