The authors acknowledge Dr. Luk H. Vandenberghe (Harvard Medical School, Department of Ophthalmology) for providing purified AAV-3. This work was supported by the National Institutes of Health and the National Cancer Institute (R01CA193578, R01CA227261, R01CA219700 to D.F.K.).

1. Loading the specimen holder for liquid-EM
<ol>
	<li>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.</li>
	<li>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.</li>
	<li>Load a dry base microchip into the tip of the specimen holder. Add ~0.2 &#181;L 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.</li>
	<li>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.</li>
</ol>
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.
<ol>
	<li>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.</li>
	<li>Add ~2 &#181;L of sample contained in 50 mM HEPES, pH 7.5; 150 mM NaCl; 10 mM MgCl2; 10 mM CaCl2&#160;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.</li>
	<li>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.</li>
	<li>The specimen is now ready to be inserted into the TEM. Examine samples placed in autoloaders under cryo conditions or at room temperature.</li>
</ol>
3. Imaging specimens using a transmission electron microscope
<ol>
	<li>Liquid-EM imaging
	<ol>
		<li>Load the&#160;specimen holder into the TEM equipped with a field-emission gun (FEG) and operating at 200 kV.</li>
		<li>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&#176; to +15&#176; 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.</li>
		<li>Record images as long-framed movies using the in situ package integrated with a direct detector having a pixel spacing of 6 &#181;m (Video 1&#160;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.</li>
		<li>Adjust exposure times (0.25-1 s) to minimize beam damage to the specimen. Use a defocus range of -1-4 &#181;m at the specified magnification. If a thick solution is encountered, use higher defocus values or select a different region of interest.</li>
		<li>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).</li>
	</ol>
	</li>
	<li>Cryo-EM imaging
	<ol>
		<li>Load the&#160;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).</li>
		<li>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 &#181;m at a magnification of 59,000x and 40 frames per second.</li>
		<li>Use a defocus range of 1-4 &#181;m at the specified magnification. If thick layers of vitreous ice are encountered, use higher defocus values, or select a different region for data collection.</li>
	</ol>
	</li>
</ol>
4. Data analysis and 3 D structure comparisons
<ol>
	<li>Analysis of adeno-associated virus (AAV) in liquid and vitreous ice
	<ol>
		<li>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.</li>
		<li>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.</li>
		<li>Calculate initial reconstructions using C1 symmetry using the program&#39;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 A&#778; for liquid specimens and 1.13 A&#778; for ice specimens.</li>
		<li>Use a mask value of 300 A&#778; 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 A&#778; or better. Use particle equivalency implementing icosahedral symmetry at 16,800 for liquid and 15,240 for ice12,14. 
		NOTE: Resolution estimates are based on the gold-standard Fourier shell correlation (FSC) criteria.</li>
		<li>Mask EM maps at ~250 A&#778; and examine results using a molecular structure analysis software package23,24. In Figure 3, slices are shown in increments of ~5 nm.</li>
		<li>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.</li>
	</ol>
	</li>
	<li>Analysis of SARS-CoV-2 sub-viral assemblies in liquid
	<ol>
		<li>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.</li>
		<li>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&#160;and 1.66 A&#778; pixel size.</li>
		<li>Perform 3D refinement in the program to obtain an EM map, an example is shown at ~8.25 A&#778; 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.</li>
	</ol>
	</li>
	<li>Analysis of rotavirus double-layered particles (DLPs) in vitreous ice
	<ol>
		<li>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.</li>
		<li>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.</li>
		<li>Calculate initial models using ab-initio options and C1 symmetry. Use refinement parameters including a pixel size of 1.47 A&#778; and a mask value of 800 A&#778;.</li>
		<li>Perform additional refinement routines while imposing I1 symmetry. Example results include a 10.15 A&#778; 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).</li>
		<li>Mask final maps at ~750 A&#778; 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.</li>
	</ol>
	</li>
</ol>

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The authors declare that they have no competing financial interests. The author, Madeline J. Dressel-Dukes, is an employee of Protochips, Inc. and Michael Spilman is an employee of DirectElectron.

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...

