Free cryo-EM method solves tilt woes


Rebecca Pool

Tuesday, July 4, 2017 - 13:30
Image: Researchers liken looking at soup tins from different angles to viewing proteins at a tilt to reveal different aspects of the structure. [Salk Institute]
US-based researchers have developed a method for tilting biological specimens during single-particle cryo-electron microscopy that allows researchers to map structures to near-atomic resolution.
Professor Dmitry Lyumkis from The Salk Institute for Biological Studies and colleagues have successfully mapped the influenza haemagglutinin trimer and ribosomal biogenesis intermediates, with associated algorithms and tools freely available online.
"People have tried to implement tilting before, but there have been a lot of challenges," says Lyumkis. "We've eliminated many of these problems with our new approach."
3D reconstruction of the haemagglutinin protein from 40°-tilted data: tilting enables recovery of near-atomic-resolution information from a data set. [Yong Zi Tan et al, Nature Methods, 03 July 2017]
According to the researcher, past tilt-based approaches used in single-particle studies have yielded reconstructions with only low-to-moderate resolution for many reasons.
For example, the electron beam has to travel through a thicker grid, samples move when tilted and analysis methods have been designed on the basis that the sample grid is always at the same distance from the cryo-EM.
However, Lyumkis and colleagues have now developed a strategy of collecting data at defined tilts, to obtain near-atomic resolution maps of single particle structures.
According to the researchers, the so-called tilt collection scheme is independent of the sample and can be universally used.
And they have also devised a tool to quantify the resulting directional resolution using 3D Fourier shell correlation volumes.
"All aspects of data collection are performed as in conventional single particle analysis, with the sole exception that the goniometer is set to a defined nominal tilt angle," highlights Lyumkis. 
"We used gold substrates to minimise beam-induced movement at tilt and motion correction of individual movie frames to account for any residual movements," he adds. "[And] to account for increased ice thickness and particle contrast reduction at tilt, we used a high-dose imaging strategy and exposure filtered the resulting frames." 
When, for example, the researchers tested the new approach on the influenza haemagglutinin protein - a notoriously hard protein to characterise using cryo-EM - they found that tilting the sample gave a more complete dataset.
"Due to the geometry of the data collection when we tilt, we [collect] much more data when characterising the molecules, giving us a more complete picture of the protein's shape" says Lyumkis.
All related algorithms and tools are openly available and the Lyumkis hopes other researchers will start using these, with the method becoming a standard metric for cryo-EM structure validation.
"One of the ideas we're looking at now is whether data collection should always be performed at a tilt rather than in the conventional way," he says. "It won't hurt and it should help."
Research is published in Nature Methods.
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