Puzzle the structure of a molecular giant

Comparison of the models of human nuclear pore complex obtained before (left) and after using AlphaFold2 combined with cryo-ET (right). The human nuclear pore complex has a total weight of 120 MDa. The previous models only covered up to 35 MDa. The new model covers 70 MDa. Credit: Agnieszka Obarska-Kosińska/EMBL and MPI of Biophysics

By combining AlphaFold2 with experimental and computational techniques, scientists have been able to uncover the architecture of the human nuclear pore complex in more detail than ever before.

The human nuclear pore complex (NPC) is a true molecular giant, sitting on the membrane that separates the nucleus from the cytoplasm. It is shaped like a donut and acts as both a gateway and a checkpoint for molecules traveling between the cytoplasm and the nucleus. In addition, the NPC facilitates fundamental processes in the cell, such as gene expression and translation. The nuclear transport system also plays a role in several diseases, including neurodegenerative disorders, cancer and viral infections.

What is the structure of the NPC? How are the proteins glued together? How does it attach to the nuclear membrane? These and other questions have now been answered by the Kosinski Group at EMBL Hamburg and Center for Structural Systems Biology (CSSB), the Beck and Hummer Labs at the Max Planck Institute of Biophysics and collaborators. They created the most complete model of the human NPC to date by combining the protein structure prediction program AlphaFold2 with techniques such as cryo-electron tomography, single-particle cryo-EM and integrative modelling.

The human nuclear pore complex (NPC) is a donut-shaped molecular complex that consists of 30 different proteins that are in about 1000 copies. It weighs 120 MDa, which is huge on a cell scale. Credit: Agnieszka Obarska-Kosińska/EMBL and MPI of Biophysics

For structural biologists, the human NPC is a challenging yet exciting 3D puzzle, with about 30 different proteins each present in multiple copies. This amounts to about 1000 puzzle pieces, which form a round core with flexible parts around it. Until now, the most accurate models of the human NPC core covered only 46% of the structure. But now, building on two decades of previous research in the field, scientists have created a new model of the NPC structure that occupies more than 90% of the core.

While previously proposed NPC models had gaps, containing some proteins only in fragments, the new model removes much of this ambiguity.

“It’s like taking an electronic device apart and putting it back together. There will always be some screws left and you just don’t know where to put them,” said EMBL group leader Jan Kosinski, who co-led the study. “We’ve finally managed to fit most of them, and now we know exactly where they are, what they’re doing and how.”

Experimentation and artificial intelligence work together

How did the scientists achieve this? The key was to develop several experimental and calculation methods† This allowed the scientists to visualize the NPC at different scales and levels of detail.

For example, to model the overall silhouette of the NPC, the researchers used cryoelectron tomography. With this technique, they were able to observe the NPC in its cellular environment, rather than in isolation. More details of the individual protein building blocks were revealed by AlphaFold2, an artificial intelligence-based program that predicts egg white structures, made by the company DeepMind.

“AlphaFold2 was a breakthrough for us,” said Agnieszka Obarska-Kosińska, postdoc who conducted the molecular modelling. “We used to not know the structure of many proteins within the NPC. You can’t put together a puzzle if you don’t know what the pieces look like. But AlphaFold2 in combination with other approaches allowed us to predict those shapes.”

To refine the picture even further, the researchers used ColabFold, a version of AlphaFold2 adapted by the scientific community to model interactions between proteins. This allowed them to visualize how the different puzzle pieces are combined to form smaller sub-complexes, and how those sub-complexes are then glued together to form the NPC.

Finally, they put all the pieces together using the Assembline software previously developed by the Kosinski Group and validated it against experimental data

The resulting model was so complete and detailed that it allowed the researchers to create time-resolved molecular simulations that explain how the NPC proteins and the nuclear membrane interact to create a stable pore and how it responds to mechanical signals.

Future Directions

This work was a great leap forward for NPC research, but much remains to be discovered.

“This work illustrates how in the future structural biology will embrace cell biology to create atomic models of ever-larger assemblies of molecules that perform different functions in different parts of the cell,” said Martin Beck. Gerhard Hummer agrees: “We can now think of building a complete dynamic model of the NPC and simulate nuclear transport in atomic detail.”

In the future, the Kosinski Group will focus on developing automatic methods for integrating structural and microscopy data using AlphaFold2 and their proprietary software Assembline. They plan to apply these approaches to studying molecular processes that cause viral infections.

The research was published in Science

Observing the secret life of molecules in the cell

More information:
Shyamal Mosalaganti et al, AI-based structure prediction enables integrative structural analysis of human nuclear pores, Science (2022). DOI: 10.1126/science.abm9506

Quote: Puzzling the Structure of a Molecular Giant (2022, June 10) retrieved June 10, 2022 from https://phys.org/news/2022-06-puzzling-molecular-giant.html

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