Integrative structural biology
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.
Workflows
Integrative structural biology workflows
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.
Fast sample quality screening of cryo-EM samples with automated native MS
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 |
|---|---|---|
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. |
High fidelity structural models from in cell cryo-electron tomography and crosslinking mass spectrometry
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 |
|---|---|---|
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. |
Insights into structural dynamics and function with hydrogen-deuterium exchange mass spectrometry and cryo-EM
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 |
|---|---|---|
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. |
Techniques
Single particle analysis
Single particle analysis is a cryo-EM technique that enables structural characterization at near-atomic resolutions of the structure of biomolecular complexes/assemblies.
Cryo-tomography
Cryo-electron tomography delivers both structural information about individual proteins as well as their spatial arrangements within the cell.
Microcrystal electron diffraction
Microcrystal electron diffraction extracts atomic details from individual nanocrystals (<200 nm in size), even in a heterogeneous mixture.
Crosslinking mass spectrometry
XL-MS is a MS technique that enables analysis of protein-protein interactions to better understand how proteins affect biological processes.
Intact protein analysis
A screening tool to obtain information on mass of a protein or to ascertain information on post-translational modifications.
Top-down proteomics
Top-down proteomics is a MS technique that allows for the analysis of intact proteins.
Affinity purification mass spectrometry
Enrichment approach to couple qualitative and quantitative mass spectrometry to examine subset of proteins.
Hydrogen deuterium exchange MS
HDX-MS is a powerful MS tool for studying protein structures, dynamics, folding, complexes, and interactions.
Native mass spectrometry
Native MS can provide information on masses of intact proteins or protein complexes, subunit identification and stoichiometry.
Peptide sequencing by MS
A MS technique to determine the original protein components of the sample. Information on post-translational modifications and stoichiometry can also be obtained.
Limited proteolysis
A mass spectrometry approach used to probe the quaternary structure of protein complexes.
Documents
Optimization of FAIMS-XL-MS workflow for phospho-enrichable crosslinkers
Higher precision 3D analysis from structure to function
Crosslinking mass spec (XL-MS) goes mainstream
Case studies in Integrative Structural Biology
Webinars
Recent advances in single-particle cryo-EM have enabled structure determination of small (<200 kDa) biological complexes, allowing researchers to study proteins that resist crystallization. In this webinar, Dr. Joost Snijder and Dr. Ieva Drulyte present the cryo-EM structure of the ~80 kDa heavily glycosylated human coronavirus HKU1 hemagglutinin esterase (HE) at a global resolution of 3.4 Å.
In this webinar, Dr. Dom Olinares discusses the recent advances in single particle cryo-EM that have enabled the structural determination of numerous flexible and conformationally heterogeneous protein assemblies at high resolution.
In this webinar, Albert Konijnenberg discusses the role of cryo-EM in vaccine development and as an assay for mutants. Learn how native and charge detection mass spectrometry can be used for Adeno-associated virus (AAV) particle characterization. Find out how combining structures from cryo-EM with glycan characterization by mass spectrometry can help scientists understand the bodies’ immune response to SARS-CoV-2.
Mass spectrometry has made significant advances in the field of structural biology. In combination with cryo-electron microscopy, a reliable and complete structure can be solved for macromolecular complexes comprised of components like proteins, post-translational protein modifications, DNA, RNA, and lipids. These two techniques have proven to be complementary methods in answering biological questions.
Vicki Wysocki, Ph.D.
Professor, Dept of Chemistry and Biochemistry
The Ohio State University
Catherine Dold
Health & Environment Writer,
C&EN Media Group
Automating native mass spectrometry through the use of online buffer exchange
This webinar focuses on the development of online buffer exchange (OBE) for native mass spectrometry (nMS) applications and describes how the experiment is implemented and how it can be extended by coupling to affinity separation (e.g., IMAC-OBE) to be used to optimize protein overexpression.
Dr. Patrick Griffin, Chair of Molecular Medicine
Patrick Griffin received his PhD in Chemistry at the University of Virginia working in Don Hunt’s lab during the birth of biological mass spectrometry and proteomics. In 2004, Patrick joined The Scripps Research Institute (TSRI), Scripps Florida as Professor and in 2007 was named founding Chair of the Department of Molecular Therapeutics. In 2017, he was named Chair of Molecular Medicine. Using mutagenesis, HDX-MS, crystallography, and NMR, Patrick’s research is focused on structure-function of nuclear receptors, enzymes, and membrane receptors.
Hijacking molecular plasticity to find tune nuclear receptor signaling: chemical biology and precision therapeutics
This webinar will highlight a new platform for structure-function analysis to dissect activation mechanisms of nuclear receptors.
Instruments
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 |
Vanquish HPLC and UHPLC Systems
Advantages
- LC systems that can be seamlessly coupled to best-in-class Thermo Scientific Orbitrap mass spectrometers to handle any separation challenge from small to large molecules, or from simple to complex samples.
FAIMS Pro Duo Interface for Mass Spectrometry
Advantages
- Differential ion mobility interface for mass spectrometry
- enhances instrument selectivity and detection limits using gas-phase fractionation. This results in reduced matrix interference and higher-quality data
- Enhances transmission of subclasses of peptides/proteins and PTMs
- Increases confidence and protein coverage
Cryo transmission electron microscopes for structural biology
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.
Tundra Cryo-TEM
Accessible & Smart
- Easy, iterative loading and imaging for rapid sample-viability determination
- AI-guided automation with results displayed progressively
- Cost effective and space efficient
Download Tundra Cryo TEM datasheet ›
| Intermediate-resolution SPA | 100 kV, <3.5 Å* |
| Medium throughput | Dataset in 24 hours |
| Sample type | Proteins |
| Applications | SPA |
Glacios 2 Cryo-TEM
Capable & Verstaile
- Maximized ease-of-use and excellent performance to offer a complete package for introducing cryo-TEM into your research
- Compact hardware footprint (minimizes installation requirements) at an affordable price
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 |
Krios Cryo TEM
Powerful & Productive
- Ultimate productivity and image quality with an integrated workflow solution
- Highest level of automation from sample vitrification to data analysis
- Compact design fits in standard room without costly renovations
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 |
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