TY - JOUR
T1 - An automated pipeline to screen membrane protein 2D crystallization
AU - Kim, Changki
AU - Vink, Martin
AU - Hu, Minghui
AU - Love, James
AU - Stokes, David L.
AU - Ubarretxena-Belandia, Iban
N1 - Funding Information:
Acknowledgments This paper is dedicated to Russ Hinchliffe, who was indispensable in the design and manufacture of the dialysis block and other components used in our pipeline. We are grateful to Ms. Kumiko Sugawara for producing the proteins YkgB-D332 and YkgB-D36 used for crystallization. We also wish to thank Ms. K. D. Derr, Dr. Ruben Diaz-Avalos and Dr. William Rice at The New York Structural Biology Center for fruitful discussions and members of the New York Consortium on Membrane Protein Structure for their supply of both proteins and ideas. Funding for this work was provided by grant R01-GM081817 from the National Institutes of Health and grant MCB-0546087 from the National Science Foundation.
PY - 2010/6
Y1 - 2010/6
N2 - Electron crystallography relies on electron cryomicroscopy of two-dimensional (2D) crystals and is particularly well suited for studying the structure of membrane proteins in their native lipid bilayer environment. To obtain 2D crystals from purified membrane proteins, the detergent in a protein-lipid-detergent ternary mixture must be removed, generally by dialysis, under conditions favoring reconstitution into proteoliposomes and formation of well-ordered lattices. To identify these conditions a wide range of parameters such as pH, lipid composition, lipid-to-protein ratio, ionic strength and ligands must be screened in a procedure involving four steps: crystallization, specimen preparation for electron microscopy, image acquisition, and evaluation. Traditionally, these steps have been carried out manually and, as a result, the scope of 2D crystallization trials has been limited. We have therefore developed an automated pipeline to screen the formation of 2D crystals. We employed a 96-well dialysis block for reconstitution of the target protein over a wide range of conditions designed to promote crystallization. A 96-position magnetic platform and a liquid handling robot were used to prepare negatively stained specimens in parallel. Robotic grid insertion into the electron microscope and computerized image acquisition ensures rapid evaluation of the crystallization screen. To date, 38 2D crystallization screens have been conducted for 15 different membrane proteins, totaling over 3000 individual crystallization experiments. Three of these proteins have yielded diffracting 2D crystals. Our automated pipeline outperforms traditional 2D crystallization methods in terms of throughput and reproducibility.
AB - Electron crystallography relies on electron cryomicroscopy of two-dimensional (2D) crystals and is particularly well suited for studying the structure of membrane proteins in their native lipid bilayer environment. To obtain 2D crystals from purified membrane proteins, the detergent in a protein-lipid-detergent ternary mixture must be removed, generally by dialysis, under conditions favoring reconstitution into proteoliposomes and formation of well-ordered lattices. To identify these conditions a wide range of parameters such as pH, lipid composition, lipid-to-protein ratio, ionic strength and ligands must be screened in a procedure involving four steps: crystallization, specimen preparation for electron microscopy, image acquisition, and evaluation. Traditionally, these steps have been carried out manually and, as a result, the scope of 2D crystallization trials has been limited. We have therefore developed an automated pipeline to screen the formation of 2D crystals. We employed a 96-well dialysis block for reconstitution of the target protein over a wide range of conditions designed to promote crystallization. A 96-position magnetic platform and a liquid handling robot were used to prepare negatively stained specimens in parallel. Robotic grid insertion into the electron microscope and computerized image acquisition ensures rapid evaluation of the crystallization screen. To date, 38 2D crystallization screens have been conducted for 15 different membrane proteins, totaling over 3000 individual crystallization experiments. Three of these proteins have yielded diffracting 2D crystals. Our automated pipeline outperforms traditional 2D crystallization methods in terms of throughput and reproducibility.
KW - Automation
KW - Dialysis
KW - Electron crystallography
KW - High-throughput
KW - Membrane protein
KW - Two-dimensional (2D) crystals
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U2 - 10.1007/s10969-010-9088-5
DO - 10.1007/s10969-010-9088-5
M3 - Article
C2 - 20349145
AN - SCOPUS:77956761912
SN - 1345-711X
VL - 11
SP - 155
EP - 166
JO - Journal of Structural and Functional Genomics
JF - Journal of Structural and Functional Genomics
IS - 2
ER -