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 detail1,2,3,4,5. 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&#160;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 imaging6,7,8,9,10,11. 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 pathogens12,13,14.
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 ice12,14,18,19. 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&#160;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.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Acetone</td>
			<td>Fisher Scientific</td>
			<td>&#160;A11-1</td>
			<td>1 Liter</td>
		</tr>
		<tr>
			<td>Autoloader clipping tool</td>
			<td>ThermoFisher Scientific</td>
			<td>N/A</td>
			<td>Also SubAngstrom supplier</td>
		</tr>
		<tr>
			<td>Autoloader grid clips</td>
			<td>ThermoFisher Scientific</td>
			<td>N/A</td>
			<td>top and bottom clips</td>
		</tr>
		<tr>
			<td>Carbon-coated gold EM grids</td>
			<td>Electron Microcopy Sciences</td>
			<td>CF400-AU-50</td>
			<td>400-mesh, 5-nm thickness</td>
		</tr>
		<tr>
			<td>COVID-19 patient serum</td>
			<td>RayBiotech</td>
			<td>CoV-Pos-S-500</td>
			<td>500 microliters of PCR+ serum</td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>Fisher Scientific</td>
			<td>&#160;A412-1</td>
			<td>1 Liter</td>
		</tr>
		<tr>
			<td>Microwell-integrad microchips</td>
			<td>Protochips, Inc.</td>
			<td>EPB-42A1-10</td>
			<td>10x10-mm window arrays</td>
		</tr>
		<tr>
			<td>TEMWindows microchips</td>
			<td>Simpore Inc.</td>
			<td>SN100-A10Q33B</td>
			<td>9 large windows, 10-nn thick</td>
		</tr>
		<tr>
			<td>TEMWindows microchips</td>
			<td>Simpore, Inc.&#160;</td>
			<td>SN100-A05Q33A</td>
			<td>9 small windows, 5-nm thick</td>
		</tr>
		<tr>
			<td>Top microchips</td>
			<td>Protochips, Inc.</td>
			<td>EPT-50W</td>
			<td>500 mm x 100 mm window</td>
		</tr>
		<tr>
			<td>Whatman #1 filter paper</td>
			<td>Whatman</td>
			<td>1001 090</td>
			<td>100 pieces, 90 mm</td>
		</tr>
		<tr>
			<td>Equipment&#160;</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>DirectView direct electron detector</td>
			<td>Direct Electron</td>
			<td></td>
			<td>6-micron pixel spacing</td>
		</tr>
		<tr>
			<td>Falcon 3 EC direct electron detector</td>
			<td>ThermoFisher Scientific</td>
			<td></td>
			<td>14-micron pixel spacing</td>
		</tr>
		<tr>
			<td>Gatan 655 Dry pump station</td>
			<td>Gatan, Inc.</td>
			<td></td>
			<td>&#160;Pump holder tip to 10-6 range</td>
		</tr>
		<tr>
			<td>Mark IV Vitrobot</td>
			<td>ThermoFisher Scientific</td>
			<td></td>
			<td>state-of-the-art specimen preparation unit&#160;</td>
		</tr>
		<tr>
			<td>PELCO easiGlow, glow discharge unit</td>
			<td>Ted Pella, Inc.</td>
			<td></td>
			<td>&#160;Negative polarity mode</td>
		</tr>
		<tr>
			<td>Poseidon Select specimen holder</td>
			<td>Protochips, Inc.</td>
			<td></td>
			<td>&#160;FEI compatible;specimen holder</td>
		</tr>
		<tr>
			<td>Talos F200C TEM</td>
			<td>ThermoFisher Scientific</td>
			<td></td>
			<td>200 kV; Liquid-TEM</td>
		</tr>
		<tr>
			<td>Titan Krios G3</td>
			<td>ThermoFisher Scientific</td>
			<td></td>
			<td>300 kV; Cryo-TEM</td>
		</tr>
		<tr>
			<td>Freely available software</td>
			<td>Website link</td>
			<td></td>
			<td>Comments (optional)</td>
		</tr>
		<tr>
			<td>cryoSPARC</td>
			<td>https://cryosparc.com/</td>
			<td></td>
			<td>other image processing software</td>
		</tr>
		<tr>
			<td>CTFFIND4</td>
			<td>https://grigoriefflab.umassmed.edu/ctffind4</td>
			<td></td>
			<td>CTF finding program</td>
		</tr>
		<tr>
			<td>MotionCorr2</td>
			<td>https://emcore.ucsf.edu/ucsf-software</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>RELION</td>
			<td>https://www3.mrc-lmb.cam.ac.uk/relion/index.php?title=Main_Page</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>SerialEM</td>
			<td>https://bio3d.colorado.edu/SerialEM/</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>UCSF Chimera</td>
			<td>https://www.cgl.ucsf.edu/chimera/</td>
			<td></td>
			<td>molecular structure analysis software package</td>
		</tr>
	</tbody>
</table>

advancing high-resolution imaging of virus assemblies in liquid and ice

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-&#197;-10 &#197;. 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.

