AI Insight
Researchers have developed Nilas, an optimized low-energy focused ion beam milling strategy that produces thinner biological samples with less damage for cryo-electron microscopy imaging. This technique generates lamellae as thin as 50 nm or less while preserving cellular structures better than conventional methods, enabling higher resolution 3D reconstructions of molecular complexes inside cells. The improved method allows detection of smaller protein complexes down to approximately 220 kDa and successfully identified additional non-ribosomal structures like RNA polymerase III that were previously difficult to visualize.
Why it matters
This advancement significantly expands the range of protein complexes that can be studied in their natural cellular environment, moving closer to comprehensive visualization of the proteome. The technique is compatible with existing equipment, making it immediately accessible to researchers worldwide without requiring specialized hardware investments.
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⚠️ Preprint – Noch nicht peer-reviewed
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Focused ion beam (FIB)-milling has been adapted to thin frozen cells for visualization of macromolecular structures in situ with cryogenic electron microscopy. However, only a few large and abundant complexes have been annotated to date. FIB-milling introduces damage which limits the recoverable information from cellular sections. Here, we present Nilas, a low-energy milling strategy optimized to minimize damage and produce thin lamellae. Nilas-milled lamellae show minimal FIB-milling damage, contain areas at or below 50 nm and produce higher resolution in situ 3D reconstructions. Nilas improves the recovery of ribosomal subunits and reduces the predicted minimal detectable molecular mass with two-dimensional template matching (2DTM) to approximately 220 kDa. Consistently, we recover additional non-ribosomal complexes including RNA polymerase III with 2DTM in Nilas-milled lamellae. Nilas is compatible with common milling hardware, making it accessible to diverse users. By extending the size limit for in situ structural biology we bring visual proteomics closer to reality.
Source: Optimized cryo-FIB milling strategy to generate thin, minimally damaged biological lamellae