Resolution map of the poly C9 pore.
Image: Brad Spicer (Spicer et al., 2018)
“These data will be crucial for the development of new molecules to regulate complement in a wide range of different immunity-related disorders.”
Our work aims to understand how immune effectors are triggered to destroy targets. In particular we are interested in three pore forming proteins – Complement component-9 (C9), Macrophage Expressed Gene-1 (MPEG-1) and Perforin. These three molecules play key roles in the destruction of pathogenic microbes, virally infected cells and malignant cells. In working on these protein complexes, we have also recognised the importance of developing new and better approaches for sample preparation for EM and for structure determination.
PROF. JAMES WHISSTOCK
AT A GLANCE
In 2018, we reported the structure of the terminal pore-forming portion (C9) of the complement Membrane Attack Complex (MAC; Spicer et al., Nature Communications 2018). Our work gave long-sought-after insight into how this crucial part of the immune system assembles into a pore. We also identified the regions of C9 that must move and interact during complex formation. These data will be crucial for the development of new molecules to regulate complement in a wide range of different immunity-related disorders.
The immune complexes our team works on are typically very hard to produce in large quantities and often are challenging to prepare for EM experiments. Accordingly, our team has been working to develop new and improved techniques to prepare a sample in the first place, and furthermore to improve our ability to rapidly analyse data from the images we collect.
In regards to developing new and improved techniques to prepare a sample, we are using nanofluidic approaches to deposit very small amounts of material onto EM grids, such that blotting away excess sample is no longer needed. With respect to data analysis, together with the MASSIVE team at Monash, we are developing software workflows that permit “on-the-fly” analysis of EM data as it is produced by the microscope. Such approaches will greatly speed up EM experiments and will further permit a greater number of samples to be processed through the microscope.
- Determine the high-resolution structures of the perforin-1 and perforin-2 pore form.
- Develop new workflows to visualise and characterise large protein complexes in situ.
- Build and further refine new sample preparation devices for single particle cryo EM.
- Develop new computational workflows for determining the structure of protein complexes using single particle cryo EM.
The atomic resolution structure of the immune effector Complement Component-9 (C9)
In 2018, Centre scientists determined the long sought-after structure of the immune effector C9, in both the soluble, monomeric state, and in the pore form. The team used X-ray crystallography to determine the structure of the C9 monomer (see figure below) and single particle cryo Electron Microscopy to determine the structure of the complete pore (see figure, right).
The structure revealed how the complement system assembles to form a pore in a stepwise fashion, with the first membrane spanning region functioning as a crucial control point to regulate the addition of new C9 monomers into the growing pore. This work demonstrates the power of combining two distinct methodologies for structure determination – both X-ray crystallography and cryo-electron microscopy – in order to yield new insights that could not be gleaned from each individual structure alone. The team anticipate that the new discoveries could lead to better approaches to control C9 in a range of different inflammatory conditions.
Top: Structure of the C9 polymer.
Bottom: Structure of monomeric C9.
A new approach for sample preparation for cryo-Transmission Electron Microscopy
Historically, the most commonly used method for cryo-EM sample preparation comprises depositing an excess of sample solution on a holey-carbon EM grid, which is reduced to a thin film (~100 nm) via mechanical blotting prior to plunge-freezing in liquid ethane. This approach is sub-optimal because more than 98% of the sample is wasted as part of the process, and can also be problematic in terms of the protein distribution on the grid. In 2018, Imaging CoE scientists developed two proof of principle approaches capable of resolving these limitations.
In order to limit the sample waste and improve grid quality, we developed an approach to atomising the sample solution iton droplets with a diameter of ~6 µm and then spraying this material onto a cryo-EM grid while it is being plunged into liquid ethane. The new system only requires 50 nL of sample per grid. Further, the time that the sample spends on the grid prior to cryo-fixation is less than 50 ms, reducing issues with preferential orientation occurring when a grid is prepared through conventional blotting. The time that the sample spends on the grid prior to cryo-fixation is less than 50 ms, reducing issues with preferential orientation occurring when a grid is prepared through conventional blotting.
A second approach we developed involved a nanofluidic chamber, which can be used for cryo-EM imaging of samples that are sensitive to the atmosphere or that need to be kept at a specific pressure while in liquid phase such as when studying nanoparticles. A thin channel sandwiched between two silicon-nitride membranes is plunge-frozen in liquid ethane and imaged under cryoTEM. This method, again only uses a tiny amount of sample that can be imaged with no losses. In parallel it completely resolves the known problem of the segregation of molecules at the air-water interface because it takes place in a sealed compartment.
Together we anticipate that these novel methods for sample preparation will lead to future advancements in the cryo-electron microscopy such as the development of time-resolved imaging and the analyses of processes extremely sensitive to the environment such as oxidation states.
1. Ashtiani D., Venugopal H., Belousoff M., Spicer B., Mak J., Neild A.*, de Marco A.* (2018) “Delivery of femtolitre droplets using surface acoustic wave based atomisation for cryo-EM grid preparation” Journal of structural biology 203(2), p.94-101.
2. Ashtiani, D; de Marco A.*; Neild, A.* (2019) “Tailoring Surface Acoustic Wave Atomisation for Cryo-Electron Microscopy Sample Preparation” (in press).
3. Gorelick S.; Alan T., Sadek A., Tjeung R., de Marco A. (2018) “Nanofluidic and monolithic environmental cells for cryogenic microscopy” Nanotechnology 30(8), 085301.
Top: Atomised protein solution being sprayed onto an EM grid.(1-3)
Middle: Ribosomes in thin ice preserved for TEM experiments.(1-3)
Bottom: High resolution structure of a ribosome obtained from the new methodology.(1-3)