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...

Watch this Scientific Journal Video about Advancing High-Resolution Imaging of Virus Assemblies in Liquid and Ice at JoVE.com

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

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

Interest in liquid-electron microscopy (liquid-EM) has skyrocketed in recent years as scientists can now observe real-time processes at the nanoscale. It ...

advancing-high-resolution-imaging-virus-assemblies-liquid

Research

JoVE Journal

Biochemistry

2.9K Views.  Pennsylvania State University. Here protocols are described to prepare virus assemblies suitable for liquid-EM and cryo-EM analysis at the nanoscale using transmission electron microscopy.

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

Identification of Plant Ice-binding Proteins Through Assessment of Ice-recrystallization Inhibition and Isolation Using Ice-affinity Purification

Ice-binding proteins (IBPs) belong to a family of stress-induced proteins that are synthesized by certain organisms exposed to subzero temperatures. In plants, freeze damage occurs when extracellular ice crystals grow, resulting in the rupture of plasma membranes and possible cell death. Adsorption of IBPs to ice crystals restricts further growth by a process known as ice-recrystallization inhibition (IRI), thereby reducing cellular damage. IBPs also demonstrate the ability to depress the freezing point of a solution below the equilibrium melting point, a property known as thermal hysteresis (TH) activity. These protective properties have raised interest in the identification of novel IBPs due to their potential use in industrial, medical and agricultural applications. This paper describes the identification of plant IBPs through 1) the induction and extraction of IBPs in plant tissue, 2) the screening of extracts for IRI activity, and 3) the isolation and purification of IBPs. Following the induction of IBPs by low temperature exposure, extracts are tested for IRI activity using a &#39;splat assay&#39;, which allows the observation of ice crystal growth using a standard light microscope. This assay requires a low protein concentration and generates results that are quickly obtained and easily interpreted, providing an initial screen for ice binding activity. IBPs can then be isolated from contaminating proteins by utilizing the property of IBPs to adsorb to ice, through a technique called &#39;ice-affinity purification&#39;. Using cell lysates collected from plant extracts, an ice hemisphere can be slowly grown on a brass probe. This incorporates IBPs into the crystalline structure of the polycrystalline ice. Requiring no a priori biochemical or structural knowledge of the IBP, this method allows for recovery of active protein. Ice-purified protein fractions can be used for downstream applications including the identification of peptide sequences by mass spectrometry and the biochemical analysis of native proteins.

This paper outlines the identification of ice-binding proteins from freeze-tolerant plants through the assessment of ice-recrystallization inhibition activity and subsequent isolation of native IBPs using ice-affinity purification.

Ice-binding proteins (IBPs) belong to a family of stress-induced proteins that are synthesized by certain organisms exposed to subzero temperatures. In ...

High-resolution Single Particle Analysis from Electron Cryo-microscopy Images Using SPHIRE

SPHIRE (SPARX for High-Resolution Electron Microscopy) is a novel open-source, user-friendly software suite for the semi-automated processing of single particle electron cryo-microscopy (cryo-EM) data. The protocol presented here describes in detail how to obtain a near-atomic resolution structure starting from cryo-EM micrograph movies by guiding users through all steps of the single particle structure determination pipeline. These steps are controlled from the new SPHIRE graphical user interface and require minimum user intervention. Using this protocol, a 3.5 &#197; structure of TcdA1, a Tc toxin complex from Photorhabdus luminescens, was derived from only 9500 single particles. This streamlined approach will help novice users without extensive processing experience and a priori structural information, to obtain noise-free and unbiased atomic models of their purified macromolecular complexes in their native state.

This paper presents a protocol for processing cryo-EM images using the software suite SPHIRE. The present protocol can be applied for nearly all single particle EM projects that target near-atomic resolution.

SPHIRE (SPARX for High-Resolution Electron Microscopy) is a novel open-source, user-friendly software suite for the semi-automated processing of single ...

