Revolutionizing Protein Structure Analysis: Cryo-EM without Purification Accelerates the Future of Structural Biology

  • New workflow enables solving protein structures via cryo-EM directly from crude cell-free lysates with no or minimal purification.
  • The PURE expression system provides minimal background, supporting atomic-resolution reconstructions in under 24 hours.
  • Miniaturized 25µl reactions are sufficient, reducing costs and labor.
  • Additional rapid cleanup steps yield modest resolution improvement with minimal extra time.
  • Approach opens doors for automation and high-throughput screening in structural biology.
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From Gene to Structure in Under 24 Hours: How Minimizing Purification Is Transforming Cryo-EM

In recent years, the pace of advancements in structural biology has accelerated dramatically, driven by breakthroughs in both computational and experimental methods. Yet, one sticking point lingered: the laborious and time-consuming purification of proteins before structural analysis. Now, a team at Pacific Northwest National Laboratory and Washington State University has pushed the boundaries by demonstrating that high-resolution cryo-electron microscopy (cryo-EM) structures can be solved directly from crude cell-free mixtures, bypassing lengthy purification steps—and all in less than a day.

Breaking the Bottleneck in Structural Biology

For decades, generating homogenous, purified protein samples has been the bottleneck in structural biology. Even with automation and modern electron microscopes capable of screening multiple samples per day, the initial sample preparation often lags far behind, taking days to months. This delay hinders the pace of fundamental research and drug discovery.

The study introduces a workflow that employs the PURE cell-free expression system. Unlike traditional cell-based methods or even wheat germ lysate systems, the PURE system utilizes a streamlined mixture of only 36 protein complexes, eliminating a vast background of interfering proteins—critical for clear imaging and accurate structure determination. Experiments using this platform achieved robust expression of target proteins at reaction volumes as small as 25µl, generating enough sample for biochemical validation and cryo-EM analysis in a matter of hours.

Direct Imaging—No Purification, No Problem

By skipping purification, the process becomes not only faster, but also far more amenable to automation and high-throughput screening. The research team tested this “structure without purification” workflow using Artemia ferritin and the plant protein PDX1.2 as benchmarks. Following rapid cell-free expression and minimal handling, lysates were applied directly to cryo-EM grids. Automated imaging and data processing rapidly filtered out non-target particles, leading to 3D reconstructions at atomic resolutions: 2.8Å for ferritin and 2.5Å for PDX1.2, surpassing or matching traditional purified samples.

Small-scale post-expression treatments—like spin filtering, benzonase digestion, or his-tag affinity cleanup—were evaluated for their incremental impact on map quality. Remarkably, these rapid interventions offered modest resolution improvements, bringing final maps as fine as 2.1Å, but at the cost of only about an hour of added prep time. Crucially, even fully crude lysates yielded side-chain level detail sufficient for confident model building and refinement.

Broad Implications for the Field

The elimination of large-scale purification unlocks significant advantages:

  • Reduced costs: Miniaturized reactions cut reagent use and waste.

  • Accelerated workflow: The entire process from expression to model can be completed in less than 24 hours.

  • Expanded accessibility: Proteins that are unstable, toxic, or otherwise intractable in traditional systems become evaluable.

  • Compatibility with automation: Small volumes and minimal handling simplify integration with automated sample preparation and imaging platforms.

Notably, the presence of ribosomes and other lysate components unexpectedly improved ice quality during vitrification, often a tricky variable in cryo-EM. The authors further note that the PURE system is compatible with modern genetic engineering to introduce post-translational modifications or folding-assisting factors, reducing concerns about protein quality in the absence of a full cellular context.

A New Era for Structural Biology

This innovative workflow demonstrates that direct-from-lysate, cell-free cryo-EM analysis is more than a proof-of-concept—it is a practical, robust, and scalable technique poised to accelerate discovery across biology and medicine. With resolutions previously achievable only through painstaking purification now accessible in hours, the field is set for a leap in throughput and impact.