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Integrative structural biology is when two analytical techniques are combined to achieve a complete and accurate determination of the 3D protein or protein complex structure. The precise and accurate characterization of protein structure and protein complexes is essential for understanding protein function and the mechanisms of action in a biological system. Solving the structure of large dynamic complexes often requires integrating several complementary techniques, such as mass spectrometry (MS) and cryo-electron microscopy (cryo-EM)—an approach known as integrative structural biology.
Cryo-EM makes it possible to reproducibly produce near-atomic resolution of proteins in all their complex conformations, structures, and modified forms. This has made cryo-EM the go-to technique for scientists around the world, generating breakthroughs in research for infectious disease, neurodegenerative disease, and cancer, among others.
MS can provide complementary information to cryo-EM by guiding downstream structure determination. For example, information on sample purity, stability and sample homogeneity can be ascertained to make intelligent decision about cryo-EM structural determination. Additionally, MS techniques can provide rich constraints to support integrative structural models that deliver greater resolution and accuracy. Adding MS data to cryo-EM techniques can provide the boost needed to generate a complete and accurate 3D structural model.
Learn about the Thermo ScientificOptiMSe workflow. A fast, automated, and intuitive way to to eliminate unpromising samples for vitrification and cryo-EM.
There are a growing number of innovative ways researchers are combining techniques to better understand the proteome. Explore pioneering integrative structural biology workflows below, and the available tools to achieve each step of the workflow.
Sample integrity, purity, and homogeneity are critical for achieving high-resolution structures using cryo-EM. Rapid and reliable screening methods for assessing sample quality are essential. Native mass spectrometry (nMS) enables direct mass measurement of macromolecular assemblies by maintaining their near-native structures and assembly states upon gas phase transfer from solution. nMS is a powerful diagnostic and a screening platform that can be used for rapid identification of whether the correct protein and/or nucleic acid components as well as bound cofactors and ligands are correctly assembled. With high resolving power, nMS can reveal protein heterogeneity arising from post-translational modifications such as glycosylation and phosphorylation as well as spot samples with contaminations or degradation.
Workflow step | Summary | Available tools |
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Sample preparation | A variety of traditional sample preparation techniques can be used to achieve sample integrity required for cryo-EM. Complete end-to-end solutions are available for even the most challenging protein. | |
Native mass spectrometry | Protein structures are kept intact and introduced into the mass spectrometer in biologically relevant conditions. Specifically designed software is used to obtain information on the intact protein or protein complex, including subunit stoichiometry, subunit identification, biomolecule binding, protein complex topology and protein dynamics can also be ascertained. | |
Identification of high-quality samples | Data analysis can be performed with Thermo Scientific BioPharma Finder software. This software produces highly accurate results, even for low-abundance proteins, and enables detection of extremely small protein modifications. | |
Single particle cryo-EM | Data collection consists of high-resolution imaging with a cryo-TEM. With advances in data collection software, individual particles can be automatically identified in the TEM image and grouped according to particle orientation. For every sample, robust, reliable automation simplifies and accelerates imaging and identification. | |
Structure visualization | Once sufficient particle data is collected the data can be recombined into a 3D representation of the protein or protein complex. This uses 2D data from tens of thousands of particles and typically involves multiple data processing steps. A number of professionally developed and open-source data processing solutions exist to simplify and expedite this process. |
Combining chemical crosslinking mass spectrometry (XL-MS) with cryo-ET can help improve structural resolution of proteins in their native state. XL-MS in combination with cryo-ET can provide confirmation of cryo-EM structural data by mapping crosslinks on proposed structures; fitting/positioning of subunits with the help of XL-derived restraints; modelling of missing regions invisible to EM due to their flexibility. More generally, combination of cryo-EM and XL-MS data with other experimentally or computationally derived information can provide a more complete integrative/hybrid model.
