Electron cryo-microscopy of TPPS4⋅2HCl tubes reveals a helical organisation explaining the origin of their chirality

Abstract

A widely studied achiral porphyrin, which is highly soluble in aqueous solutions (TPPS4), is shown to self-assemble into helical nanotubes. These were imaged by electron cryo-microscopy and a state-of-the-art image analysis allows building a map at ∼5 Å resolution, one of the highest obtained so far for molecular materials. The authors were able to trace the apparent symmetry breaking to existing nuclei in the "as received samples", while carefully purified samples show that both handnesses occur in equal amounts.

Keywords: J‐aggregates/H‐aggregates; chirality; electron cryo‐microscopy; helical reconstruction; porphyrinoids; self‐assembly.

Scheme 1

Protonation of the inner pyrrolenic nitrogen atoms and of two sulphonic groups at low pH (100 mm HCl) leads to an intermediate I which self‐assembles forming aggregates in water. At pH<1 all four sulphonic groups are protonated.7k Numbers indicate absorption maxima in nm.

Figure 1

A) Aggregation behaviour after injection of 300 μL of a solution 5.3 mg TPPS₄⋅2 HCl dissolved in 1.0 mL deionized water (pH 7) into 10 mL aqueous HCl at pH 1. M denotes monomer maxima, J and H denote aggregate maxima. B,C) Absorption and ECD spectra, respectively, of samples prepared from similar aggregation experiments, ripened for 24 h. The rarely encountered negative exciton couplet (red trace) has the longest Cotton effect slightly truncated due to saturation of the detector. The different cuvette pathlengths (red trace—2 mm; green trace—1 mm) were chosen to obtain similarly intense Cotton effects and were not responsible for the mirror imaged traces.

Figure 2

A) Cryo‐EM image of a collection of TPPS4 tubes prepared by the injection method of a concentrated aqueous solution (pH 7 water) into a much larger volume of pH 1.0 aqueous HCl solution. B) TPPS₄ tubes were concentrated by centrifugation and pipetted on the Quantifoil holey carbon grid for freezing. In this low magnification image some of the 1 μm holes are empty, while bundles of tubes are visible in the ice across the others. One with best alignment was chosen for diffraction and the area circled and arrowed was selected. C) Electron diffraction pattern taken of the selected area showing arcs characteristic of helical organisation with layer lines arrowed at a: 4.8, b: 3.5, and c: 2.1 Å⁻¹ spacings while d is an equatorial reflection at 3.7 Å⁻¹. D) X‐ray diffraction pattern of a partially aligned preparation of TPPS4 tubes showing (arrows a–c) 9.4, 5.2 and 3.5 Å⁻¹, arrows d and e show equatorial spacings at 7.4 and 3.7 Å⁻¹. Close inspection of the small angle data around the beam stop (inset) shows how the alignment sharpens the rings of diffraction, consistent with the sample being packed tubes of a narrow range of diameters.

Figure 3

Part of a single tube which was boxed (A), masked and Fourier filtered (B) and Fourier transformed (C). The lattice is shown in (C) as a dashed red line. Three‐dimensional reconstruction (D) computed by helical Fourier‐Bessel analysis of both sides of four tubes averaged together shows the packing of the porphyrin molecules. A single section of the map viewed down the cylinder axis shows the 26 helices (inset). Layer lines at 122 Å⁻¹,10.7 Å⁻¹ and 5.1 Å⁻¹ were included in the calculation. The resolution is ∼5 Å. This is a P (+) 26‐start helix with 72 subunits in each turn of the helix.