Microscopy 101
as LCAT. LCAT is a plasma enzyme that interacts with Apo A on the HDL particle surface and esterifies free cholesterol (FC) to its stored form cholesteryl ester (CE). As it converts FC to EC, LCAT facilitates the movement of EC into the core of the HDL particle, driving its maturation from a dis- coidal particle to a spherical particle (Fig- ure 1). We have generated two-dimensional class averages of HDL particles crosslinked to LCAT, which clearly defines the associa- tion of LCAT (arrow) to the surface of the HDL (Figure 8).
Discussion Two-dimensional class averages of par-
ticles visualized with our modified nega- tive stain procedure show greater detail on the HDL particle surface, making three- dimensional reconstruction more attainable than with the PTA staining method. Te class averages of the LpA-I plasma particle from our modified negative stain procedure clearly show reproducible areas of density differences that we interpret as distinguish- ing surface proteins from surrounding lip- ids (arrow, Figure 6F). Further, from our 3D reconstruction, we believe the areas of higher density versus lower density repre- sent protein and lipid, respectively (Figure 7B), based on orientation in the particle and molecular weight. Te new staining method also provides more defined particle edges for more accurate particle diameter measure- ments. Te greater edge definition can also be used for thresholding for automated mea- surement of particles in the future. Te improved surface detail available
Figure 6: Comparison of our previous traditional negative staining method using PTA to our modified method using UF. A) A micrograph of isolated plasma HDL particles stained with 2% PTA. Image taken at 150,000× magnification. B) Plasma HDL particles stained with 0.7% UF using our modified negative staining procedure. Image taken at 150,000×. Scale bar = 100 nm for both 6A and 6B. C) A histogram of HDL particle distribution of ~100 particle diameters from the dataset shown in A. Mean and standard deviation = 9.98 nm +/- 1.129 nm. D) A histogram from measuring ~100 particle diameters from the dataset shown in B. Mean and standard deviation = 10.61 nm +/- 1.319 nm. E) Two-dimensional class averages generated from the dataset shown in A. F) Two-dimensional class averages generated from the dataset shown in B.
our modified staining procedure (Figure 6F) showed greater surface detail than those from our previous procedure (Fig- ure 6E). Chosen class averages from the modified procedure were then arranged into a three-dimensional display, which was viewed and adjusted using the volume viewer tool in UCSF Chimera [9] (Figure 7A). Further, we were able to separate areas of higher density (brighter areas in images) from those areas of lower density (darker areas in images) not seen with the PTA staining procedure (Figure 7B). Besides whole-particle reconstructions, we have also exam-
ined interactions between HDL particles and proteins impor- tant in the assembly and maturation of the HDL particle such
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with our modified procedure also allowed visualization of
the interactions between
the HDL particles and LCAT. Tese images are aiding us in discovering unique bind- ing sites between LCAT and Apo A on the HDL particle surface predicted by molecu- lar modeling. While the additional image detail has moved our work forward, resolu- tion from negative stain data continues to
be a limiting factor due to the drying and flattening of the par- ticles on the grid as well as stain artifact. However, we are now using our improved negative stain-generated models to help us refine more detail from cryo-EM images of similar particles lacking the negative stain drying artifacts.
Conclusion Our new negative staining procedure using uranyl for-
mate has allowed us to obtain more surface detail in HDL preparations and more accurate diameter measurements with- out significantly altering particle morphology. Te additional surface detail helps to determine how proteins such as Apo A
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