Single-protein detection in crowded molecular environments in cryo-EM images
- Howard Hughes Medical Institute, Janelia Research Campus, United States
Abstract
We present an approach to study macromolecular assemblies by detecting component proteins' characteristic high-resolution projection patterns, calculated from their known 3D structures, in single electron cryo-micrographs. Our method detects single apoferritin molecules in vitreous ice with high specificity and determines their orientation and location precisely. Simulations show that high spatial-frequency information and-in the presence of protein background-a whitening filter are essential for optimal detection, in particular for images taken far from focus. Experimentally, we could detect small viral RNA polymerase molecules, distributed randomly among binding locations, inside rotavirus particles. Based on the currently attainable image quality, we estimate a threshold for detection that is 150 kDa in ice and 300 kDa in 100 nm thick samples of dense biological material.
Data availability
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Horse spleen apoferritinPublicly available at the RCSB Protein Data Bank (accession no: 2W0O).
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THE CRYSTAL STRUCTURE OF THE BACTERIAL CHAPERONIN GROEL AT 2.8 ANGSTROMSPublicly available at the RCSB Protein Data Bank (accession no: 1GRL).
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Location of the dsRNA-dependent polymerase, VP1, in rotavirus particlesPublicly available at the RCSB Protein Data Bank (accession no: 4F5X).
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Crystal Structure of VP1 apoenzyme of Rotavirus SA11 (N-terminal hexahistidine-tagged)Publicly available at the RCSB Protein Data Bank (accession no: 2R7O).
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Crystal Structure of the Rotavirus Double Layered ParticlePublicly available at the RCSB Protein Data Bank (accession no: 3KZ4).
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Crystal Structure of Bovine Serum AlbuminPublicly available at the RCSB Protein Data Bank (accession no: 4F5S).
Article and author information
Author details
Nikolaus Grigorieff
Funding
Howard Hughes Medical Institute (Internal)
- J Peter Rickgauer
- Nikolaus Grigorieff
- Winfried Denk
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Copyright
© 2017, Rickgauer et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
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Single-protein detection in crowded molecular environments in cryo-EM images
eLife 6:e25648.
https://doi.org/10.7554/eLife.25648
Further reading
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- Structural Biology and Molecular Biophysics
The aggregation of the protein α-synuclein is closely associated with several neurodegenerative disorders and as such the structures of the amyloid fibril aggregates have high scientific and medical significance. However, there are dozens of unique atomic-resolution structures of these aggregates, and such a highly polymorphic nature of the α-synuclein fibrils hampers efforts in disease-relevant in vitro studies on α-synuclein amyloid aggregation. In order to better understand the factors that affect polymorph selection, we studied the structures of α-synuclein fibrils in vitro as a function of pH and buffer using cryo-EM helical reconstruction. We find that in the physiological range of pH 5.8–7.4, a pH-dependent selection between Type 1, 2, and 3 polymorphs occurs. Our results indicate that even in the presence of seeds, the polymorph selection during aggregation is highly dependent on the buffer conditions, attributed to the non-polymorph-specific nature of secondary nucleation. We also uncovered two new polymorphs that occur at pH 7.0 in phosphate-buffered saline. The first is a monofilament Type 1 fibril that highly resembles the structure of the juvenile-onset synucleinopathy polymorph found in patient-derived material. The second is a new Type 5 polymorph that resembles a polymorph that has been recently reported in a study that used diseased tissues to seed aggregation. Taken together, our results highlight the shallow amyloid energy hypersurface that can be altered by subtle changes in the environment, including the pH which is shown to play a major role in polymorph selection and in many cases appears to be the determining factor in seeded aggregation. The results also suggest the possibility of producing disease-relevant structure in vitro.
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- Biochemistry and Chemical Biology
- Structural Biology and Molecular Biophysics
Degradation of heparan sulfate (HS), a glycosaminoglycan (GAG) comprised of repeating units of N-acetylglucosamine and glucuronic acid, begins in the cytosol and is completed in the lysosomes. Acetylation of the terminal non-reducing amino group of α-D-glucosamine of HS is essential for its complete breakdown into monosaccharides and free sulfate. Heparan-α-glucosaminide N-acetyltransferase (HGSNAT), a resident of the lysosomal membrane, catalyzes this essential acetylation reaction by accepting and transferring the acetyl group from cytosolic acetyl-CoA to terminal α-D-glucosamine of HS in the lysosomal lumen. Mutation-induced dysfunction in HGSNAT causes abnormal accumulation of HS within the lysosomes and leads to an autosomal recessive neurodegenerative lysosomal storage disorder called mucopolysaccharidosis IIIC (MPS IIIC). There are no approved drugs or treatment strategies to cure or manage the symptoms of, MPS IIIC. Here, we use cryo-electron microscopy (cryo-EM) to determine a high-resolution structure of the HGSNAT-acetyl-CoA complex, the first step in the HGSNAT-catalyzed acetyltransferase reaction. In addition, we map the known MPS IIIC mutations onto the structure and elucidate the molecular basis for mutation-induced HGSNAT dysfunction.
