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Cryo‐EM in the Study of Membrane Transport Proteins

Laura Yaunhee Kim, Matthew C. Johnson, Ingeborg Schmidt‐Krey

10.1002/cphy.c110028

Source: Volume 2, Issue 1, January 2012

Published online: January 2012

Full Article on Wiley Online Library



Abstract

Electron cryomicroscopy (cryo‐EM) has evolved as a widely used approach to understand a range of structure‐function questions, particularly of membrane proteins. Studies by both electron crystallography and single particle analysis have provided a wealth of information on membrane transport proteins. Cryo‐EM methods with an emphasis on electron crystallography, which has yielded the most membrane transport protein structural information of any of the cryo‐EM techniques, are described here. Two‐dimensional crystallization approaches are outlined, as well as advances in cryo‐EM specimen preparation, data collection, and image processing. Examples of membrane transport protein structure described serve to illustrate some of the advances in both structural understanding and methods. Further examples outline impressive results that were obtained by a combination of electron crystallography and X‐ray crystallography as well as additional complementary methods. © 2012 American Physiological Society. Compr Physiol 2:283‐293, 2012.

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Figure 1. Figure 1.

A two‐dimensional (2D) crystal usually consists of protein (black/dark gray ovals) within a lipid bilayer. The schematic representation shows a cutout of such a 2D crystal.
Figure 2. Figure 2.

The membrane morphologies encountered in electron crystallography are (A) collapsed vesicles, (B) planar‐tubular crystals, which basically correspond to an extended vesicle, and (C) sheets, which do not necessarily have a square shape and ideally consist of a single 2D crystal layer, composed of the reconstituted membrane protein molecules within one lipid bilayer. A highly ordered 2D crystal sheet composed of a single layer offers the great advantage of allowing for electron diffraction data collection. The schematic figure in (D) depicts a helical crystal, which offers all orientations of the protein in each micrograph. Thus, it is not necessary to collect data of tilted specimens of a helical crystal. The size of 2D crystals can be as small as 50 nm on the very low end up to one or more microns in the ideal situation.
Figure 3. Figure 3.

Single particle analysis involves the distribution of the detergent‐solubilized membrane protein on an EM grid (A). Certain samples will be randomly oriented while others, such as shown in (B), will have preferred orientations. The dark oval represents the protein solublilized in detergent (white).
Figure 4. Figure 4.

Electron crystallography involves screening of negatively stained samples to identify order, size, and morphology during 2D crystallization trials. Electron cryomicroscopy (Cryo‐EM) data is collected at low electron doses, evaluated by optical diffraction, scanned, and data is processed to obtain structural information.
Figure 5. Figure 5.

Single particle analysis requires screening in negative stain. Cryo‐EM data is collected with digital cameras, or recorded photographic film is evaluated by optical diffraction and subsequently scanned. CCD cameras used for data collection eliminate these steps. Image processing reveals novel structural in the form of projection and/or 3D maps.
Figure 6. Figure 6.

(A) A side view of a grid prepared by regular vitrification or backinjection with the carbon film in dark gray and the vitreous ice in light gray. (B) A carbon‐sandwich grid side view, where the specimen is enclosed in the vitreous ice between two carbon film layers. (C) A holey or perforated grid is used for single particles and, sometimes, helical crystals. The upper panel of (C) shows the top view of such a holey or perforated grid.
Figure 7. Figure 7.

The carbon‐sandwich grid allows for even highly sensitive 2D crystals to be maintained under optimal hydration conditions and vitreous ice thickness. It is particularly valuable to avoid charging effects at high tilt angles 45.


Figure 1.

A two‐dimensional (2D) crystal usually consists of protein (black/dark gray ovals) within a lipid bilayer. The schematic representation shows a cutout of such a 2D crystal.



Figure 2.

The membrane morphologies encountered in electron crystallography are (A) collapsed vesicles, (B) planar‐tubular crystals, which basically correspond to an extended vesicle, and (C) sheets, which do not necessarily have a square shape and ideally consist of a single 2D crystal layer, composed of the reconstituted membrane protein molecules within one lipid bilayer. A highly ordered 2D crystal sheet composed of a single layer offers the great advantage of allowing for electron diffraction data collection. The schematic figure in (D) depicts a helical crystal, which offers all orientations of the protein in each micrograph. Thus, it is not necessary to collect data of tilted specimens of a helical crystal. The size of 2D crystals can be as small as 50 nm on the very low end up to one or more microns in the ideal situation.



Figure 3.

Single particle analysis involves the distribution of the detergent‐solubilized membrane protein on an EM grid (A). Certain samples will be randomly oriented while others, such as shown in (B), will have preferred orientations. The dark oval represents the protein solublilized in detergent (white).



Figure 4.

Electron crystallography involves screening of negatively stained samples to identify order, size, and morphology during 2D crystallization trials. Electron cryomicroscopy (Cryo‐EM) data is collected at low electron doses, evaluated by optical diffraction, scanned, and data is processed to obtain structural information.



Figure 5.

Single particle analysis requires screening in negative stain. Cryo‐EM data is collected with digital cameras, or recorded photographic film is evaluated by optical diffraction and subsequently scanned. CCD cameras used for data collection eliminate these steps. Image processing reveals novel structural in the form of projection and/or 3D maps.



Figure 6.

(A) A side view of a grid prepared by regular vitrification or backinjection with the carbon film in dark gray and the vitreous ice in light gray. (B) A carbon‐sandwich grid side view, where the specimen is enclosed in the vitreous ice between two carbon film layers. (C) A holey or perforated grid is used for single particles and, sometimes, helical crystals. The upper panel of (C) shows the top view of such a holey or perforated grid.



Figure 7.

The carbon‐sandwich grid allows for even highly sensitive 2D crystals to be maintained under optimal hydration conditions and vitreous ice thickness. It is particularly valuable to avoid charging effects at high tilt angles 45.

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Article Title: Cryo‐EM in the Study of Membrane Transport Proteins
 

How to Cite

Laura Yaunhee Kim, Matthew C. Johnson, Ingeborg Schmidt‐Krey. Cryo‐EM in the Study of Membrane Transport Proteins. Compr Physiol 2012, 2: 283-293. doi: 10.1002/cphy.c110028