Advancing High-Resolution Imaging of Virus Assemblies in Liquid and Ice

Summary

Here protocols are described to prepare virus assemblies suitable for liquid-EM and cryo-EM analysis at the nanoscale using transmission electron microscopy.

Abstract

Interest in liquid-electron microscopy (liquid-EM) has skyrocketed in recent years as scientists can now observe real-time processes at the nanoscale. It is extremely desirable to pair high-resolution cryo-EM information with dynamic observations as many events occur at rapid timescales - in the millisecond range or faster. Improved knowledge of flexible structures can also assist in the design of novel reagents to combat emerging pathogens, such as SARS-CoV-2. More importantly, viewing biological materials in a fluid environment provides a unique glimpse of their performance in the human body. Presented here are newly developed methods to investigate the nanoscale properties of virus assemblies in liquid and vitreous ice. To accomplish this goal, well-defined samples were used as model systems. Side-by-side comparisons of sample preparation methods and representative structural information are presented. Sub-nanometer features are shown for structures resolved in the range of ~3.5-Å-10 Å. Other recent results that support this complementary framework include dynamic insights of vaccine candidates and antibody-based therapies imaged in liquid. Overall, these correlative applications advance our ability to visualize molecular dynamics, providing a unique context for their use in human health and disease.

Introduction

Biomedical research improves our understanding of human health and disease through the development of new technologies. High-resolution imaging is transforming our view of the nanoworld - permitting us to study cells and molecules in exquisite detail^1,2,3,4,5. Static information of dynamic components such as soft polymers, protein assemblies, or human viruses reveals only a limited snapshot of their complex narrative. To better understand how molecular entities operate, their structure and function must be jointly investigated.

Recent advances in the production of materials such as atomically thin graphene or silicon-based microchips provide new opportunities for real-time structure-function analysis using transmission electron microscopes (TEMs). These materials can create hermetically sealed chambers for live EM imaging^{6,7,8,9,10,11}. The new field of liquid-EM, the room temperature correlate to cryo-EM, provides unprecedented views of hard or soft materials in solution, allowing scientists to simultaneously study the structure and dynamics of their specimen. Liquid-EM applications include real-time recordings of therapeutic nanoparticles interacting with cancer stem cells as well as changes in the molecular intricacies of viral pathogens^12,13,14.

Just as methodological advances spurred the resolution revolution in the cryo-EM field, new techniques and methods are needed to extend the use of liquid-EM as a high-throughput tool for the scientific community. The overall goal of the methods presented here is to streamline liquid-EM specimen preparation protocols. The rationale behind the developed techniques is to employ new microchip designs and autoloader devices, suitable for both liquid- and cryo-EM data collection (Figure 1)^{7,14,15,16,17}. The assemblies are mechanically sealed using standard grid clips for automated instruments, such as the Krios, which can accommodate multiple samples per session or a F200C TEM (Figure 2). This methodology expands the use of high-resolution imaging beyond standard cryo-EM applications demonstrating broader purposes for real-time materials analysis.

In the current video article, protocols are presented for preparing virus assemblies in liquid with and without commercially available specimen holders. Using the specialized specimen holder for liquid-EM, thin liquid specimens can provide structural information comparable to cryo-EM samples, as well as dynamic insights of the specimens. Also demonstrated are methods for preparing liquid specimens using autoloader tools for high-throughput routines. The major advantage over other techniques is that automated specimen production allows the user to quickly assess their samples for optimum thickness and electron dosage prior to data collection. This screening technique quickly identifies ideal areas for real-time recordings in liquid or ice^12,14,18,19. For purposes of 3D structure determination, liquid-EM may complement the long-established cryo-EM methods implemented in cryo-EM. Readers employing conventional TEM or cryo-EM technologies may consider using liquid-EM workflows to provide new, dynamic observations of their samples in a manner that complements their current strategies.

Virus samples used in this protocol include purified adeno-associated virus subtype 3 (AAV) obtained as a gift and cultured under standard conditions¹². Also used were non-infectious SARS CoV-2 sub-viral assemblies derived from the serum of COVID-19 patients¹² and obtained from a commercial source. Finally, purified simian rotavirus (SA11 strain) double-layered particles (DLPs) were obtained from the laboratory of Dr. Sarah M. McDonald Esstman at Wake Forest University and cultured using standard conditions ^6,17. Software packages described here are freely available and the links have been provided in the Table of Materials section.

Protocol

1. Loading the specimen holder for liquid-EM

1. Clean the silicon nitride (SiN) microchips by incubating each chip in 150 mL of acetone for 2 min followed by incubation in 150 mL of methanol for 2 min. Allow chips to dry in laminar airflow.
2. Plasma clean the dried chips using a glow-discharge instrument operating under standard conditions of 30 W, 15 mA for 45 s using Argon gas.
3. Load a dry base microchip into the tip of the specimen holder. Add ~0.2 µL of sample (0.2-1 mg/mL of virus assemblies in 50 mM HEPES, pH 7.5; 150 mM NaCl; 10 mM MgCl₂; 10 mM CaCl₂) to the base chip. Following a 1-2 min incubation step, place the top chip on the wet base chip containing the sample.
4. Clamp the assembly together to form a hermetically sealed enclosure, held in place mechanically by three brass screws. Upon sealing the assembly, pump the tip to 10^-6 Torr using a turbo-pumped dry pumping station. The holder is now ready to be inserted into the TEM.
   NOTE: The select holder has no cooling capabilities and is not used for cryo-EM.