Atomic Scale Structural Studies of Macromolecular Assemblies by Solid-state Nuclear Magnetic Resonance Spectroscopy

Supramolecular protein assemblies play fundamental roles in biological processes ranging from host-pathogen interaction, viral infection to the propagation of neurodegenerative disorders. Such assemblies consist in multiple protein subunits organized in a non-covalent way to form large macromolecular objects that can execute a variety of cellular functions or cause detrimental consequences. Atomic insights into the assembly mechanisms and the functioning of those macromolecular assemblies remain often scarce since their inherent insolubility and non-crystallinity often drastically reduces the quality of the data obtained from most techniques used in structural biology, such as X-ray crystallography and solution Nuclear Magnetic Resonance (NMR). We here present magic-angle spinning solid-state NMR spectroscopy (SSNMR) as a powerful method to investigate structures of macromolecular assemblies at atomic resolution. SSNMR can reveal atomic details on the assembled complex without size and solubility limitations. The protocol presented here describes the essential steps from the production of 13C/15N isotope-labeled macromolecular protein assemblies to the acquisition of standard SSNMR spectra and their analysis and interpretation. As an example, we show the pipeline of a SSNMR structural analysis of a filamentous protein assembly.

Structures of supramolecular protein assemblies at atomic resolution are of high relevance because of their crucial roles in a variety of biological phenomena. Herein, we present a protocol to perform high-resolution structural studies on insoluble and non-crystalline macromolecular protein assemblies by magic-angle spinning solid-state nuclear magnetic resonance spectroscopy (MAS SSNMR).

Supramolecular protein assemblies play fundamental roles in biological processes ranging from host-pathogen interaction, viral infection to the ...

High-Resolution Complexome Profiling by Cryoslicing BN-MS Analysis

Proteins generally exert biological functions through interactions with other proteins, either in dynamic protein assemblies or as a part of stably formed complexes. The latter can be elegantly resolved according to molecular size using native polyacrylamide gel electrophoresis (BN-PAGE). Coupling of such separations to sensitive mass spectrometry (BN-MS) has been well-established and theoretically allows for exhaustive assessment of the extractable complexome in biological samples. However, this approach is rather laborious and provides limited complex size resolution and sensitivity. Also, its application has remained restricted to abundant mitochondrial and plastid proteins. Thus, for a majority of proteins, information regarding integration into stable protein complexes is still lacking. Presented here is an optimized approach for complexome profiling comprising preparative-scale BN-PAGE separation, sub-millimeter sampling of broad gel lanes by cryomicrotome slicing, and mass spectrometric analysis with label-free protein quantification. The procedures and tools for critical steps are described in detail. As an application, the report describes complexome analysis of a solubilized endosome-enriched membrane fraction from mouse kidneys, with 2,545 proteins profiled in total. The results demonstrate identification of uniform, low-abundance membrane proteins such as intracellular ion channels as well as high resolution, complex protein assembly patterns, including glycosylation isoforms. The results are in agreement with independent biochemical analyses. In summary, this methodology allows for comprehensive and unbiased identification of protein (super)complexes and their subunit composition, providing a basis for investigating stoichiometry, assembly, and interaction dynamics of protein complexes in any biological system.

A versatile cryoslicing BN-MS protocol using a microtome is presented for high-resolution complexome profiling.

Proteins generally exert biological functions through interactions with other proteins, either in dynamic protein assemblies or as a part of stably formed ...

Untargeted Liquid Chromatography-Mass Spectrometry-Based Metabolomics Analysis of Wheat Grain

Understanding the interactions between genes, the environment and management in agricultural practice could allow more accurate prediction and management of product yield and quality. Metabolomics data provides a read-out of these interactions at a given moment in time and is informative of an organism&#39;s biochemical status. Further, individual metabolites or panels of metabolites can be used as precise biomarkers for yield and quality prediction and management. The plant metabolome is predicted to contain thousands of small molecules with varied physicochemical properties that provide an opportunity for a biochemical insight into physiological traits and biomarker discovery. To exploit this, a key aim for metabolomics researchers is to capture as much of the physicochemical diversity as possible within a single analysis. Here we present a liquid chromatography-mass spectrometry-based untargeted metabolomics method for the analysis of field-grown wheat grain. The method uses the liquid chromatograph quaternary solvent manager to introduce a third mobile phase and combines a traditional reversed-phase gradient with a lipid-amenable gradient. Grain preparation, metabolite extraction, instrumental analysis and data processing workflows are described in detail. Good mass accuracy and signal reproducibility were observed, and the method yielded approximately 500 biologically relevant features per ionization mode. Further, significantly different metabolite and lipid feature signals between wheat varieties were determined.

