doi:10.1016/j.jsb.2006.08.002 
Copyright © 2006 Elsevier Inc. All rights reserved.
Crystallization note
Two-dimensional crystallization of human vitamin K-dependent γ-glutamyl carboxylase
Ingeborg Schmidt-Kreya, 
, 
, Winfried Haaseb, Vasantha Mutucumaranac, Darrel W. Staffordc and Werner Kühlbrandtb
aGeorgia Institute of Technology, School of Biology, 310 Ferst Drive, Atlanta, GA 30332-0230, USA
bMax-Planck-Institute of Biophysics, Department of Structural Biology, Max-von-Laue-Str. 3, 60438 Frankfurt am Main, Germany
cThe University of North Carolina at Chapel Hill, Department of Biology and Center for Thrombosis and Hemostasis, Chapel Hill, 27599-3280, USA
Received 15 June 2006;
revised 8 August 2006;
accepted 8 August 2006.
Available online 15 August 2006.
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Abstract
Planar-tubular two-dimensional (2D) crystals of human vitamin K-dependent γ-glutamyl carboxylase grow in the presence of dimyristoyl phosphatidylcholine (DMPC). Surprisingly, these crystals form below the phase transition temperature of DMPC and at the unusually low molar lipid-to-protein (LPR) ratio of 1, while 2D crystals are conventionally grown above the phase transition temperature of the reconstituting lipid and significantly higher LPRs. The crystals are up to 0.75 μm in the shorter dimension of the planar tubes and at least 1 μm in length. Due to the planar-tubular nature of the crystals, two lattices are present. These are rotated by nearly 90° in respect to each other. The ordered arrays exhibit p121 plane group symmetry with unit cell dimensions of a = 83.7 Å, b = 76.6 Å, γ = 91°. Projection maps calculated from images of negatively stained and electron cryo-microscopy samples reveal the human vitamin K-dependent γ-glutamyl carboxylase to be a monomer.
Keywords: Human vitamin K-dependent γ-glutamyl carboxylase; Two-dimensional crystallization; Electron crystallography; Blood coagulation; Anti-coagulation
Fig. 1. A freeze-fracture image of a mosaic carboxylase 2D crystalline sheet grown below the phase transition temperature of the added phospholipids (DMPC). The freeze-fracture results demonstrate that the carboxylase crystals are indeed constituted of ordered protein. The scale bar is 100 nm.
Fig. 2. Small, stacked crystals form at relatively high molar lipid-to-protein ratios of 15. The scale bar is 50 nm.
Fig. 3. When the molar lipid-to-protein ratio is reduced to 1, a change in crystal morphology is observed over time: (A) Crystalline sheets and vesicles form as a first step during the reconstitution and crystallization process. As the size of the ordered arrays increases with time a transition in morphology occurs from these sheets and vesicles to (B) triangular vesicles, which finally become (C) planar-tubular crystals with continuous crystalline arrays spanning the entire membrane. The scale bar is 200 nm (A–C). (D) shows the transitional stages (scale bar = 1 μm) and (E) is an enlarged ordered section of the planar-tubular crystal (C). The scale bar is 50 nm.
Fig. 4. Morphological changes due to salt concentration. A change in the salt concentration to (A) 100 mM results in folded sheets and to (B) 400 mM NaCl produces much smaller planar tubes than obtained under the standard conditions of 250 mM NaCl. The scale bar is 200 nm.
Fig. 5. (A) A plot depicting the quality of the combined phase error for individual reflections of the merged data set including three images. The sizes of the boxes correspond to phase errors with 1 < 8°, 2 < 14°, 3 < 20°, 4 < 30°, 5 < 40°, 6 < 50°, 7 < 70°, 8 < 90°. The larger boxes are labeled. The rings correspond to resolutions of 20, 15, 10, 8 Å, respectively. (B) A projection map of merged image data at a resolution of 20.4 Å of negatively stained crystals. The unit cell is marked and contains two monomers in opposite membrane orientations. (C) A cryo-EM projection map at a resolution of 12 Å. The unit cell dimensions are a = 83.7 Å, b = 76.6 Å, γ = 91°.
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