Current Research

Nikolaus Grigorieff develops high-resolution electron cryo-microscopy techniques to study the three-dimensional structure of large macromolecules and their assemblies.

Our laboratory uses high-resolution electron cryo-microscopy (cryo-EM) to study the three-dimensional (3D) structure of proteins and protein complexes. We generally focus on proteins that are difficult to study by more traditional techniques such as x-ray crystallography and nuclear magnetic resonance (NMR). For example, large protein assemblies, such as the spliceosome, pose problems because they undergo constant changes in composition and conformation when performing their task. They are too large for NMR analysis, difficult to crystallize for x-ray crystallography, but amenable to electron microscopy.

Using EM, we can visualize individual protein molecules and complexes if their total molecular weight is larger than ~200 kDa, thus avoiding the need for crystals. This "single-particle" approach requires extremely small amounts of material, typically only a few tens of picomoles. Images from single protein particles are usually dominated by noise because the sample exposure to a high-energy beam must be kept small to limit protein damage. To obtain a well-defined structure of the particle, we must align many thousands of images with each other and use computer image processing to average the images. Depending on the protein particle, sample preparation technique, and instrumentation, a resolution better than 5 Å can be obtained. We are developing new image-processing methods to push the current limit to higher resolution.

The Spliceosome
An important goal in my laboratory is to understand the structural underpinnings of gene splicing. In collaboration with Melissa Moore (HHMI, University of Massachusetts Medical School), we are working 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 complexes (50–60 S) composed of pre-mRNA, four small nuclear ribonucleoprotein (snRNP) particles, as well as 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 more than 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 to use cryo-EM of unstained specimens to improve this structure. The 3D structure of one or more of the spliceosomal complexes, at a resolution of ~10 Å or higher, will be invaluable for a better understanding of the inner workings of this large molecular machine.

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 Aβ peptide and are associated with neurodegeneration. Amyloid formation is also observed with other diseases, such as type II diabetes and Creutzfeldt-Jakob disease. Amyloid structures often 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 (Leibniz Institute for Age Research, Jena, Germany), we study the molecular architecture of amyloid fibrils associated with human disease (Figure 1). Our goal is to understand the folding pathway of fibril formation that leads to the substantial polymorphism observed for most amyloid aggregates, and to identify potential targets for disease treatment.

Rotavirus is the main agent of severe gastroenteritis in children worldwide, leading to more than half a million deaths every year. The virus genome is composed of 11 segments of double-stranded RNA (dsRNA), which encode the 12 viral proteins. Four of these form the viral capsid (VP2, VP4, VP6, and glycoprotein VP7), two compose the transcription apparatus inside the capsid (VP1 and VP3), and the remaining proteins are involved in genome packing and a number of cellular functions promoting viral replication. The fully assembled capsid (Figure 2) consists of an outer layer (VP4 and VP7), a middle layer (VP6), and an inner layer (VP2). When the virus enters the cell, the reduced calcium concentration inside the cell appears to induce loss of the outer layer, activating transcription.

Using material provided by Stephen Harrison's lab (HHMI, Harvard Medical School), we imaged different stages of assembly of rotavirus at near-atomic resolution (Figure 3). With these images and a crystal structure of a VP7 trimer determined in the Harrison laboratory, we built an atomic model of the outer two layers of a rotavirus particle missing VP4. The structure shows that the main interactions between the VP6 layer and VP7 occur at the N termini of the VP7 trimers, which are disordered in the crystal structure and were built de novo using the cryo-EM map. The three N termini act as a clamp that is released when the VP7 trimer is destabilized upon removal of calcium, which binds at the interface between monomers in the VP7 trimer. Furthermore, comparison of viral capsids with and without VP7 present reveals a translocation of VP6 and VP2 away from the particle center and an increase of the diameter of the pores at the 5-fold axes upon removal of the outer VP7 layer. The opening of the 5-fold channels is likely related to activation of the transcription apparatus (VP1 and VP3) located near the 5-fold axes inside the particle. Thus, VP7 may act as a calcium ''sensor,'' which transmits a signal to coordinate the onset of transcription with entry into the host-cell cytoplasm.

As of September 16, 2013

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