2. Production of microchip sandwich assemblies

NOTE: Different SiN or silicon dioxide (SiO) microchips can be used directly from the shipped gel packs. Carbon-coated gold grids may also be used directly as supplied.

1. Plasma clean the microchips and carbon grids using a glow discharge instrument operating under standard conditions of 30 W, 15 mA for 45 s using Argon gas.
2. Add ~2 µL of sample contained in 50 mM HEPES, pH 7.5; 150 mM NaCl; 10 mM MgCl₂; 10 mM CaCl₂ to a glow-discharged microchip placed on a gel pack. Remove excess solution (~50%) using a filter paper or a pipette. Following a 1-2 min incubation step, add the glow-discharged carbon grid to the wet microchip containing the sample.
3. Clamp the assembly together using a single-tilt specimen holder or autoloader grid clips at room temperature to form a hermetically sealed enclosure. For the auto-loader clamps, place the sandwich assembly on the bottom C-clip, place the top clip on top of the assembly and use the standard clamping tool to seal the assembly together.
   NOTE: The clamping procedure performed here uses the same steps as for cryo-EM grid clamping, but at room temperature. Clamped samples may be stored for 2 months or longer prior to imaging while maintaining liquid in the enclosure. No leak check is used with a room temperature specimen holder or the autoloader.
4. The specimen is now ready to be inserted into the TEM. Examine samples placed in autoloaders under cryo conditions or at room temperature.

3. Imaging specimens using a transmission electron microscope

1. Liquid-EM imaging
   1. Load the specimen holder into the TEM equipped with a field-emission gun (FEG) and operating at 200 kV.
   2. Turn on the gun and adjust the eucentric height of the microscope stage with respect to the specimen by using the wobbler function, tilting the sample from -15° to +15° in the column. This procedure adjusts the stage in the Z-direction to accommodate the sample thickness. And helps ensure an accurate magnification during image recording.
   3. Record images as long-framed movies using the in situ package integrated with a direct detector having a pixel spacing of 6 µm (Video 1 and Video 2). Individual images may also be recorded using the serial data collection software package²⁰, implementing automated imaging routines. Acquire images under low-dose conditions at magnifications ranging from 28,000x-92,000x and 40 frames per second.
   4. Adjust exposure times (0.25-1 s) to minimize beam damage to the specimen. Use a defocus range of -1-4 µm at the specified magnification. If a thick solution is encountered, use higher defocus values or select a different region of interest.
   5. Ensure the solution is present in the samples throughout the imaging session by focusing the electron beam on a sacrificial area not used for data collection, until bubbles are formed (Supplementary Figure 1).
2. Cryo-EM imaging
   1. Load the clipped EM grids or microchip sandwiches into the TEM equipped with a FEG and operating at 300 kV. Turn on the gun and adjust the eucentric height of the microscope stage, using a similar procedure described for the liquid-TEM above (step 3.1.2).
   2. Record individual images using the single particle analysis system integrated within the microscope system while implementing automated imaging routines. Record images under low-dose conditions using the single particle analysis direct electron detector having a pixel spacing of 14 µm at a magnification of 59,000x and 40 frames per second.
   3. Use a defocus range of 1-4 µm at the specified magnification. If thick layers of vitreous ice are encountered, use higher defocus values, or select a different region for data collection.

