Human protein complex captured in action
Image: Human ORC protein. [CSHL]
Using cryo-electron microscopy and x-ray crystallography, US-based biologists have produced atomic resolution images of the multi-part protein complex - origin recognition complex - in its active mode.
ORC complexes self-assemble in the cell nucleus and bind at specific spots called start sites or origins along the double helix in chromosomes, to perform the very first step in the human DNA replication dance.
But as Professor Leemor Joshua-Tor from the Cold Spring Harbor Laboratory and Howard Hughes Medical Institute highlights, earlier images of ORC were lower resolution.
The researchers obtained higher resolution images using an FEI Titian Krios electron microscope operated at a high tension of 300 kV, semi-automatically collecting images via SerialEM under low-dose mode at a magnification of ×29,000 and pixel size of 1.01 Å.
A Gatan K2 summit direct electron detector was also used under super-resolution mode for image recording.
A view of the human ORC protein, showing 4 of 5 ORC subunits. A missing subunit makes it possible to see how DNA (grey) fits through the "ring". [CSHL]
Crucially, the new images make clear how ATP binds at positions in one major part of the ORC assembly, consisting of protein subunits.
According to Joshua-Tor, these high-resolution images of the active human ORC solve three major mysteries.
“They help us understand how DNA might bind with ORC; how the ATP fuel is used, and how mutations in proteins in the ORC complex give rise to human disease,” she says.
In human cells, ORC assembles at literally thousands of origin sites across the entire genome, to form an initial configuration called the pre-replication complex, or pre-RC.
The images of the human version of this complex, called ORC—for origin recognition complex—show it in its active mode.
In ORC’s active phase, the researchers showed that a subassembly containing ORC subunits 1,2,3,4 and 5 engages multiple ATP molecules and forms a partial ring-shaped complex.
ATP is also used to recruit another protein component called CDC6, transforming the open ring into a closed ring.
By this time, the multi-part assembly has engaged and bound to the double helix, which passes through the centre of the ring; the ring is designed to snugly fit the DNA.
Research is published in e-Life.