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Integrative Structural Biology

When one technique is not enough to achieve a complete and accurate 3D structure

To understand protein functioning in the cellular environment, it is essential that researchers determine protein complex assembly and structure beyond that of 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 advanced significantly, impacting the field of structural biology. Technology developments in mass analyzers are at the forefront of driving the growing number of structural biology studies enabled by increased performance in 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 of macromolecular complexes, including components like protein, post-translational protein modification, DNA, RNA, and lipids.

Mass spectrometry–cryo-EM combined workflow

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 check these parameters in a quantitative way in a simple, quick experiment.

Native MS tells you if the intact complex matches the mass of the protein subunits, if the complete protein might exist in dimer or trimer or other forms, if a subunit is present in multiple copies, or if a small molecule might be binding to the complex, like ATP, but also post-translational modifications (PTMs) or ligands that were not anticipated to be caught that can block the active site of the complex.

Native MS images showing stable molecule (top), a molecule that has higher charge states, indicative for protein unfolding (middle), and many low-mass, high-charge peptides, indicative for denaturation (bottom). Image courtesy of NovAliX.

Identification of structural components

MS can also provide the subunit composition of a protein complex (denaturing MS), reveal interaction partners in the cellular environment (interactomics), and reveal subunit stoichiometry (the amount of copies that are present of each subunit; by native MS). This information gives a structure identity, which aids in its interpretation and downstream modeling.

Cryo-electron microscopy imaging

Cryo-EM, particularly single particle analysis (SPA), is becoming an essential technique for structure determination of viruses and protein complexes. SPA is a structural technique wherein 2D transmission electron micrographs of individual, randomly orientated protein complexes or viruses can be mathematically aligned through image processing techniques 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); an alternative that can directly visualize complete macromolecular complexes instead of only selected parts.

Besides SPA, Cryo-electron tomography and sub-tomogram averaging result in particle structures at lower resolution, however typically in a more native environment like the intact cell. These EM applications benefit from an integrative structural approach with MS techniques, which are equally suitable to perform accurate measurements even with crude and complex sample compositions. 

Building a 3D model

Chemical crosslinking followed by MS provides information on the global protein subunit topology within a large complex. The topology helps locate the subunits within the large density map resulting from cryo-EM. 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 given spatial constraints from the crosslinks, an atomic model can be validated and refined.

Proteins of the 28S subunit. CX-MS crosslinks used to identify mitochondrial-specific ribosomal proteins or confirm their locations (Cα of crosslinked residues shown as spheres).

RNA, post-translational modifications and lipids

As a final data point for a complete atomic model, MS can localize flexible domains and weak densities within macromolecular complexes. Most notably, post-translational modifications, like phosphorylations and glycosylations, can be localized with amino acid resolution. Next to PTMs, structural lipids can be identified, along with bound ribonucleic acids like RNA and DNA, which aids their modeling within the less well-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 applications of these synergistic approaches, integrative structural biology will continue accelerating the path to the acquisition of more reliable, more complete, and higher precision 3D structures.

Calcium channel. Image courtesy of Nature.

Final result.

The combined results from mass spectrometry and cryo-EM result in a more accurate, more complete and more reliable atomic model of biomolecular complexes.

Thermo Fisher Scientific Mass Spectrometry

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Featured Document

Integrative Structural Biology eBook

In collaboration with the editors of Science Magazine, we invite you to download this comprehensive booklet, containing articles from the Science family of journals, perspectives on integrative structural biology, and interviews with leading researchers.

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Publication list

1.
A. Heuer, M. Gerovac, C. Schmidt, S. Trowitzsch, A. Preis, P. Kötter, O. Berninghausen, T. Becker, R. Beckmann, R. Tampé   (2017)   Structure of the 40S-ABCE1 post-splitting complex in ribosome recycling and translation initiation.   Nature structural & molecular biology   

DOI:  10.1038/nsmb.3396

References PDB protein(s):  5LL6

Read abstract

Structure of the 40S-ABCE1 post-splitting complex in ribosome recycling and translation initiation.

