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.

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 stepSummaryAvailable 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. 

Protein expression

Protein sample preparation

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. 

Q Exactive UHMR Hybrid Quadrupole-Orbitrap MS

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.  

Biopharma Finder Software

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.   

Tundra Cryo TEM- 100 kV

Glacios Cryo TEM- 200 kV

Krios Cryo TEM- 300 kV

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.

Smart EPU Software with embedded CryoSparc Live

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 stepOverviewAvailable tools

Cell culture

Cells prepared by routine culture methods are grown on carbon-coated gold electron microscopy (EM) grids for cryo-TEM analysis.

Cell culture media

 

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.

Aquilos 2 Cryo-FIB

Arctis Cryo-PFIB

Helios Hydra 5 DualBeam

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.  

Glacios Cryo TEM- 200 kV

Krios Cryo TEM- 300 kV

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.   

 

Amira Software

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)

DSBU (Disuccinimidyl Dibutyric Urea)

DSPP  (Disuccinimidyl Phenyl Phosphonic Acid, PhoX)

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.

Orbitrap Eclipse Tribrid MS

Proteome Discoverer Software with XlinkX node

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 stepOverviewAvailable 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. 

Protein expression

Protein sample preparation

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.   

Tundra Cryo TEM- 100 kV

Glacios Cryo TEM- 200 kV

Krios Cryo TEM- 300 Kv

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.

Orbitrap Exploris 240 or 480 MS

Orbitrap Eclipse Tribrid MS

Glycoproteomics

Mass spectrometry based glycoproteomics can be used to ascertain information such as glycosylation sites and types and number of glycoforms that are present.   

Orbitrap Eclipse Tribrid MS

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.

 

Orbitrap Exploris 480 Mass Spectrometer

Orbitrap Ascend Tribrid Mass Spectrometer

Q Exactive UHMR Hybrid Quadrupole-Orbitrap MS System

 Orbitrap Exploris 480 Mass SpectrometerOrbitrap Ascend Tribrid Mass SpectrometerQ Exactive UHMR Hybrid Quadrupole-Orbitrap MS System
Advantages
  • A compact footprint as space is always a premium regardless of lab. 

  • Superior ion transmission, HRAM and FAIMS Pro option. Ideal for examining  large proteins, protein complexes, PTM under biological conditions, 

  • An analytical framework that meets the needs of most structural research. 

  • Most versatile mass spectrometer for any structural biology experiment. 

  • High resolution accurate mass (HRAM), multiple fragmentation, MSn capability and FAIMS Pro option. 

  • Provides insights on the most complex molecules and biological systems, from whole proteome profiling, protein complexes to individual protein structural characterization.

  • Ideal mass spectrometer for native intact mass, top-down analyses and screening sample quality for cryo-EM. 

  • Enables the investigation of heterogeneity of protein complexes. HRAM, multiple fragmentations option, and direct mass technology capability.

Resources

 Download Orbitrap Exploris 480 MS spec sheet ›

 Orbitrap Ascend Tribrid MS spec sheet ›

 Download Q Exactive UHMR MS spec sheet ›

Resolving power480,000 at m/z 195 7,500-480,000 FWHM at m/z 200Up to 240,000 at m/z 200 
Scan speedUp to 40HzOrbitrap mass analyzer MSⁿ up to 45 Hz
Ion trap MSⁿ up to 50 Hz
18Hz
Mass range40 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 accuracyInternal <1 ppm RMS; External: <3 ppm RMSInternal <1 ppm RMS; External: <3 ppm RMSInternal <1 ppm RMS; External: <3 ppm RMS

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

 Download FAIMS Pro Duo brochure ›

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 SPA100 kV, <3.5 Å*
Medium throughputDataset in 24 hours
Sample typeProteins
ApplicationsSPA

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 SPA200 kV, <2.5 Å*
High throughputDataset in 30 minutes
Sample typeProteins, crystals, cells
ApplicationsSPA, 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 SPA300 kV, <2 Å*
Highest throughputDataset in minutes
Sample typeProteins, crystals, cells
ApplicationsSPA, Micro-ED, tomography

* Indicates a required field

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