The His-tag as a decoy modulating preferred orientation in cryoEM

Abstract

The His-tag is a widely used affinity tag that facilitates purification by means of affinity chromatography of recombinant proteins for functional and structural studies. We show here that His-tag presence affects how coproheme decarboxylase interacts with the air-water interface during grid preparation for cryoEM. Depending on His-tag presence or absence, we observe significant changes in patterns of preferred orientation. Our analysis of particle orientations suggests that His-tag presence can mask the hydrophobic and hydrophilic patches on a protein's surface that mediate the interactions with the air-water interface, while the hydrophobic linker between a His-tag and the coding sequence of the protein may enhance other interactions with the air-water interface. Our observations suggest that tagging, including rational design of the linkers between an affinity tag and a protein of interest, offer a promising approach to modulating interactions with the air-water interface.

Keywords: AWI; His-tag; air-water interface; cryo-electron microscopy single particle reconstruction; cryoEM SPR; preferred orientation; visualization.

FIGURE 1

(A) The electrostatic potential map for 6VSA.pdb, which represents the cryoEM reconstruction of the cleaved version of coproheme decarboxylase. Three different orientations that stress a highly polarized charge distribution for this protein that explains observed patterns of preferred orientation (top). In the bottom row, each orientation of the electrostatic potential map is shown with 40% of transparency to show the orientations of the hydrophobic loop between residues 110–120 (green spheres) based on 1T0T.pdb and its proximity to the N-terminus (dark blue speres). (B) The model with the His-tags and their linkers added to show how they can extend from the surface of the protein up to ∼60–65 Å.

FIGURE 2

Histograms of distributions for particles of coproheme decarboxylase with His-tag cleaved (A), His-tag partially cleaved (B), and His-tag uncleaved (C). The histograms show the number of particles as the function of cosine Θ, where Θ is the angle between the 5-fold axis of the particle and the direction of the beam. The map of the electrostatic potential for each orientation is shown above the specific peaks corresponding to the most frequent orientations.

FIGURE 3

Preferred orientations (heat plots) shown in polar coordinates for protein with: (A) His-tag cleaved protein, (B) His-tag partially cleaved, and (C) His-tag uncleaved. To stress the difference in the polarity of the particles, we show on the left traditional plots for all particles, while the middle and right figures show (+) and (-) polarities (particles tilted away from or toward the beam). The different polarities result from different angles of interactions with the AWI, but also may result from interactions with two separate AWIs, which would symmetrize the histogram, as the cosine of the angle between the particle and the beam changes sign for the same geometric interaction with the AWI. We do not directly know how much these two effects contribute to our data.

FIGURE 4

3D reconstructions for three samples. (A) 3D reconstruction with C5 (top) and C1 (bottom) symmetries for the sample with His-tag cleaved; (B) 3D reconstruction with C5 symmetry for the sample with His-tag partially cleaved. The reconstruction with C1 symmetry did not provide the interpretable density. (C) 3D reconstruction with C5 (top) and C1 (bottom) symmetries for the sample with His-tag uncleaved. The blue stars denote the 2 loops that have the best density in C1 reconstructions.

FIGURE 5

Tomographic reconstruction of the sample with His-tag present in all copies. The upper part of the figure shows the distribution of orientations for that sample, while the two panels at the bottom part of the figure represent the patterns on two different AWIs. One of the AWIs (left) shows very few particles in comparison with the second AWI (right). This is consistent with the results of our data analysis performed on a different subset of particles from the same type of sample. The particles in the sample are also forming “groups” that are not present in the sample with His-tags cleaved (Supplementary Figure S3A–C).

FIGURE 6

Proposed models of interactions with the AWI for each sample. (A) Particles with His-tag cleaved interact with the AWI using predominantly a hydrophobic “bottom” surface. (B) Particles with His-tag partially cleaved interact with the AWI using a hydrophobic loop with interactions most likely enhanced by the hydrophobic linker between the N-terminus of the protein and the His-tag. (C) Particles with His-tag present in all monomers interact with the AWI using the predominantly negatively charged side of the molecule. This can be achieved if the positively charged His-tag compensates the negative charge on the other surface, forming a new interface that can interact with the AWI.

FIGURE 7

Schematic representation of an incomplete cleavage problem for a pentameric protein such as coproheme decarboxylase. The incomplete cleavage may make partially labelled particles escape binding to the affinity column and flow through together with oligomers having all His-tags cleaved. This is why for multimeric proteins, it is worth performing detailed analysis of the applicable ranges of eluting agent concentrations.