Powerful microscopy technique brings proteins into focus

Cryo-electron microscopy (cryo-EM) as a technology has become more important because the field that it revolutionised – structural biology – has become more important. The international scientific community had this rise in fortunes, so to speak, acknowledged when the Nobel Prize for chemistry was awarded to three people in 2017 for perfecting its use to study important biomolecules and molecular processes.

(Who received the prize is immaterial, considering more than just three people are likely to have contributed to the development of cryo-EM; however, the prize-giving committee’s choice of field to spotlight is a direction worth following.)

In 2015, two separate groups of scientists used cryo-EM to image objects 2.8 Å and 2.2 Å (1 nm is one-billionth of a metre; 1 Å is one-tenth of this) wide. These distances are considered to be atomic because they represent the ability to image features about as big as a smallish atom, comparable to that of, say, sodium. Before cryo-EM, scientists could image such distances only with X-ray crystallography, which requires the samples to be studied to be crystallised first. This isn’t always possible.

But though cryo-EM didn’t require specimens to be crystallised, they had to be placed in a vacuum first. In vacuum, water evaporates, and when water evaporates from biological objects like tissue, the specimen could lose its structural integrity and collapse or deform. The trio that won the chemistry prize in 2017 developed multiple workarounds for this and other problems. Taken together, their innovations allowed scientists to find cryo-EM to be more and more valuable for research.

One of the laureates, Joachim Frank, developed computational techniques in the 1970s and 1980s to enhance, correct and in other ways modify images obtained with cryo-EM. And one of these techniques in turn was particularly important.

An object will reflect a wave if the object’s size is comparable to the wave’s wavelength. Humans see a chair or table because the chair or table reflects visible light, and our eyes detect the reflected electromagnetic waves. A cryo-EM ‘sees’ its samples using electrons, which have a smaller wavelength than photons and can thus reveal even smaller objects.

However, there’s a catch. The more energetic an electron is, the lower its wavelength is, and the smaller the feature it can resolve – but a high-energy electron can also damage the specimen altogether. Frank’s contributions allowed scientists to reduce the number of electrons or their energy to obtain equally good images of their specimens, leading to resolutions of 2.2 Å.

Today, structural biology continues to be important, but its demands have become more exacting. To elucidate the structures of smaller and smaller molecules, scientists need cryo-EM and other tools to be able to resolve smaller and smaller features, but come up against significant physical barriers.

For example, while Frank’s techniques allowed scientists to reduce the number of electrons required to obtain the image of a sample, using fewer probe particles also meant a lower signal-to-noise ratio (SNR). So the need for new techniques, new solutions, to these old problems has become apparent.

In a paper published online on October 21, a group of scientists from Belgium, the Netherlands and the UK describe “three technological developments that further increase the SNR of cryo-EM images”. These are a new kind of electron source, a new energy filter and a new electron camera.

The electron source is something the authors call a cold field emission electron gun (CFEG). Some electron microscopes use field emission guns (FEGs) to shoot sharply focused, coherent beams of electrons optimised to have energies that will produce a bright image. A CFEG is a FEG that reduces the brightness in favour of reducing the average difference in energies between electrons. The higher this difference – or the energy spread – is, the more blur there will be in the image.

The authors’ pitch is that FEGs help produce brighter but more blurred images than CFEGs, and that CFEGs help produce significantly better images when the goal is to image features smaller than 2 Å. Specifically, they write, the SNR increases 2.5x at a resolution of 1.5 Å and 9.5x at 1.2 Å.

The second improvement has to do with the choice of electrons used to compose the final image. The electrons fired by the gun (CFEG or otherwise) go on to have one of two types of collisions with the specimen. In an elastic collision, the electron’s kinetic energy doesn’t change – i.e. it doesn’t impart its kinetic energy to the specimen. In an inelastic collision, the electron’s kinetic energy changes because the electron has passed on some of it to the specimen itself. This energy transfer can produce noise, lower the SNR and distort the final image.

The authors propose using a filter that removes electrons that have undergone inelastic collisions from the final assessment. In simple terms, the filter comprises a slit through which only electrons of a certain energy can pass and a prism that bends their path towards a detector. This said, they do acknowledge that it will be interesting to explore in future whether inelastically scattered electrons can be be better accounted for instead of being eliminated altogether – akin to silencing a classroom by expelling unruly children versus retaining them and teaching them to keep quiet.

The final improvement is to use the “next-generation” Falcon 4 direct-electron detector. This is the latest iteration in a line of products developed by Thermo Fisher Scientific, to count the number of electrons impinging on a surface as accurately as possible, their relative location and at a desirable exposure. The Falcon 4 has a square detection area 14 µm to a side, a sampling frequency of 248 Hz and a “sub-pixel accuracy” (according to the authors) that allows the device to not lose track of electrons even if they impinge close to each other on the detector.

A schematic overview of the experimental setup. Credit: https://doi.org/10.1038/s41586-020-2829-0

Combining all three improvements, the authors write that they were able to image a human membrane protein called ß3 GABA_A R with a resolution of 1.7 Å and mouse apoferritin at 1.22 Å. (The protein called ferritin binds to iron and stores/releases it; apoferritin is ferritin sans iron.)

A reconstructed image of GABA_A R. The red blobs are water molecules. NAG is N-acetyl glucosamine. Credit: https://doi.org/10.1038/s41586-020-2829-0

“The increased SNR of cryo-EM images enabled by the technology described here,” the authors conclude, “will expand [the technique] to more difficult samples, including membrane proteins in lipid bilayers, small proteins and structurally heterogeneous macromolecular complexes.”