Workflow step | Overview | Available tools |
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Cell culture | Cells prepared by routine culture methods are grown on carbon-coated gold electron microscopy (EM) grids for cryo-TEM analysis. |
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Cryo-FIB with correlative light microscopy | Using cryo-correlative microscopy the structures of interest are identified. A dedicated cryo-FLM stage keeps the sample in its vitrified state during cryofluorescence imaging. All Thermo Scientific cryo-FIBs and cryo-PFIBs can be equipped with an iFLM Correlative System, allowing samples to be imaged directly within the high vacuum without additional transfer steps from an external cryo-light microscope. The dedicated cryo-FIB prepares a thin, uniform lamella at the vitreous temperature. | |
Imaging by cryo TEM | During cryo-ET, the sample is tilted in known increments about an axis. The individual projection images from the tomographic tilt series are then combined computationally in a procedure known as back projection, which creates the 3D tomographic volume. | |
Reconstruction | The 3D tomogram featuring cellular structures can be segmented and colored in a variety of ways to enhance its display and presentation. From the tomogram small subsets of data containing the structures of interest can be computationally extracted and subjected to image processing methods. |
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Protein crosslinking | Crosslinking reagents are used to covalently link interacting proteins that are in close proximity. If the crosslinking is done at the protein level, the samples are digested to peptides with an appropriate enzyme. An enrichment step is incorporated upon digestion to isolate crosslinked peptides. | DSSO (disuccinimidyl sulfoxide) |
Crosslinking mass spectrometry | The samples are separated and introduced into the mass spectrometer for analysis. Proteome Discoverer software with XlinkX node is used for data interpretation. This workflow enables the identification of interacting regions thereby enabling creation of protein-protein interaction maps. |
Cryo-EM captures a snapshot of a biological process frozen in time, when in fact biological processes are highly dynamic. By combining structural cryo-em data with MS data, additional information on protein dynamics can be identified. For example, for protein complexes that can be studied ex vivo or ex situ, hydrogen-deuterium exchange-MS (HDX-MS) is a powerful technique that can provide information on protein-protein or protein-ligand interaction sites and conformational changes induced by PTMs.
Workflow step | Overview | Available tools |
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Sample preparation | A variety of traditional sample preparation techniques can be used to achieve sample integrity required for cryo-EM. Complete end-to-end solutions are available for even the most challenging protein. | |
Single particle cryo-EM | With advances in data collection software, individual particles can be automatically identified in the TEM image and grouped according to particle orientation. Near-atomic structural determination of single particles can be achieved with Thermo Scientific Cryo-TEMs. Once sufficient particle data is collected the data can be recombined into a 3D representation of the protein or protein complex. A number of professionally developed and open-source data processing solutions exist to simplify and expedite this process. | |
Hydrogen deuterium exchange mass spectrometry (HDX-MS) | HDX-MS can be used to obtain protein structure and conformation information. Additionally, for protein complexes, HDX-MS can provide information on protein-protein or protein-ligand interaction sites and conformational changes induced by PTMs. | |
Glycoproteomics | Mass spectrometry based glycoproteomics can be used to ascertain information such as glycosylation sites and types and number of glycoforms that are present. |
Single particle analysis is a cryo-EM technique that enables structural characterization at near-atomic resolutions.
Cryo-electron tomography delivers both structural information about individual proteins as well as their spatial arrangements within the cell.
Microcrystal electron diffraction extracts atomic details from individual nanocrystals (<200 nm in size), even in a heterogeneous mixture.
MS technique that enables analysis of protein-protein interactions to better understand how proteins affect biological processes.
A screening tool to obtain information on mass of a protein or to ascertain information on post-translational modifications.
Provides information on protein mass, PTMs, proteoforms and protein sequence via fragmentation of the intact proteins in the mass spectrometer.
Enrichment approach that can be coupled to qualitative and quantitative mass spectrometry to examine subset of proteins in complex samples.
HDX-MS is a powerful tool for studying protein structures, interactions, intrinsic disorder, and conformational changes induced by PTMs.
Provides information on proteins or protein complexes in their biological state such as mass, subunit stoichiometry, protein complex topology, and dynamics.
