While the revolution will not be crystallized, biochemistry reigns supreme

Abstract

Single-particle cryo-electron microscopy (EM) is currently gaining attention for the ability to calculate structures that reach sub-5 Å resolutions; however, the technique is more than just an alternative approach to X-ray crystallography. Molecular machines work via dynamic conformational changes, making structural flexibility the hallmark of function. While the dynamic regions in molecules are essential, they are also the most challenging to structurally characterize. Single-particle EM has the distinct advantage of being able to directly visualize purified molecules without the formation of ordered arrays of molecules locked into identical conformations. Additionally, structures determined using single-particle EM can span resolution ranges from very low- to atomic-levels (>30-1.8 Å), sometimes even in the same structure. The ability to accommodate various resolutions gives single-particle EM the unique capacity to structurally characterize dynamic regions of biological molecules, thereby contributing essential structural information needed for the development of molecular models that explain function. Further, many important molecular machines are intrinsically dynamic and compositionally heterogeneous. Structures of these complexes may never reach sub-5 Å resolutions due to this flexibility required for function. Thus, the biochemical quality of the sample, as well as, the calculation and interpretation of low- to mid-resolution cryo-EM structures (30-8 Å) remains critical for generating insights into the architecture of many challenging biological samples that cannot be visualized using alternative techniques.

Keywords: biochemistry; cryo-electron microscopy; negative stain; spliceosomes.

Figure 1

General flowchart showing the basic steps required to analyze biological samples using single‐particle electron microscopy (EM). While it is possible to structurally characterize small (<200 kDa) and dynamic samples via single‐particle cryo‐EM, larger molecular mass (>300 kDa) samples that are structurally homogenous have a better chance at reaching near‐atomic to atomic resolutions (sub‐5Å). There are many places in the process where sample optimization is critical for success. This requires biochemically improving the purification and/or finding the proper conditions to visualize the sample in vitrified ice. Unfortunately, these optimization steps involve labor‐intensive trial and error that is not hypothesis driven.

Figure 2

The negative stain uranyl formate does not alter the conformation of pH sensitive pore‐forming toxins. A: Clostridium difficile toxin TcdA does not change structural conformation when stained with uranyl formate. B: For pH induced structural rearrangement to occur, TcdA must first be washed with a low pH buffer (pH 4.5) before staining. Scale bars, 50 nm. Representative class averages for each condition are shown in corner. Side length of averages, 57.3 nm. Averages published in Ref. 68. C: Helicobacter pylori VacA oligomers remain oligomeric when stained with uranyl formate.59, 66 D: H. pylori VacA oligomers dissociate when incubated in a low pH buffer (pH 3.5).59, 125 Scale bars, 50 nm.

Figure 3

Changing the types of support grids and buffer conditions can dramatically alter the appearance of particles in vitrified ice. A: Image of S. pombe U5.U2/U6 spliceosome complexes vitrified on Quantifoil holey carbon grids covered with a thin layer of carbon using an FEI vitrobot.73 B: Image of S. pombe U5.U2/U6 spliceosome complexes that were isolated using GraFix,74 concentrated using a Millipore Microcon centrifugal filter (100 kDa cut‐off), and frozen on Quantifoil holey carbon grids using an FEI vitrobot. Scale bars, 50 nm.

Figure 4

Crosslinking improves the homogeneity of a U5.U2/U6 spliceosome complex. Image of S. pombe U5.U2/U6 complexes before (A) or after (B) crosslinking for 1.5 hours with 0.1% glutaraldehyde at 4°C. Scale bars, 100 nm. (C) Averages representing a common spliceosome view found when negatively stained. No crosslinking (upper), crosslinking (lower). Number of particles, lower right corner. Side length of panel, 56 nm. Averages were generated using the program Spider47.

Figure 5

GraFix can be used to improve both the compositional and structural homogeneity of purifications.74 A: A S. pombe U5.U2/U6 spliceosome purification was resolved in a 10–30% glycerol gradient. Immunoblot of fractions collected from bottom (30%) to top (10%) of gradient and probed with anti‐Cdc5 antibodies. The migration of fatty acid synthase (40S) collected from parallel gradients is indicated. B: Negatively stained image of spliceosomes found in fraction 7 collected from a 10–30% GraFix glycerol gradient that contains a 0–0.15% glutaraldehyde gradient. C: Negatively stained image of spliceosome particles pooled from fractions 6, 7, and 8 from the GraFix gradient described in B, buffer exchanged to remove glycerol, and concentrated using a Millipore Microcon centrifugal filter (100 kDa cut‐off). Estimated glutaraldehyde concentration in fractions 6‐8 is ∼0.1%. Scale bar, 100 nm.

Figure 6

DID‐Dyn2 labels are clearly visible in DID‐labeled S. pombe spliceosome complexes. (A‐B) Negatively stained images of S. pombe U5.U2/U6 spliceosome labeled with DID‐Dyn2.106 U5.U2/U6 particles tagged with Lea1‐DID1 (A) and Smg1‐DID1 (B). The rod‐shapes of the DID‐Dyn2 tag are easily visible in the raw images. White arrows highlight the position of a few of the DID‐Dyn2 rods. Scale bar, 50 nm.