A method for the untargeted analysis of wheat grain metabolites and lipids is presented. The protocol includes an acetonitrile metabolite extraction method and reversed phase liquid chromatography-mass spectrometry methodology, with acquisition in positive and negative electrospray ionization modes.

Understanding the interactions between genes, the environment and management in agricultural practice could allow more accurate prediction and management ...

Semi-Quantitative Analysis of Peptidoglycan by Liquid Chromatography Mass Spectrometry and Bioinformatics

Peptidoglycan is an important component of bacterial cell walls and a common cellular target for antimicrobials. Although aspects of peptidoglycan structure are fairly conserved across all bacteria, there is also considerable variation between Gram-positives/negatives and between species. In addition, there are numerous known variations, modifications, or adaptations to the peptidoglycan that can occur within a bacterial species in response to growth phase and/or environmental stimuli. These variations produce a highly dynamic structure that is known to participate in many cellular functions, including growth/division, antibiotic resistance, and host defense avoidance. To understand the variation within peptidoglycan, the overall structure must be broken down into its constitutive parts (known as muropeptides) and assessed for overall cellular composition. Peptidoglycomics uses advanced mass spectrometry combined with high-powered bioinformatic data analysis to examine peptidoglycan composition in fine detail. The following protocol describes the purification of peptidoglycan from bacterial cultures, the acquisition of muropeptide intensity data through a liquid chromatograph&#8212;mass spectrometer, and the differential analysis of peptidoglycan composition using bioinformatics.

This protocol covers a detailed analysis of peptidoglycan composition using liquid chromatography mass spectrometry coupled with advanced feature extraction and bioinformatic analysis software.

Peptidoglycan is an important component of bacterial cell walls and a common cellular target for antimicrobials. Although aspects of peptidoglycan ...

Generation and Assembly of Virus-Specific Nucleocapsids of the Respiratory Syncytial Virus

The use of an authentic RNA template is critical to advance the fundamental knowledge of viral RNA synthesis that can guide both mechanistic discovery and assay development in virology. The RNA template of nonsegmented negative-sense (NNS) RNA viruses, such as the respiratory syncytial virus (RSV), is not an RNA molecule alone but rather a nucleoprotein (N) encapsidated ribonucleoprotein complex. Despite the importance of the authentic RNA template, the generation and assembly of such a ribonucleoprotein complex remain sophisticated and require in-depth elucidation. The main challenge is that the overexpressed RSV N binds non-specifically to cellular RNAs to form random nucleocapsid-like particles (NCLPs). Here, we established a protocol to obtain RNA-free N (N0) first by co-expressing N with a chaperone phosphoprotein (P), then assembling N0 with RNA oligos with the RSV-specific RNA sequence to obtain virus-specific nucleocapsids (NCs). This protocol shows how to overcome the difficulty in the preparation of this traditionally challenging viral ribonucleoprotein complex.

For in-depth mechanistic analysis of the respiratory syncytial virus (RSV) RNA synthesis, we report a protocol of utilizing the chaperone phosphoprotein (P) for coexpression of the RNA-free nucleoprotein (N0) for subsequent in vitro assembly of the virus-specific nucleocapsids (NCs).

The use of an authentic RNA template is critical to advance the fundamental knowledge of viral RNA synthesis that can guide both mechanistic discovery and ...

F&#246;rster Resonance Energy Transfer Measurements in Living Plant Cells

Sensitized emission-based F&#246;rster resonance energy transfer (FRET) experiments are easily done but depend on the microscopic setup. Confocal laser scanning microscopes have become a workhorse for biologists. Commercial systems offer high flexibility in laser power adjustment and detector sensitivity and often combine different detectors to obtain the perfect image. However, the comparison of intensity-based data from different experiments and setups is often impossible due to this flexibility. Biologist-friendly procedures are of advantage and allow for simple and reliable adjustment of laser and detector settings. 
Furthermore, as FRET experiments in living cells are affected by the variability in protein expression and donor-acceptor ratios, protein expression levels must be considered for data evaluation. Described here is a simple protocol for reliable and reproducible FRET measurements, including routines for the estimation of protein expression and adjustment of laser intensity and detector settings. Data evaluation will be performed by calibration with a fluorophore fusion of known FRET efficiency. To improve simplicity, correction factors have been compared that have been obtained in cells and by measuring recombinant fluorescent proteins.

