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Unlocking the nuclear pore complex

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Unlocking the nuclear pore complex

Pietro Fontana is part of a global effort piecing together one of biology’s hardest puzzles

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When Pietro Fontana joined the Wu Lab at Harvard Medical School and Boston’s Children Hospital in May 2019, he had in front of him what has been called one of the world’s hardest, giant jigsaw puzzles – the task of piecing together a model of the nuclear pore complex, one of the largest molecular machines in human cells.

“It was very challenging from the start,” he explains; a modest assertion perhaps. The complex has been called a behemoth for good reason: it is made up of more than 30 different protein subunits, termed nucleoporins, and in total contains more than 1,000 of them, all intricately weaved.

I think AlphaFold has completely changed the idea of structural biology —Pietro Fontana

So when he sat down to use AlphaFold in his work for the first time two years later – together with Alex Tong of UC Berkeley who was more familiar with the AI system at the time – he was uncertain, if not sceptical, that it would help. But what followed in the summer of 2021 was a somewhat unexpected breakthrough moment. AlphaFold predicted the structures of nucleoporins that had not been previously determined, unveiling more of the nuclear pore complex in the process. Thanks to the AI, the lab could generate a near-complete model of the cytoplasmic ring.

Model of the nuclear pore complex cytoplasmic ring. Credit: Fontana et al. Science 2022

“Many components were already very well known, but with AlphaFold, we also built the ones that were structurally unknown,” he says. “I started to realise how it’s actually a big and useful tool for us. I think AlphaFold has completely changed the idea of structural biology.”

Molecular scientists like Fontana have dedicated themselves to deciphering the nuclear pore complex for decades. It acts as a gatekeeper for everything that goes in and out of the nucleus and is thought to hold answers to a growing number of serious human diseases, including amyotrophic lateral sclerosis and other neurodegenerative illnesses, and to questions we might not have even asked yet. Knowing how the complex is assembled could open the door to other groundbreaking, even life-saving discoveries.

Pietro Fontana

The sheer size alone is challenging, but its heterogeneous nature is an added complication. “That’s one major difficulty in achieving a resolution [clear enough] that we can interpret the sequence and structure of the complex,” says Hao Wu, the lab’s principal investigator. Even with a lot of data, the team only managed images of medium resolution in the past.

Every subunit we predicted clearly represented how the density looks like... that was pretty remarkable —Hao Wu

Missing puzzle pieces also hamstrung progress. Without the full set, it’s hard to tell how the jigsaw fits, says Wu. When it comes to the nuclear pore complex, “in order to figure out how the different protein subunits come together, you really need to have some assistance on their individual structures,” she explains.

This is where AlphaFold changed the game for the Wu Lab. Running it on proteins found in African clawed frog eggs – used as a model system as the oocytes are enriched in nuclear pore complex particles – the team, which also included Ying Dong and Xiong Pi in the Wu lab, managed to map all the different subunit structures, which were unknown up until then. “When we started trying, we didn’t really know if the predictions would fit the map nicely,” Wu recalls. “But that’s what happened. Every subunit we predicted clearly represented how the density looks like... That was pretty remarkable.” 

Of course, science is a collaborative effort. When it comes to a riddle as intricate as the nuclear pore complex, it’s not just teamwork, but the culmination of the diligence – and tenacity – of many teams across the world. Across the Atlantic, scientists from Max Planck Institute of Biophysics (MPIBP) and European Molecular Biology Laboratory (EMBL) in Germany have used AlphaFold in combination with cryo-electron tomography to model the human nuclear pore complex. What they have achieved so far is a new model twice as complete as the old one. Now covering two-thirds of the nuclear pore complex, a huge part of the puzzle has been solved, and a significant step towards understanding how the complex controls what goes in and out of the cell nucleus has been taken. 

The model of the human nuclear pore complex by scientists from MPIBP and EMBL in Germany. Credit: Agnieszka Obarska-Kosinska

There is still a way to go – the final third remains. And while AlphaFold will make the remaining puzzle easier to solve, scientists are also aware of its limitations. According to Wu, the AI software worked well in the case of the nuclear pore complex because its subunits contained repeated helical structures, which tend to be easier to predict. But it might not be as straightforward for other proteins.

Not treating AlphaFold – or any other AI tools for that matter – as a be-all and end-all and understanding how they work are key, cautions Alexander Tong, who was part of the Harvard Medical School team Wu led. “In fact, AlphaFold can give you some very strange results,” the latter adds. “But if you understand how it predicts, you can then take that into account [in the analysis].”

Still, it’s clear AlphaFold has not just expanded the limits of science, but in a timeframe previously not thought possible. “Honestly, I didn’t believe in it much myself, despite spending all my time on it,” Fontana says. “I’m glad AlphaFold came out at the right moment because it sped everything up significantly.” 

Fontana P., Dong Y., Pi X., Tong A.B., Hecksel C.W., Wang L., Fu TM., Bustamante C., Wu H. Structure of cytoplasmic ring of nuclear pore complex by integrative cryo-EM and AlphaFold. Science 376, 6598, (2022). DOI:10.1126/science.abm9326.