A. Heuer, M. Gerovac, C. Schmidt, S. Trowitzsch, A. Preis, P. Kötter, O. Berninghausen, T. Becker, R. Beckmann, R. Tampé

The essential ATP-binding cassette protein ABCE1 splits 80S ribosomes into 60S and 40S subunits after canonical termination or quality-control-based mRNA surveillance processes. However, the underlying splitting mechanism remains enigmatic. Here, we present a cryo-EM structure of the yeast 40S-ABCE1 post-splitting complex at 3.9-Å resolution. Compared to the pre-splitting state, we observe repositioning of ABCE1's iron-sulfur cluster domain, which rotates 150° into a binding pocket on the 40S subunit. This repositioning explains a newly observed anti-association activity of ABCE1. Notably, the movement implies a collision with A-site factors, thus explaining the splitting mechanism. Disruption of key interactions in the post-splitting complex impairs cellular homeostasis. Additionally, the structure of a native post-splitting complex reveals ABCE1 to be part of the 43S initiation complex, suggesting a coordination of termination, recycling, and initiation.

2.
K. van Pee, A. Neuhaus, E. D'Imprima, D. Mills, W. Kühlbrandt, Ö. Yildiz   (2017)   CryoEM structures of membrane pore and prepore complex reveal cytolytic mechanism of Pneumolysin.   eLife   6

DOI:  10.7554/eLife.23644

References PDB protein(s):  5LY6

Read abstract

CryoEM structures of membrane pore and prepore complex reveal cytolytic mechanism of Pneumolysin.

K. van Pee, A. Neuhaus, E. D'Imprima, D. Mills, W. Kühlbrandt, Ö. Yildiz

Many pathogenic bacteria produce pore-forming toxins to attack and kill human cells. We have determined the 4.5 Å structure of the ~2.2 MDa pore complex of pneumolysin, the main virulence factor of Streptococcus pneumoniae, by cryoEM. The pneumolysin pore is a 400 Å ring of 42 membrane-inserted monomers. Domain D3 of the soluble toxin refolds into two ~85 Å β-hairpins that traverse the lipid bilayer and assemble into a 168-strand β-barrel. The pore complex is stabilized by salt bridges between β-hairpins of adjacent subunits and an internal α-barrel. The apolar outer barrel surface with large sidechains is immersed in the lipid bilayer, while the inner barrel surface is highly charged. Comparison of the cryoEM pore complex to the prepore structure obtained by electron cryo-tomography and the x-ray structure of the soluble form reveals the detailed mechanisms by which the toxin monomers insert into the lipid bilayer to perforate the target membrane.

3.
K. Škubník, J. Nováček, T. Füzik, A. Přidal, R. Paxton, P. Plevka   (2017)   Structure of deformed wing virus, a major honey bee pathogen.   Proceedings of the National Academy of Sciences of the United States of America   114

DOI:  10.1073/pnas.1615695114

References PDB protein(s):  5L7Q, 5L8Q, 5MUP, 5MV5, 5MV6

Read abstract

Structure of deformed wing virus, a major honey bee pathogen.

K. Škubník, J. Nováček, T. Füzik, A. Přidal, R. Paxton, P. Plevka

The worldwide population of western honey bees (Apis mellifera) is under pressure from habitat loss, environmental stress, and pathogens, particularly viruses that cause lethal epidemics. Deformed wing virus (DWV) from the family Iflaviridae, together with its vector, the mite Varroa destructor, is likely the major threat to the world's honey bees. However, lack of knowledge of the atomic structures of iflaviruses has hindered the development of effective treatments against them. Here, we present the virion structures of DWV determined to a resolution of 3.1 Å using cryo-electron microscopy and 3.8 Å by X-ray crystallography. The C-terminal extension of capsid protein VP3 folds into a globular protruding (P) domain, exposed on the virion surface. The P domain contains an Asp-His-Ser catalytic triad that is, together with five residues that are spatially close, conserved among iflaviruses. These residues may participate in receptor binding or provide the protease, lipase, or esterase activity required for entry of the virus into a host cell. Furthermore, nucleotides of the DWV RNA genome interact with VP3 subunits. The capsid protein residues involved in the RNA binding are conserved among honey bee iflaviruses, suggesting a putative role of the genome in stabilizing the virion or facilitating capsid assembly. Identifying the RNA-binding and putative catalytic sites within the DWV virion structure enables future analyses of how DWV and other iflaviruses infect insect cells and also opens up possibilities for the development of antiviral treatments.

