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Grigorieff Lab

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Current Research
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We apply cryo-EM to non-crystalline preparations and need only very little material, making this technique uniquely suitable for large assemblies that are responsible for cellular functions.

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Our new methods are designed to achieve the highest possible resolution (4 Å or better). We have recently established a new movie imaging protocol that makes use of a new type of camera, the direct electron detector. Using this detector, the total electron dose used for an image can be distributed over many short movie frames rather than a single long exposure. Any sample movement that occurs during the exposure (for example, beam-induced motion) can subsequently be tracked in the movies and “undone” by aligning the movie frames to each other, thereby producing a final image that is free of blurring.

Amyloid Fibrils

Amyloid fibrils are peptide or protein aggregates that form under certain conditions in vitro or in vivo. For example, the amyloid fibril plaques found in brain tissue of Alzheimer patients are formed from the peptide Aβ and are associated with neurodegeneration. Amyloid formation is also observed with other diseases, such as type II diabetes and Creutzfeldt-Jakob disease.

Amyloid structures represent an alternative to the native folding pattern of many peptides and proteins. A characteristic motif of this folding pattern is the cross-β structure in which the peptides or proteins associate by β-sheet formation within protofilaments making up a fibril.

In collaboration with Marcus Fändrich (Max Planck Research Unit for Enzymology of Protein Folding, Halle, Germany), we study the molecular architecture of amyloid fibrils associated with human disease. Our goal is to identify fundamental principles of amyloid formation, and potential targets for disease treatment.

The Spliceosome

An important goal in my laboratory is to understand the structural underpinnings of gene splicing. This work is carried out in collaboration with the Moore laboratory at HHMI, UMass Worcester to obtain purified, homogeneous splicing complexes that are suitable for single particle EM. The spliceosome removes introns from nascent transcripts, an essential step in eukaryotic gene expression. Most introns interrupt precursors to messenger RNAs (pre-mRNAs), and their precise excision is required to create readable mRNAs. Spliceosomes are ribosome-sized (50 - 60 S) complexes composed of pre-mRNA, four small nuclear ribonucleoprotein (snRNP) particles, and a host of associated protein factors. The snRNPs (U1, U2, U4/6, and U5) are, in turn, multicomponent complexes, each containing at least one small stable RNA molecule (snRNA) and five or more tightly bound polypeptides. In all, it has been estimated that nuclear pre-mRNA splicing requires the action of over 100 different gene products. We have recently obtained images of purified spliceosomes (C complex) that have been used to determine an initial 3D structure of the spliceosome. Our goal is now to improve this structure using cryo-electron microscopy of unstained specimens. The 3D structure of one or more of the spliceosomal complexes, at a resolution of about 20 Angstroms or higher, will be invaluable for a better understanding of the inner workings of this large molecular machine.

The catalytically competent C complex stands at the end of an ordered pathway by which the snRNPs assemble to form spliceosomes. To better understand this assembly, and how splice sites are recognized, we are also working on earlier splicing complexes. Finally, together with the Moore laboratory, we study post-splicing complexes that remains on the spliced mRNA substrate, such as the exon junction complex (EJC). The EJC targets the spliced mRNA for nuclear export and is involved in determining its fate in subsequent processing, such as translation by the ribosome.

Viruses at High Resolution

One of the main limitations of the single-particle technique in cryo-EM is the attainable resolution.

We are collaborating with Stephen Harrison (HHMI, Harvard Medical School) to use virus particles as test specimens to develop better single particle image-processing methods. Virus particles have a high degree of symmetry and are stable in an aqueous solution, making them ideal for EM imaging.

We have recently used rotavirus particles (see Figure) to determine a structure of one of its capsid proteins to a resolution of 2.6 Å.

Furthermore, we use helical viruses, such as TMV, to test new algorithms for helical particle processing.

Methods Development

A major interest of the lab is the development and optimization of methods for high-resolution cryo-EM. This has led us to investigate:

  • Electron exposure during data collection
  • Refinement of 3D reconstructions using single-particle images
  • Particle selection from micrographs
  • Resolution measurement
  • Maximum-likelihood methods
  • High-resolution 3D reconstruction of helical specimens
  • CTF estimation
  • Movie processing
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Interested in Learning More about the Lab?

As a group, we strive to develop new and improved methods in cryo-electron microscopy (cryo-EM) that improve the resolution of three-dimensional reconstructions of biological structures. 

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Available Positions

Postdoctoral Position in Computational Biology/Image Processing

Our group has a postdoctoral position available to develop new image processing algorithms for electron cryo-microscopy. We are primarily interested in candidates with:

  • a strong background in computational image processing and algorithm development
  • an energetic and interactive personality
  • experience in electron cryo-microscopy (preferred but not required)

For details of the projects and other information about the lab please contact us.

Paper Rejection Repository

Nobody likes to receive a letter from the editor of your favorite journal letting you know that your paper was rejected. Some journals have begun including reviewers’ comments with accepted papers to make the views of experts available to the reader. However, often the paper has been submitted to several journals and rejected before it is finally accepted. Our forum offers a place to publish these letters and comments to educate others.

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