Cryo-EM maps critical DNA complex


Rebecca Pool

Friday, September 15, 2017 - 11:30
Eva Nogales: At the forefront of cryo-EM [Marilyn Chung/Berkeley Lab]
Using cryo-electron microscopy, US-based researchers at Berkeley Lab have obtained the highest resolution map yet of a large assembly of human proteins, critical to DNA function.
Professor Eva Nogales from Molecular Biophysics and Integrated Bioimaging, and colleagues, resolved the 3D structure of the protein complex, transcription factor IIH (TFIIH), to 4.4 angstroms, near-atomic resolution.
This protein complex is used to unzip the DNA double helix so that genes can be accessed and read during transcription or repair.
“When TFIIH goes wrong, DNA repair can’t occur, and that malfunction is associated with severe cancer propensity, premature aging, and a variety of other defects,” says Nogales. “Using this structure, we can now begin to place mutations in context to better understand why they give rise to misbehavior in cells.”
The cryo-EM structure of Transcription Factor II Human (TFIIH). The atomic coordinate model, coloured according to the different TFIIH subunits, is shown inside the semi-transparent cryo-EM map. [Basil Greber/Berkeley Lab and UC Berkeley]
TFIIH’s critical role in DNA function has made it a prime target for research, but it is considered a difficult protein complex to study, especially in humans.
TFIIH exists in minute amounts in a cell, so the researchers had to grow 50 litres of human cells in culture to yield just a few micrograms of the purified protein.
What's more, human TFIIH is fragile and prone to falling apart during flash-freezing, so the researchers used an optimized buffer solution to help protect the protein structure.
“These compounds that protect the proteins also work as antifreeze agents, but there’s a trade-off between protein stability and the ability to produce a transparent film of ice needed for cryo-EM,” explains Nogales's colleague, Dr Basil Greber. "Once you have that sample inside the microscope, you keep collecting data as long as you can; this process can take four days straight.”
"The fact that we resolved this protein structure from human cells makes this even more relevant to disease research," adds Nogales. "There’s no need to extrapolate the protein’s function based upon how it works in other organisms."
According to the researchers, they collected 1.5 million images of individual molecules.
The researchers used a FEI low-base Titan TEM with a Gatan side-entry holder, operating at 300 kV acceleration voltage.
The TEM was equipped with a Gatan K2 Summit direct electron detector camera operating in counting mode.
Data were collected semi-automatically using the Leginon package at 37,879× magnification, resulting in a pixel size of 1.32 Å.
“We [had to] select particles that were representative of an intact complex," says Greber. "After 300,000 central processing unit hours at the National Energy Research Scientific Computing Center, we ended up with 120,000 images of individual particles that were used to compute the 3D map of the protein.”
To obtain an atomic model of the protein complex based on this 3-D map, the researchers used the software program, PHENIX - Python-based Hierarchical ENvironment for Integrated Xtallography - developed by Paul Adams, Director of Berkeley Lab’s Molecular Biophysics and Integrated Bioimaging Division.
“This work is a prime example of what structural biologists can do," highlights Nogales. "We establish the framework for understanding how the molecules function. And with that information, researchers can develop finely targeted therapies with more predictive power.”
Research is published in Nature.
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