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Electron Microscopy of Large Molecular Assemblies and Membrane Proteins
Summary: Nikolaus Grigorieff uses electron microscopy to visualize the three-dimensional structure of large molecular complexes in cells. His aim is to understand how the individual components of these machines function and interact with one another.
Our laboratory uses high-resolution electron microscopy (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, membrane proteins are usually too large for NMR analysis or are difficult to crystallize for x-ray crystallography. Large protein assemblies, such as the spliceosome, pose additional problems because they undergo constant changes in composition and conformation.
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. Single-particle EM is therefore ideally suited for the structural analysis of larger proteins and protein assemblies. 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 6 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, 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 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 (Figure 1). 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 ~20 Å or higher, will be invaluable for a better understanding of the inner workings of this large molecular machine.
N-Ethylmaleimide–Sensitive Factor
NSF, which belongs to the family of AAA ATPases, is an essential component of the protein machinery that regulates vesicle fusion with target membranes, for example, at synaptic terminals. NSF associates with α-SNAP (soluble NSF attachment protein) to disassemble SNARE (soluble NSF attachment protein receptor) complexes. SNAREs, together with other proteins, facilitate docking and fusion of vesicles, and they are recycled and reactivated through disassembly by NSF. NSF functions as a homohexamer, and each protomer contains three domains. The N-terminal domain of NSF is essential for the binding of α-SNAP and is followed by ATPase domains D1 and D2. Binding and hydrolysis of ATP by the D1 domain induces conformational changes in NSF leading to disassembly of the SNARE complex. Axel Brunger (HHMI, Stanford University) determined the crystal structures of the N and D2 domains and of α-SNAP and a SNARE complex. In collaboration with the Brunger laboratory, we recently obtained a structure at 11-Å resolution of NSF bound to α-SNAP and a SNARE that reveals the arrangement of the D1 and D2 domains within the NSF hexamer. Other parts of the structure, including the N domain and α-SNAP/SNARE complex, appear to be disordered and were not resolved at the same level of detail. Our goal is to use improved preparations of the complex and novel image-processing techniques that can accommodate sample heterogeneity to visualize these parts of the structure at higher resolution.
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 (Leibniz-Institute for Age Research, Jena, 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.
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 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 double-layer particles to determine a structure of one of the capsid proteins, VP6, to a resolution of 3.8 Å (Figure 2). We are using these data to investigate limitations in the image alignment and reconstruction algorithms. (This work is supported by a grant from the National Institutes of Health.)
Last updated: June 18, 2008
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