A protocol is provided for setting up a standard confocal laser-scanning microscope for in vivo F&#246;rster resonance energy transfer measurements, followed by data evaluation.

Sensitized emission-based F&#246;rster resonance energy transfer (FRET) experiments are easily done but depend on the microscopic setup. Confocal laser ...

Routine Collection of High-Resolution cryo-EM Datasets Using 200 KV Transmission Electron Microscope

Cryo-electron microscopy (cryo-EM) has been established as a routine method for protein structure determination during the past decade, taking an ever-increasing share of published structural data. Recent advances in TEM technology and automation have boosted both the speed of data collection and quality of acquired images while simultaneously decreasing the required level of expertise for obtaining cryo-EM maps at sub-3 &#197; resolutions. While most of such high-resolution structures have been obtained using state-of-the-art 300 kV cryo-TEM systems, high-resolution structures can be also obtained with 200 kV cryo-TEM systems, especially when equipped with an energy filter. Additionally, automation of microscope alignments and data collection with real-time image quality assessment reduces system complexity and assures optimal microscope settings, resulting in increased yield of high-quality images and overall throughput of data collection. This protocol demonstrates the implementation of recent technological advances and automation features on a 200 kV cryo-transmission electron microscope and shows how to collect data for the reconstruction of 3D maps that are sufficient for de novo atomic model building. We focus on best practices, critical variables, and common issues that must be considered to enable the routine collection of such high-resolution cryo-EM datasets. Particularly the following essential topics are reviewed in detail: i) automation of microscope alignments, ii) selection of suitable areas for data acquisition, iii) optimal optical parameters for high-quality, high-throughput data collection, iv) energy filter tuning for zero-loss imaging, and v) data management and quality assessment. Application of the best practices and improvement of achievable resolution using an energy filter will be demonstrated on the example of apo-ferritin that was reconstructed to 1.6 &#197;, and Thermoplasma acidophilum 20S proteasome reconstructed to 2.1-&#197; resolution using a 200 kV TEM equipped with an energy filter and a direct electron detector.

High-resolution cryo-EM maps of macromolecules can be also achieved by using 200 kV TEM microscopes. This protocol shows the best practices for setting accurate optics alignments, data acquisition schemes, and selection of imaging areas that are all essential for the successful collection of high-resolution datasets using a 200 kV TEM.

Cryo-electron microscopy (cryo-EM) has been established as a routine method for protein structure determination during the past decade, taking an ...

High-Resolution Neutron Spectroscopy to Study Picosecond-Nanosecond Dynamics of Proteins and Hydration Water

Neutron scattering offers the possibility to probe the dynamics within samples for a wide range of energies in a nondestructive manner and without labeling other than deuterium. In particular, neutron backscattering spectroscopy records the scattering signals at multiple scattering angles simultaneously and is well suited to study the dynamics of biological systems on the ps-ns timescale. By employing D2O-and possibly deuterated buffer components-the method allows monitoring of both center-of-mass diffusion and backbone and side-chain motions (internal dynamics) of proteins in liquid state.
Additionally, hydration water dynamics can be studied by employing powders of perdeuterated proteins hydrated with H2O. This paper presents the workflow employed on the instrument IN16B at the Institut Laue-Langevin (ILL) to investigate protein and hydration water dynamics. The preparation of solution samples and hydrated protein powder samples using vapor exchange is explained. The data analysis procedure for both protein and hydration water dynamics is described for different types of datasets (quasielastic spectra or fixed-window scans) that can be obtained on a neutron backscattering spectrometer.
The method is illustrated with two studies involving amyloid proteins. The aggregation of lysozyme into &#181;m sized spherical aggregates-denoted particulates-is shown to occur in a one-step process on the space and time range probed on IN16B, while the internal dynamics remains unchanged. Further, the dynamics of hydration water of tau was studied on hydrated powders of perdeuterated protein. It is shown that translational motions of water are activated upon the formation of amyloid fibers. Finally, critical steps in the protocol are discussed as to how neutron scattering is positioned regarding the study of dynamics with respect to other experimental biophysical methods.

Neutron backscattering spectroscopy offers a nondestructive and label-free access to the ps-ns dynamics of proteins and their hydration water. The workflow is presented with two studies on amyloid proteins: on the time-resolved dynamics of lysozyme during aggregation and on the hydration water dynamics of tau upon fiber formation.