4.
C. Engel, T. Gubbey, S. Neyer, S. Sainsbury, C. Oberthuer, C. Baejen, C. Bernecky, P. Cramer   (2017)   Structural Basis of RNA Polymerase I Transcription Initiation.   Cell   169

DOI:  10.1016/j.cell.2017.03.003

References PDB protein(s):  5N5Y, 5N5Z, 5N60, 5N61

Read abstract

Structural Basis of RNA Polymerase I Transcription Initiation.

C. Engel, T. Gubbey, S. Neyer, S. Sainsbury, C. Oberthuer, C. Baejen, C. Bernecky, P. Cramer

Transcription initiation at the ribosomal RNA promoter requires RNA polymerase (Pol) I and the initiation factors Rrn3 and core factor (CF). Here, we combine X-ray crystallography and cryo-electron microscopy (cryo-EM) to obtain a molecular model for basal Pol I initiation. The three-subunit CF binds upstream promoter DNA, docks to the Pol I-Rrn3 complex, and loads DNA into the expanded active center cleft of the polymerase. DNA unwinding between the Pol I protrusion and clamp domains enables cleft contraction, resulting in an active Pol I conformation and RNA synthesis. Comparison with the Pol II system suggests that promoter specificity relies on a distinct "bendability" and "meltability" of the promoter sequence that enables contacts between initiation factors, DNA, and polymerase.

5.
J. Gu, M. Wu, R. Guo, K. Yan, J. Lei, N. Gao, M. Yang   (2016)   The architecture of the mammalian respirasome.   Nature   537

DOI:  10.1038/nature19359

References PDB protein(s):  5GPN

Read abstract

The architecture of the mammalian respirasome.

J. Gu, M. Wu, R. Guo, K. Yan, J. Lei, N. Gao, M. Yang

The respiratory chain complexes I, III and IV (CI, CIII and CIV) are present in the bacterial membrane or the inner mitochondrial membrane and have a role of transferring electrons and establishing the proton gradient for ATP synthesis by complex V. The respiratory chain complexes can assemble into supercomplexes (SCs), but their precise arrangement is unknown. Here we report a 5.4 Å cryo-electron microscopy structure of the major 1.7 megadalton SCI1III2IV1 respirasome purified from porcine heart. The CIII dimer and CIV bind at the same side of the L-shaped CI, with their transmembrane domains essentially aligned to form a transmembrane disk. Compared to free CI, the CI in the respirasome is more compact because of interactions with CIII and CIV. The NDUFA11 and NDUFB9 supernumerary subunits of CI contribute to the oligomerization of CI and CIII. The structure of the respirasome provides information on the precise arrangements of the respiratory chain complexes in mitochondria.

