7A projection map of the S-layer protein sbpA obtained with trehalose-embedded monolayer crystals.
Two-dimensional crystallization on lipid monolayers is a versatile tool to obtain structural information of proteins by electron microscopy. An inherent problem with this approach is to prepare samples in a way that preserves the crystalline order of the protein array and produces specimens that are sufficiently flat for high-resolution data collection at high tilt angles. As a test specimen to optimize the preparation of lipid monolayer crystals for electron microscopy imaging, we used the S-layer protein sbpA, a protein with potential for designing arrays of both biological and inorganic materials with engineered properties for a variety of nanotechnology applications. Sugar embedding is currently considered the best method to prepare two-dimensional crystals of membrane proteins reconstituted into lipid bilayers. We found that using a loop to transfer lipid monolayer crystals to an electron microscopy grid followed by embedding in trehalose and quick-freezing in liquid ethane also yielded the highest resolution images for sbpA lipid monolayer crystals. Using images of specimens prepared in this way we could calculate a projection map of sbpA at 7A resolution, one of the highest resolution projection structures obtained with lipid monolayer crystals to date.
Norville
Julie E
JE
MIT Computer Science and Artificial Intelligence Laboratory, 32 Vassar Street, Cambridge, MA 02139, USA.
Kelly
Deborah F
DF
Knight
Thomas F
TF
Belcher
Angela M
AM
Walz
Thomas
T
D001407:Bacillus / Q000737:chemistry*; D001426:Bacterial Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000648:ultrastructure*; D002244:Carbon; D020285:Cryoelectron Microscopy / Q000295:instrumentation / Q000379:methods*; D003460:Crystallization / Q000379:methods*; D003461:Crystallography / Q000295:instrumentation / Q000379:methods*; D004134:Dimyristoylphosphatidylcholine; D004980:Ethane; D009004:Monosaccharide Transport Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000648:ultrastructure*; D016624:Negative Staining / Q000379:methods; D016062:Porosity; D011487:Protein Conformation; D000644:Quaternary Ammonium Compounds; D013048:Specimen Handling / Q000295:instrumentation / Q000379:methods*; D014199:Trehalose
D001426:Bacterial Proteins; D009004:Monosaccharide Transport Proteins; D000644:Quaternary Ammonium Compounds; C120455:SbpA protein, bacteria; C046112:didodecyldimethylammonium; D002244:Carbon; D014199:Trehalose; D004980:Ethane; D004134:Dimyristoylphosphatidylcholine