X-ray diffraction from membrane protein nanocrystals

Abstract

Membrane proteins constitute > 30% of the proteins in an average cell, and yet the number of currently known structures of unique membrane proteins is < 300. To develop new concepts for membrane protein structure determination, we have explored the serial nanocrystallography method, in which fully hydrated protein nanocrystals are delivered to an x-ray beam within a liquid jet at room temperature. As a model system, we have collected x-ray powder diffraction data from the integral membrane protein Photosystem I, which consists of 36 subunits and 381 cofactors. Data were collected from crystals ranging in size from 100 nm to 2 μm. The results demonstrate that there are membrane protein crystals that contain < 100 unit cells (200 total molecules) and that 3D crystals of membrane proteins, which contain < 200 molecules, may be suitable for structural investigation. Serial nanocrystallography overcomes the problem of x-ray damage, which is currently one of the major limitations for x-ray structure determination of small crystals. By combining serial nanocrystallography with x-ray free-electron laser sources in the future, it may be possible to produce molecular-resolution electron-density maps using membrane protein crystals that contain only a few hundred or thousand unit cells.

Figure 1

Overview of the serial powder crystallography experiment. An aqueous solution containing many PSI crystallites passes through an incident x-ray beam, creating a powder diffraction pattern.

Figure 2

(Left) Diffraction pattern from 500 nm PSI crystals with an on-axis detector and x-ray energy of 1560 eV (λ = 0.8 nm). The resolution at the corner of the detector is 2.8 nm. A total of 11520 PSI trimers are in each crystal. The pattern is wavelength-limited. (Top right) A schematic view of the determined unit cell for PSI showing the unit-cell dimensions. (Bottom right) A drawing of a PSI crystal used for single-crystal diffraction studies showing the number of unit cells in a (500-nm)³ crystal.

Figure 3

Diffraction patterns from 500-nm PSI crystals using an off-axis detector to increase the measurable scattering angle. (a) The crystals were irradiated with 1560-eV x-rays (λ = 0.8 nm) and had a resolution of 1.3 nm in the upper-left corner. (b) The crystals were irradiated with 520-eV (λ = 2.4 nm) x-rays and had a resolution of 4.0 nm in the upper left corner. Arrows and associated numbers indicate the d-spacing of the given powder peak.

Figure 4

Diffraction pattern from 100-nm PSI crystals with an off-axis detector and an x-ray energy of 520 eV (λ = 2.4 nm). The resolution at the corner of the detector is 4.0 nm. There are a total of 36 PSI trimers in each crystal.

Figure 5

A comparison of the scattering power of the 100-nm, 220-nm, and 500-nm crystals at specific Bragg reflections using the scattered intensity as a function of scattering angle.