Protein Data Bank Structures

(Click on images for more details)
  • Life in the extremes: atomic structure of Sulfolobus Turreted Icosahedral Virus
    Author: Veesler, D. et al.
    Taken on a FEI TITAN KRIOS at 4.5 Å resolution
  • Cryo-EM structure of Dengue virus serotype 3 at 28 degrees C
    Author: Fibriansah, G. et al.
    Taken on a FEI TITAN KRIOS at 6.0 Å resolution
  • Structure of beta-galactosidase at 3.2-A resolution obtained by cryo-electron microscopy
    Author: Bartesaghi, A. et al.
    Taken on a FEI TITAN KRIOS at 3.2 Å resolution
  • CryoEM single particle reconstruction of anthrax toxin protective antigen pore at 2.9 Angstrom resolution
    Author: Jiang, J. et al.
    Taken on a FEI TITAN KRIOS at 2.9 Å resolution
  • Atomic structure of a non-enveloped virus reveals pH sensors for a coordinated process of cell entry
    Author: Zhang, X. et al.
    Taken on a FEI TITAN KRIOS at 3.3 Å resolution
  • Structure of alpha-1 glycine receptor by single particle electron cryo-microscopy, strychnine-bound state
    Author: Du, J. et al.
    Taken on a FEI TITAN KRIOS at 3.9 Å resolution
  • Electron cryo-microscopy of the IST1-CHMP1B ESCRT-III copolymer
    Author: McCullough, J. et al.
    Taken on a FEI TITAN KRIOS at 4.0 Å resolution
  • Structure of Escherichia coli EF4 in posttranslocational ribosomes (Post EF4)
    Author: Zhang, D. et al.
    Taken on a FEI TITAN KRIOS at 3.700 Å resolution
  • Cryo-EM structure of the magnesium channel CorA in the closed symmetric magnesium-bound state
    Author: Matthies, D. et al.
    Taken on a FEI TITAN KRIOS at 3.8 Å resolution
  • Structure of the yeast 60S ribosomal subunit in complex with Arx1, Alb1 and C-terminally tagged Rei1
    Author: Greber, B.J. et al.
    Taken on a FEI TITAN KRIOS at 3.410 Å resolution
  • Structure of a Chaperone-Usher pilus reveals the molecular basis of rod uncoilin
    Author: Hospenthal, M.K. et al.
    Taken on a FEI TITAN KRIOS at 3.8 Å resolution
  • Cryo-EM structure of the flagellar hook of Campylobacter jejuni
    Author: Matsunami, H. et al.
    Taken on a FEI TITAN KRIOS at 3.5 Å resolution
  • Structure of F-ATPase from Pichia angusta, state1
    Author: Vinothkumar, K.R. et al.
    Taken on a FEI TITAN KRIOS at 7 Å resolution
  • Cryo-EM structure of a BG505 Env-sCD4-17b-8ANC195 complex
    Author: Wang, H. et al.
    Taken on a FEI TITAN KRIOS at 8.9 Å resolution
  • Ca2+ bound aplysia Slo1
    Author: MacKinnon, R. et al.
    Taken on a FEI TITAN KRIOS at 3.8 Å resolution
  • Dephosphorylated, ATP-free cystic fibrosis transmembrane conductance regulator (CFTR) from zebrafish
    Author: Zhang, Z. et al.
    Taken on a FEI TITAN KRIOS at 3.73 Å resolution
  • The 2.8 A Electron Microscopy Structure of Adeno-Associated Virus-DJ Bound by a Heparanoid Pentasaccharide
    Author: Xie, Q. et al.
    Taken on a FEI TITAN KRIOS at 2.8 Å resolution
  • Volta phase plate cryo-electron microscopy structure of a calcitonin receptor-heterotrimeric Gs protein complex
    Author: Liang, Y.L. et al.
    Taken on a FEI TITAN KRIOS at 4.1 Å resolution
  • Cryo-EM structure of the human ether-a-go-go related K+ channel
    Author: Wang, W.W. et al.
    Taken on a FEI TITAN KRIOS at 3.7 Å resolution
  • CryoEM structure of type II secretion system secretin GspD in E.coli K12
    Author: Yan, Z. et al.
    Taken on a FEI TITAN KRIOS at 3.04 Å resolution
  • Structure of a Pancreatic ATP-sensitive Potassium Channel
    Author: Li, N. et al.
    Taken on a FEI TITAN KRIOS at 5.6 Å resolution
  • The resting state of yeast proteasome
    Author: Ding, Z. et al.
    Taken on a FEI TITAN KRIOS at 6.3 Å resolution
  • Structure of a eukaryotic voltage-gated sodium channel at near atomic resolution
    Author: Shen, H. et al.
    Taken on a FEI TITAN KRIOS at 3.8 Å resolution
  • Prefusion structure of MERS-CoV spike glycoprotein, three-fold symmetry
    Author: Yuan, Y. et al.
    Taken on a FEI TITAN KRIOS at 3.2 Å resolution
  • Prefusion structure of MERS-CoV spike glycoprotein, conformation 2
    Author: Yuan, Y. et al.
    Taken on a FEI TITAN KRIOS at 3.2 Å resolution
  • Structure of SARS-CoV spike glycoprotein
    Author: Gui, M. et al.
    Taken on a FEI TITAN KRIOS at 3.8 Å resolution
  • Nucleosome : Class 1
    Author: Bilokapic, S. et al.
    Taken on a FEI TITAN at 3.7 Å resolution
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