At one time or another, most of us have played with a flipbook, using our thumbs to rapidly flip a series of pictures to create an illusion of movement.
Scientists use a similar technique to study the ultrafast molecular processes inside cells. By piecing together high-resolution pictures of molecules at different time points, researchers can create a molecular flipbook to see their movement — a key part of understanding how cells function.
Over the years, more and more pages of these flipbooks have been filled in for different molecules, but there are still many pages missing, providing an incomplete view of how molecules move inside cells.
Now, a new technique developed at HHMI’s Janelia Research Campus is allowing scientists to fill in these missing pages and reveal the motion of molecules inside cells like never before. A team led by the Lippincott-Schwartz Lab at Janelia used the technique, called high-resolution template matching, or HRTM, to uncover in unprecedented detail the movement of ribosomes — the molecular structures that synthesize proteins inside cells.
Ribosomes undergo changes in conformational state, striking different, well-orchestrated poses that allow strands of RNA to be fed through the structure’s two subunits where instructions carried by the RNA are read and translated into proteins — a process called elongation.
Using HRTM, the researchers were able to detect ribosomes in 41 different conformational states that cover the entire elongation cycle. By combining these sequences into a flipbook, the researchers created a 3D movie that allowed them to see the ribosome moving through the elongation process, revealing never-before-seen movements that provide clues about how elongation happens.
“What we are seeing is the motion of the ribosome and its binding partners at near atomic detail,” says Janelia Senior Group Leader Jennifer Lippincott-Schwartz, head of Janelia’s 4D Cellular Physiology research area and senior author on the new research.
Getting a full picture
HRTM was developed in 2017 by Janelia Research Scientist Peter Rickgauer, former Janelia Senior Fellow Winfried Denk, and former Janelia Group Leader and current HHMI Investigator Nikolaus Grigorieff.
While current imaging techniques have enabled researchers to obtain 3D images of molecules, these methods either captured molecules outside of cells or were unable to detect very small molecular features. To reconstruct ribosomes’ different conformational states, researchers had to average together many images, which would miss the faster, rarer configurations. As a result, these methods allowed researchers to see only a handful of the ribosomes’ conformational states, even though they knew there were more.
“You weren’t able to get a full picture,” says Rickgauer, who also led the new research. “It’s like you have every tenth page of the flipbook.”
Like these other methods, HRTM utilizes electron microscopy images of intact frozen cells. But instead of trying to capture 3D images of the molecules inside these cells, HRTM detects molecular features in 2D images of different regions of the cell.
To find the molecules of interest in each image, the researchers create simulated targets of what they are looking for, based on known information about the molecule’s 3D structure. They then use a computer to search the 2D images for these targets, in any location or in any orientation.
When a match is found, the structure and its location and orientation are recorded. The researchers then start piecing these matches together. In the new research, these matches were used to construct ribosomes in different configurations. Eventually, the researchers combined these images to create a seamless 3D movie of the ribosome moving through all the different conformations in the elongation cycle.
The flipbook movie of the elongation cycle allowed the researchers to track the movements of the ribosome and its bound ligands. They were able to observe the smooth bending motion of inter-subunit bridge proteins and the spring-like transitions of tRNA between ribosome-binding sites, which had not been previously seen in cells and could reveal clues about the molecular mechanisms behind elongation.
Along with helping the researchers better understand how ribosomes function, the new research also provides a first test of using HRTM to detect molecular motion inside cells. The technique could be used to track other types of molecular movement, and to study interactions like the binding of pharmaceutical targets in a cellular environment.
“I think this is really exciting because it provides a roadmap for studying the structural dynamics of a variety of molecular complexes in cells,” Lippincott-Schwartz says.