At these resolutions, scientists are closing in on images not just of macromolecules of biological importance but of parts of these molecules – and can in effect elucidate the structures that correspond to specific functions or processes. This is somewhat like going from knowing that viruses infect cells to determining the specific parts of a virus and a cell implicated in the infiltration process.

A very germane example is that of the novel coronavirus. In April this year, a group of researchers from France and the US reported the cryo-EM structure of the virus’s spike glycoprotein, which binds to the ACE2 protein on the surface of some cells to gain entry. By knowing this structure, other researchers can design the more perfect inhibitors to disrupt the glycoprotein’s function, as well as vaccines that mimic its presence to provoke the desired immune response.

In this regard, a resolution of 1-2 Å corresponds to the dimensions of individual covalent bonds. So by extending the cryo-EM’s ability to decipher smaller and smaller features, researchers can strike at smaller, more precise molecular mechanisms to produce more efficient, perhaps more closely controlled and finely targeted, effects.

Featured image: Scientists using a 300-kV cryo-EM at the Max Planck Institute of Molecular Physiology, Dortmund. Credit: MPI Dortmund.

‘Weak charge’ measurement holds up SM prediction

Various dark matter detectors around the world, massive particle accelerators and colliders, powerful telescopes on the ground and in space all have their distinct agendas but ultimately what unites them is humankind’s quest to understand what the hell this universe is on about. There are unanswered questions in every branch of scientific endeavour that will keep us busy for millennia to come.

Among them, physics seems to be sufferingly uniquely, as it stumbles even as we speak through a ‘nightmare scenario’: the most sensitive measurements we have made of the physical reality around us, at the largest and smallest scales, don’t agree with what physicists have been able to work out on paper. Something’s gotta give – but scientists don’t know where or how they will find their answers.

The Qweak experiment at the Jefferson Lab, Virginia, is one of scores of experiments around the world trying to find a way out of the nightmare scenario. And Qweak is doing that by studying how the rate at which electrons scatter off a proton is affected by the electrons’ polarisation (a.k.a. spin polarisation: whether the spin of each electron is “left” or “right”).

Unlike instruments like the Large Hadron Collider, which are very big, operate at much higher energies, are expensive and are used to look for new particles hiding in spacetime, Qweak and others like it make ultra-precise measurements of known values, in effect studying the effects of particles both known and unknown on natural phenomena.

And if these experiments are able to find that these values deviate at some level from that predicted by the theory, physicists will have the break they’re looking for. For example, if Qweak is the one to break new ground, then physicists will have reason to suspect that the two nuclear forces of nature, simply called strong and weak, hold some secrets.

However, Qweak’s latest – and possibly its last – results don’t break new ground. In fact, they assert that the current theory of particle physics is correct, the same theory that physicists are trying to break free of.

Most of us are familiar with protons and electrons: they’re subatomic particles, carry positive and negative charges resp., and are the stuff of one chapter of high-school physics. What students of science find out quite later is that electrons are fundamental particles – they’re not made up of smaller particles – but protons are not. Protons are made up of quarks and gluons.

Interactions between electrons and quarks/gluons is mediated by two fundamental forces: the electromagnetic and the weak nuclear. The electromagnetic force is much stronger than the aptly named weak nuclear force. On the other hand, it is agnostic to the electron’s polarisation while the weak nuclear force is sensitive to it. In fact, the weak nuclear force is known to respond differently to left- and right-handed particles.

When electrons are bombarded at protons, the electrons are scattered off. Scientists at measure how often this happens and at what angle, together with the electrons’ polarisation – and try to find correlations between the two sets of data.

An illustration showing the expected outcomes when left- and right-handed electrons, visualised as mirror-images of each other, scatter off of a proton. Credit: doi:10.1038/s41586-018-0096-0
An illustration showing the expected outcomes when left- and right-handed electrons, visualised as mirror-images of each other, scatter off of a proton. Credit: doi:10.1038/s41586-018-0096-0

At Qweak, the electrons were accelerated to 1.16 GeV and bombarded at a tank of liquid hydrogen. A detector positioned near the tank picked up on electrons scattered at angles between 5.8º and 11.6º. By finely tuning different aspects of this setup, the scientists were able to up the measurement precision to 10 parts per billion.

For example, they were able to achieve a detection rate of 7 billion per second, a target luminosity of 1.7 x 1039 cm-2 s-1 and provide a polarised beam of electrons at 180 µA – all considered high for an experiment of this kind.

The scientists were looking for patterns in the detector data that would tell them something about the proton’s weak charge: the strength with which it interacts with electrons via the weak nuclear force. (Its notation is Qweak, hence the experiment’s name.)

At Qweak, they’re doing this by studying how the electrons are scattered versus their polarisation. The Standard Model (SM) of particle physics, the theory that physicists work with to understand the behaviour of elementary particles, predicts that the number of left- and right-handed electrons scattered should differ by one for every 10 million interactions. If this number is found to be bigger or smaller than usual when measured in the wild, then the Standard Model will be in trouble – much to physicists’ delight.

SM’s corresponding value for the proton’s weak charge is 0.0708. At Qweak, the value was measured to be 0.0719 ± 0.0045, i.e. between 0.0674 and 0.0764, completely agreeing with the SM prediction. Something’s gotta give – but it’s not going to be the proton’s weak charge for now.

Paper: Precision measurement of the weak charge of the proton

Featured image credit: Pexels/Unsplash.