How scientists are using X-rays and electrons to visualize protein microcrystals, the molecular machines powering every process of life.
Imagine trying to reverse-engineer the most complex machine ever built, but you're only allowed to study a handful of its components, each smaller than a wavelength of light. This is the monumental challenge faced by structural biologists who study proteins—the molecular machines that power every process of life.
Welcome to the frontier of microcrystallography, a field where scientists are wielding two powerful but different beams—X-rays and electrons—to visualize the invisible.
Proteins are the workhorses of biology, and their function is dictated by their shape. For decades, the gold standard for determining this shape has been X-ray crystallography.
However, a major bottleneck is crystallization. For many proteins, especially those embedded in cell membranes (crucial for drug discovery), growing large, perfect crystals is impossible. They only form microcrystals, thinner than a human hair. This is where the battle of the beams begins.
To study these microcrystals, scientists have developed two advanced techniques, each with unique strengths and weaknesses.
X-ray sources have gotten incredibly powerful and focused.
Electrons are fundamental particles with a much stronger interaction with matter than X-rays.
| Parameter | Micro-Focus Synchrotron | XFEL (SFX) | MicroED |
|---|---|---|---|
| Typical Crystal Size | 5 - 50 µm | 0.1 - 10 µm | 0.01 - 5 µm |
| Radiation Damage | High | Eliminated (by outrunning) | Low |
| Sample Throughput | 1 crystal/data set | 10,000s of crystals/data set | 1 - 10 crystals/data set |
| Best Resolution | Very High (<1.5 Å) | Very High (<1.5 Å) | High (1.5 - 2.5 Å) |
A comparative overview highlighting the niche each technique occupies, with MicroED excelling at the smallest crystal sizes and requiring minimal sample consumption.
To understand how these techniques work in practice, let's examine a pivotal MicroED experiment that determined the structure of a key pharmaceutical target.
Determine the atomic structure of the enzyme catechol O-methyltransferase (COMT), a target for drugs treating Parkinson's disease. Despite extensive efforts, researchers could only produce needle-like microcrystals a few micrometers in size—far too small for standard X-ray analysis.
The COMT protein was crystallized, resulting in a slurry of tiny, needle-shaped crystals suspended in solution.
A drop of this slurry was applied to a tiny, gold-finished EM grid. The excess liquid was blotted away, leaving a thin film of sample.
The grid was instantly plunged into liquid ethane, freezing the sample so rapidly that water formed a glass-like solid (vitreous ice), preserving the crystal structure perfectly.
The frozen grid was loaded into a cryo-electron microscope. Scientists navigated the grid under the beam, identified promising single microcrystals, and centered them.
While the electron beam was on, the crystal was rotated continuously at a controlled rate, collecting a full 180-degree sweep of diffraction data in just 30-60 seconds.
The thousands of diffraction images were indexed, integrated, and merged using specialized software to produce an electron density map. This map was then used to build and refine the atomic model of the COMT protein.
The experiment was a resounding success. The MicroED data yielded a high-resolution structure (1.9 Ångströms) of COMT, clearly showing the protein's active site where drug molecules bind.
| Resolution | 1.9 Å |
| Space Group | P 2₁ 2₁ 2₁ |
| Total Frames Collected | 2,400 |
| Rotation per Frame | 0.075° |
| Total Rotation Range | 180° |
| Data Completeness | 99.8% |
This table shows the high quality and completeness of the diffraction data obtained from a single COMT microcrystal, enabling a high-resolution structure determination.
The purified protein of interest (e.g., COMT), produced in bacterial or insect cells.
Chemical cocktails containing precipitants, salts, and buffers to coax the protein into forming ordered crystals.
A tiny metal mesh (often gold or copper) that holds the sample for loading into the electron microscope.
A device (plunger) that rapidly freezes the grid in a cryogen like liquid ethane to preserve the crystal in a glass-like ice.
The core instrument that generates the electron beam, controls the sample stage, and detects the diffraction patterns.
Specialized computer programs (e.g., XDS, DIALS, PHASER, Coot) used to convert raw diffraction images into a 3D atomic model.
The story of microcrystallography is not a winner-takes-all battle between X-rays and electrons. Instead, it's a story of a powerful and complementary toolkit. XFELs provide unparalleled speed and resolution for the most challenging experiments, while MicroED offers an accessible and gentle method for the tiniest of crystals.
As both technologies continue to advance, they are converging. The lessons learned from optimizing sample delivery for XFELs are being applied to MicroED, and vice-versa. Together, they are pushing the boundaries of what is possible, allowing us to see the invisible architects of life in stunning detail and paving the way for the next generation of medicines and a deeper understanding of biology itself.