TY - JOUR
T1 - A high-throughput strategy to screen 2D crystallization trials of membrane proteins
AU - Vink, Martin
AU - Derr, KD
AU - Love, James
AU - Stokes, David L.
AU - Ubarretxena-Belandia, Iban
N1 - Funding Information:
We are indebted to Dr. C. Wu at the Skirball Institute of Biomolecular Medicine (New York, USA) for his generous gift of CopA and to Drs. J.A. Timney and C.N. Hunter at Sheffield University (Sheffield, UK) for their kind gift of LH2 complexes. We also wish to thank Mr. R. Hinchliffe at the City College of New York (New York, USA) for help with manufacturing the crystallization block and the staining plate, and Dr. K. Mitra at the Wadsworth Center (Albany, USA) and Mr. J. Riley at the New York Structural Biology Center for help with their design. We are also grateful to Drs. A. Siebert and W. Rice at the New York Structural Biology Center for fruitful discussions. National Institutes of Health Grants U54 GM075026 to the New York Consortium on Membrane Protein Structure, R01 GM56960 and R01 GM081817 to DLS, and National Science Foundation Grant MCB-0546087 to IU-B supported this work.
PY - 2007/12
Y1 - 2007/12
N2 - Electron microscopy of two-dimensional (2D) crystals has demonstrated potential for structure determination of membrane proteins. Technical limitations in large-scale crystallization screens have, however, prevented a major breakthrough in the routine application of this technology. Dialysis is generally used for detergent removal and reconstitution of the protein into a lipid bilayer, and devices for testing numerous conditions in parallel are not readily available. Furthermore, the small size of resulting 2D crystals requires electron microscopy to evaluate the results and automation of the necessary steps is essential to achieve a reasonable throughput. We have designed a crystallization block, using standard microplate dimensions, by which 96 unique samples can be dialyzed simultaneously against 96 different buffers and have demonstrated that the rate of detergent dialysis is comparable to those obtained with conventional dialysis devices. A liquid-handling robot was employed to set up 2D crystallization trials with the membrane proteins CopA from Archaeoglobus fulgidus and light-harvesting complex II (LH2) from Rhodobacter sphaeroides. For CopA, 1 week of dialysis yielded tubular crystals and, for LH2, large and well-ordered vesicular 2D crystals were obtained after 24 h, illustrating the feasibility of this approach. Combined with a high-throughput procedure for preparation of EM-grids and automation of the subsequent negative staining step, the crystallization block offers a novel pipeline that promises to speed up large-scale screening of 2D crystallization and to increase the likelihood of producing well-ordered crystals for analysis by electron crystallography.
AB - Electron microscopy of two-dimensional (2D) crystals has demonstrated potential for structure determination of membrane proteins. Technical limitations in large-scale crystallization screens have, however, prevented a major breakthrough in the routine application of this technology. Dialysis is generally used for detergent removal and reconstitution of the protein into a lipid bilayer, and devices for testing numerous conditions in parallel are not readily available. Furthermore, the small size of resulting 2D crystals requires electron microscopy to evaluate the results and automation of the necessary steps is essential to achieve a reasonable throughput. We have designed a crystallization block, using standard microplate dimensions, by which 96 unique samples can be dialyzed simultaneously against 96 different buffers and have demonstrated that the rate of detergent dialysis is comparable to those obtained with conventional dialysis devices. A liquid-handling robot was employed to set up 2D crystallization trials with the membrane proteins CopA from Archaeoglobus fulgidus and light-harvesting complex II (LH2) from Rhodobacter sphaeroides. For CopA, 1 week of dialysis yielded tubular crystals and, for LH2, large and well-ordered vesicular 2D crystals were obtained after 24 h, illustrating the feasibility of this approach. Combined with a high-throughput procedure for preparation of EM-grids and automation of the subsequent negative staining step, the crystallization block offers a novel pipeline that promises to speed up large-scale screening of 2D crystallization and to increase the likelihood of producing well-ordered crystals for analysis by electron crystallography.
KW - 96-Well format
KW - Crystallization block
KW - Dialysis block
KW - Electron crystallography
KW - High-throughput screening
KW - Membrane protein reconstitution
KW - Membrane proteins
KW - Negative staining
KW - Two-dimensional (2D) crystals
UR - http://www.scopus.com/inward/record.url?scp=36049011365&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=36049011365&partnerID=8YFLogxK
U2 - 10.1016/j.jsb.2007.09.003
DO - 10.1016/j.jsb.2007.09.003
M3 - Article
C2 - 17951070
AN - SCOPUS:36049011365
SN - 1047-8477
VL - 160
SP - 295
EP - 304
JO - Journal of Structural Biology
JF - Journal of Structural Biology
IS - 3
ER -