Jared A Delmar1, Jani Reddy Bolla2, Chih-Chia Su1, Edward W Yu3. 1. Department of Physics and Astronomy, Iowa State University, Ames, Iowa, USA. 2. Department of Chemistry, Iowa State University, Ames, Iowa, USA. 3. Department of Physics and Astronomy, Iowa State University, Ames, Iowa, USA; Department of Chemistry, Iowa State University, Ames, Iowa, USA. Electronic address: ewyu@iastate.edu.
Abstract
X-ray crystallography remains the most robust method to determine protein structure at the atomic level. However, the bottlenecks of protein expression and purification often discourage further study. In this chapter, we address the most common problems encountered at these stages. Based on our experiences in expressing and purifying antimicrobial efflux proteins, we explain how a pure and homogenous protein sample can be successfully crystallized by the vapor diffusion method. We present our current protocols and methodologies for this technique. Case studies show step-by-step how we have overcome problems related to expression and diffraction, eventually producing high-quality membrane protein crystals for structural determinations. It is our hope that a rational approach can be made of the often anecdotal process of membrane protein crystallization.
X-ray crystallography remains the most robust method to determine protein structure at the atomic level. However, the bottlenecks of protein expression and purification often discourage further study. In this chapter, we address the most common problems encountered at these stages. Based on our experiences in expressing and purifying antimicrobial efflux proteins, we explain how a pure and homogenous protein sample can be successfully crystallized by the vapor diffusion method. We present our current protocols and methodologies for this technique. Case studies show step-by-step how we have overcome problems related to n class="Species">expression and diffraction, eventually producing high-quality membrane protein crystals for structural determinations. It is our hope that a rational approach can be made of the often anecdotal process of membrane protein crystallization.
Authors: Pil Seok Chae; Søren G F Rasmussen; Rohini R Rana; Kamil Gotfryd; Richa Chandra; Michael A Goren; Andrew C Kruse; Shailika Nurva; Claus J Loland; Yves Pierre; David Drew; Jean-Luc Popot; Daniel Picot; Brian G Fox; Lan Guan; Ulrik Gether; Bernadette Byrne; Brian Kobilka; Samuel H Gellman Journal: Nat Methods Date: 2010-10-31 Impact factor: 28.547