Complete and accurate 3D structural determination when one technique is not enough

To understand protein function within the cellular environment, it is essential to determine protein complex assembly and structure beyond just individual proteins. Solving the structure of large dynamic complexes often requires integrating several complementary techniques, such as biomolecular mass spectrometry (MS) and cryo-electron microscopy (cryo-EM) – an approach known as integrative structural biology.

MS has made significant advances in the field of structural biology, enabled by increased speed, sensitivity, selectivity, and a variety of MS fragmentation techniques. Similarly, recent developments in cryo-EM sample preparation, microscope and detector technology, automation in data collection, and image processing make it possible to reproducibly reach near-atomic resolution. Combined, a reliable and complete structure can be solved for macromolecular complexes composed of components like proteins, post-translational protein modifications, DNA, RNA, and lipids.

Integrative structural biology process (mass spec and electron microscopy).
Schematic of the integrative structural biology process including mass spec and cryo-EM data.
Electron Microscopy

Integrative Structural Biology

Mass spectrometry for 3D structural determination with cryo electron microscopy.

Integrative structural biology workflow

Native mass spectrum showing protein complex and complex subunits.
Native MS can reveal if the intact complex matches the mass of the protein subunits, if it might exist as an oligomer, if a subunit is present in multiple copies, or if a small molecule might be binding to the complex.

Sample screening and optimization

A sample that is likely to result in a high-resolution cryo-EM structure must consist of biomolecules that are intact, stable and compositionally homogeneous. Native MS can quickly, easily, and quantitatively check these parameters.

Identification of structural components

Mass spec can provide the subunit composition of a protein complex via denaturing MS and reveal interaction partners in the cellular environment (interactomics) as well as subunit stoichiometry. This information gives structural identity, which aids in later interpretation and modeling.


Native mass spectrum showing folded, denatured and misfolded protein. Native mass spectrometry provides a fast and accurate readout of protein integrity and stability. Three preparations of the same protein are shown; correctly folded native state (top), denatured during preparation (middle) and misfolded (bottom). The state relates directly to the number of charges the protein complex acquires during ionization.

CryoEM structure of the KaiCBA protein complex that regulates the cyanobacterial circadian clock.

Cryo-electron microscopy imaging

Cryo-EM, particularly single particle analysis (SPA), is becoming an essential technique for structural determination of viruses and protein complexes. With SPA, 2D transmission electron micrographs of individual, randomly orientated viruses/protein complexes can be computationally aligned to generate a 3D volume of the specimen. This has allowed cryo-EM to emerge as an alternative to traditional techniques, such as X-ray crystallography and nuclear magnetic resonance (NMR), and it can even visualize complete macromolecular complexes rather than simply selected parts.

Building a 3D model

Chemical crosslinking, followed by mass spec, provides information on the global protein subunit topology within a large complex. This topology helps locate the subunits within the large cryo-EM density map. Beyond global subunit topology, the same crosslinks also allow de novo atomic model building within the EM density map when combined with the amino acid sequence. Lastly, facilitated by the spatial constraints of the crosslinks, an atomic model can be validated and refined.

28S subunit protein structure generated by cryo electron microscopy. Proteins of the 28S subunit. CX-MS crosslinking was used to identify mitochondrial-specific ribosomal proteins and confirm their locations (Cα of crosslinked residues shown as spheres).
KaiCBA protein complex imaged with cryo electron microscopy. Structure of the KaiCBA circadian clock protein complex at 4.7 Å resolution obtained by cryo electron microscopy after native MS screening for optimal sample conditions.

RNA, post-translational modifications and lipids

For further refinement and a complete atomic model, MS can localize flexible domains and weak densities within a macromolecular complex. Post-translational modifications, structural lipids and ribonucleic acids like RNA and DNA can all be identified and localized, aiding in the modeling of less defined areas of the EM density map.

When information from different sources and methods is integrated, an atomic model becomes much more reliable. With continued advancements in both MS and cryo-EM, and further application of these synergistic approaches, integrative structural biology will continue to produce more reliable, more complete and higher precision 3D structures.



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Proteins Analysis

Cryo-electron microscopy provides near-atomic resolution 3D protein structure. It can determine structural information for complexes and crystallization-resistant samples, as well as vital cellular context.

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Biopharmaceutical Research

Structural drug discovery is enabled by cryo-electron microscopy, as the method provides near-atomic-resolution detail for small molecules and protein biologics in their fully hydrated state.

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Virus Analysis

Cryo-EM enables the 3D structural visualization of virus particles, and the antigen-antibody interface, at near-atomic resolutions. A virus’s inherent structural symmetry makes it the ideal target for cryo-EM analysis.

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