Andres LeschzinerDepartment of Molecular & Cellular Biology
Northwest Building, Room 311.15
52 Oxford Street, Cambridge, MA 02138
tel: (617) 496-2717
Research in my group will focus on two major areas over the next few years:
“Imaging Biochemistry”: Automating the Initial Three-Dimensional Reconstruction of Novel Macromolecules by Electron Microscopy
Electron microscopy of frozen-hydrated samples (cryo-EM) has emerged as a powerful technique for obtaining three-dimensional reconstructions of large macromolecular assemblies under physiological conditions. Furthermore, it has the potential for visualizing them in a variety of states (conformations, reaction intermediates, etc.) in the same sample, at atomic or near-atomic resolution, on a routine basis. Yet a major bottleneck in the process has kept the field from progressing in that direction: the generation of the first, low-resolution model of a novel structure (the initial combination of 2D images into a 3D volume). The two standard approaches available for the last 20 years require significant expertise from the user to implement and to avoid potential artifacts and are time consuming. We have developed a novel reconstruction method that combines the strengths of the two existing methods while avoiding their limitations. Importantly, this makes it the only method amenable to full automation, a critical step if cryo-EM is to be applied to the visualization of complex reactions. Our goal is to develop tools that will make this automation a reality. We are working on software that will implement our reconstruction method and will interface with already existing automated data-collection packages generating, in a way transparent to the user, initial 3D reconstructions that can be refined to high resolution. This will allow us, in time, to “visualize” entire reactions.
The Mechanism of ATP-Dependent Chromatin Remodeling
The amazing degree of compaction necessary to package a cell’s DNA into its nucleus makes DNA inaccessible to processes such as replication, transcription, recombination and repair. Cells deal with this obstacle with an array of factors that modify chromatin (the nucleoprotein assembly responsible for this compaction) and turn it into a highly dynamic and tunable device. Among these factors are the ATP-dependent chromatin remodeling complexes (“remodelers”), large (often > 1 megadalton), multi-subunit macromolecular machines that use the energy from ATP hydrolysis to non-covalently modify the structure of nucleosomes, the basic unit of compaction in chromatin consisting of an octamer of histone proteins and ~147bp of DNA wrapped around it. Remodelers are present in all eukaryotes and show conservation of their catalytic ATPase subunit, suggesting the existence of a common remodeling mechanism. At the same time, the diversity of outcomes the remodelers are capable of both in vitro and in vivo indicates that this mechanism must undergo significant tuning. Despite extensive biochemical and genetic characterization of these complexes the basic remodeling mechanism remains elusive, as do any modifications to it that allow remodelers to perform their specific biological functions. We are using cryo-EM to probe existing mechanistic hypotheses and understand the ways in which different remodeling outcomes are achieved. We are currently focusing on two model systems: the yeast remodelers RSC and SWR1. The former is capable of sliding a histone octamer along the DNA (and sometimes of ejecting it) while the latter is unique in its ability to catalyze the exchange of a variant histone dimer for the native one without fully disassembling the nucleosome. A detailed structural analysis of the two systems will reveal the common underlying remodeling mechanism and how complexes tune it to achieve their specific biological functions.
Leschziner AE, Saha A, Wittmeyer J, Zhang Y, Bustamante C, Cairns BR and Nogales E (2007). Conformational flexibility in the chromatin remodeler RSC observed by electron microscopy and the orthogonal tilt reconstruction method. Proc. Natl. Acad. Sci. USA 104(12): 4913-8.
Leschziner AE and Nogales E (2007). Visualizing flexibility at molecular resolution: Analysis of heterogeneity in single-particle electron microscopy reconstructions. Annu. Rev. Biophys. Biomol. Struct. Vol.36: 43-6.
Leschziner AE and Nogales E (2006). The Orthogonal Tilt Reconstruction method: an approach to generating single-class volumes with no missing cone for ab initio reconstruction of asymmetric particles. J Struct Biol. 153(3):284-99.
Leschziner AE, Lemon B, Tjian R and Nogales E (2005). Structural studies of the human PBAF chromatin-remodeling complex. Structure (Camb) 13(2): 267-75.
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