4. Data analysis and 3 D structure comparisons

1. Analysis of adeno-associated virus (AAV) in liquid and vitreous ice
   1. Process movies for AAV particles in liquid and ice using RELION-3.08 program²¹ or other image processing software²². Perform motion correction using MotionCor2 v1.2.3.
   2. Once corrected, extract particles using the auto-picking tool in the program software package. Typical box sizes are 330 pixels for liquid specimens and 350 pixels for ice specimens.
   3. Calculate initial reconstructions using C1 symmetry using the program's 3D initial model routine and/or the ab-initio model options in the data processing software package. In the program, use a regularization parameter of T = 4 and a pixel size of 1.01 Å for liquid specimens and 1.13 Å for ice specimens.
   4. Use a mask value of 300 Å throughout the refinement procedures. Perform refinement protocols in the data processing software using I1 symmetry to obtain multiple EM maps. Expected structural resolution may be in the range of 4 Å or better. Use particle equivalency implementing icosahedral symmetry at 16,800 for liquid and 15,240 for ice^12,14.
      NOTE: Resolution estimates are based on the gold-standard Fourier shell correlation (FSC) criteria.
   5. Mask EM maps at ~250 Å and examine results using a molecular structure analysis software package^23,24. In Figure 3, slices are shown in increments of ~5 nm.
   6. Extract capsid protein (VP1) subunits from the EM maps for comparison. Quantify dynamic changes among EM structures based on the particle diameter measurements (Supplementary Figure 2), and then visualize using the Morph map function in the software.
2. Analysis of SARS-CoV-2 sub-viral assemblies in liquid
   1. Process movies using the RELION-3.08 program. Correct for image drift and beam-induced motion using MotionCor2 v1.2.3. Correct for the contrast transfer function (CTF) of the microscope.
   2. Use the auto-picking tool in the program to select viral assemblies with a box size of 800 pixels. For computational efficiency, extracted particles can be rescaled to 256 pixels. Obtain an initial model using C1 symmetry in the program with a regularization parameter of T = 2 and 1.66 Å pixel size.
   3. Perform 3D refinement in the program to obtain an EM map, an example is shown at ~8.25 Å according to standard FSC criteria (Figure 4). Visualize EM maps using the molecular structure analysis software with slices incremented at 25 nm as demonstrated in Figure 4.
3. Analysis of rotavirus double-layered particles (DLPs) in vitreous ice
   1. Process movies for rotavirus DLPs using other image processing software. Use MotionCor2 v1.2.3 to correct for drift in the images. Use Patch CTF estimation to correct for lens effects on the images.
   2. Extract particles using the auto-picking tool in the software with a box size of 950 pixels. Down-sample the box size as needed for computing purposes.
   3. Calculate initial models using ab-initio options and C1 symmetry. Use refinement parameters including a pixel size of 1.47 Å and a mask value of 800 Å.
   4. Perform additional refinement routines while imposing I1 symmetry. Example results include a 10.15 Å EM map, according to the gold-standard FSC criteria. Total particles used were 2050, which equates to 123,000 protomer units due to the icosahedral symmetry operator (Figure 5).
   5. Mask final maps at ~750 Å and visualize results in the molecular structure analysis software package. Example slices through the EM map are shown in Figure 5 in increments of ~10 nm.

Results

A liquid-TEM operating at 200 kV was used for all liquid-EM imaging experiments and a cryo-TEM operating at 300 kV was used for all cryo-EM data collection. Representative images and structures of multiple viruses are presented to demonstrate the utility of the methods across various test subjects. These include recombinant adeno-associated virus subtype 3 (AAV), SARS-CoV-2 sub-viral assemblies derived from the patient serum, and simian rotavirus double-layered particles (DLPs), SA11 strain. First, comparisons are demons...

Discussion

New opportunities are presented to streamline current liquid-EM workflows by using new automated tools and technologies adapted from the cryo-EM field. Applications involving the new microchip sandwich technique are significant with respect to other methods because they enable high-resolution imaging analysis in liquid or vitreous ice. One of the most critical steps in the protocol is producing specimens with the ideal liquid thickness to visualize exquisite details at the nanoscale level. Ideal regions of interest are i...

Materials

Name	Company	Catalog Number	Comments
Acetone	Fisher Scientific	A11-1	1 Liter
Autoloader clipping tool	ThermoFisher Scientific	N/A	Also SubAngstrom supplier
Autoloader grid clips	ThermoFisher Scientific	N/A	top and bottom clips
Carbon-coated gold EM grids	Electron Microcopy Sciences	CF400-AU-50	400-mesh, 5-nm thickness
COVID-19 patient serum	RayBiotech	CoV-Pos-S-500	500 microliters of PCR+ serum
Methanol	Fisher Scientific	A412-1	1 Liter
Microwell-integrad microchips	Protochips, Inc.	EPB-42A1-10	10x10-mm window arrays
TEMWindows microchips	Simpore Inc.	SN100-A10Q33B	9 large windows, 10-nn thick
TEMWindows microchips	Simpore, Inc.	SN100-A05Q33A	9 small windows, 5-nm thick
Top microchips	Protochips, Inc.	EPT-50W	500 mm x 100 mm window
Whatman #1 filter paper	Whatman	1001 090	100 pieces, 90 mm
Equipment
DirectView direct electron detector	Direct Electron		6-micron pixel spacing
Falcon 3 EC direct electron detector	ThermoFisher Scientific		14-micron pixel spacing
Gatan 655 Dry pump station	Gatan, Inc.		Pump holder tip to 10-6 range
Mark IV Vitrobot	ThermoFisher Scientific		state-of-the-art specimen preparation unit
PELCO easiGlow, glow discharge unit	Ted Pella, Inc.		Negative polarity mode
Poseidon Select specimen holder	Protochips, Inc.		FEI compatible;specimen holder
Talos F200C TEM	ThermoFisher Scientific		200 kV; Liquid-TEM
Titan Krios G3	ThermoFisher Scientific		300 kV; Cryo-TEM
Freely available software	Website link		Comments (optional)
cryoSPARC	https://cryosparc.com/		other image processing software
CTFFIND4	https://grigoriefflab.umassmed.edu/ctffind4		CTF finding program
MotionCorr2	https://emcore.ucsf.edu/ucsf-software
RELION	https://www3.mrc-lmb.cam.ac.uk/relion/index.php?title=Main_Page
SerialEM	https://bio3d.colorado.edu/SerialEM/
UCSF Chimera	https://www.cgl.ucsf.edu/chimera/		molecular structure analysis software package