A MS technique to determine the original protein components of the sample. Information on PTMs and stoichiometry can also be obtained.
A MS approach that relies on limited amount of enzyme for digestion. It is used to probe the quaternary structure of protein complexes.
MS tool that provides accurate mass determination to decipher protein complexes that are too complex to resolve using ensemble methods.
The dynamic and flexible nature of biomolecules poses a major challenge for structural biologists. Thus, the full characterization of a macromolecular system requires the combination of many structure-determination techniques, an approach known as integrative structural biology. In this context, advances in MS techniques in combination with cryo-EM and other structural tools is revolutionizing the understanding of protein structure, function and dynamics. Download this eBook to learn more about: Tools for integrative structural biology. The benefits of combining cryo-EM, cryo-ET and mass spectrometry. The advantages integrated modeling provides.
Over the past five years, the work of Dr. John Janetzko and colleagues in Dr. Brian Kobilka’s lab have provided considerable advances in understanding how β-arrestins recognize GPCRs and how these complexes are regulated inside cells. Using cryo-EM, they obtained one of the first structures of a GPCR-β-arrestin complex. This structure showed a bound membrane phosphoinositide, suggesting a possible mode of regulation for these complexes. Using cell-based and in vitro biochemical and biophysical approaches, including hydrogen-deuterium exchange (HDX) MS, they showed that membrane phosphoinositides act as allosteric modulators of GPCR-β-arrestin complexes, gating their assembly and disassembly. Together these studies provide high-resolution molecular details into the mechanism by which these proteins of critical importance for cellular physiology function.
Patrick Griffin, The Scripps Research Institute
Pascal Albanese, Utrecht University
Vicki Wysocki, The Ohio State University
Orbitrap Exploris 480 Mass Spectrometer | Orbitrap Ascend Tribrid Mass Spectrometer | Q Exactive UHMR Hybrid Quadrupole-Orbitrap MS System | |
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Resolving power | 480,000 at m/z 195 | 7,500-480,000 FWHM at m/z 200 | Up to 240,000 at m/z 200 |
Scan speed | Up to 40Hz | Orbitrap mass analyzer MSⁿ up to 45 Hz Ion trap MSⁿ up to 50 Hz | 18Hz |
Mass range | 40 to 6,000 m/z (up to 8,000 m/z with the BioPharma option) | Standard mass range m/z 40–2000, mid-mass range m/z 200–6000, and optional HMRn+ mass range m/z 500–16,000 | 50 to 8,000 m/z |
Dynamic range | >5000:1 | >5,000 within a single MS acquisition | >5000:1 |
Mass accuracy | Internal <1 ppm RMS; External: <3 ppm RMS | Internal <1 ppm RMS; External: <3 ppm RMS | Internal <1 ppm RMS; External: <3 ppm RMS |
The Thermo Scientific Cryo TEMs are revolutionizing life science research through innovation and accessibility. By combining automation, artificial intelligence, and an improved user experience, Thermo Scientific Cryo-TEMs allows you to harness the power of single particle analysis, MicroED and Cryo-ET at resolutions that are accelerating our breakthroughs in structural biology.
Accessible & Smart
Download Tundra Cryo TEM datasheet ›
Intermediate-resolution SPA | 100 kV, <3.5 Å* |
Medium throughput | Dataset in 24 hours |
Sample type | Proteins |
Applications | SPA |
Capable & Verstaile
Download Glacios Cryo TEM datasheet ›
High-resolution SPA | 200 kV, <2.5 Å* |
High throughput | Dataset in 30 minutes |
Sample type | Proteins, crystals, cells |
Applications | SPA, Micro-ED, tomography |
Powerful & Productive
Download Krios Cryo TEM datasheet ›
Ultra-high resolution SPA | 300 kV, <2 Å* |
Highest throughput | Dataset in minutes |
Sample type | Proteins, crystals, cells |
Applications | SPA, Micro-ED, tomography |
* Indicates a required field