RESEARCH
Molecular Resolution
The model system we study is the assembly and maturation process of complex double stranded DNA viruses. We use the phages T7 and phi29 as model systems offering complementary aspects to cover the most interesting features shared by this virus family during their assembly, as the production of intermediates, the expansion of the shell by a complex subunit rearrangement, the transformation of inner capsid components (core, scaffolding), and the changes involved in the function of the DNA packaging machinery. We also improve the single particle reconstruction methods to get higher resolution data suitable to apply hybrid approaches, by a combination of 3D-cryo-EM and modelling methods, to compare quasi-atomic 3D maps of proheads and mature heads of T7.
Cryo-electron tomography is becoming a extremely useful al powerful technique. The output 3D volumes comes from a hydrated state, therefore suitable for measurements, alignments and statistics. In viruses or in cell structures, several structures are repetitive but anchored or surrounded to pleiomorphic surfaces. Subvolume averaging exploit repetitive structures within the sample to enhance the signal to noise ratio to visualize unique structures as the influenza virus ribonucleoprotein assembly inside the native virion. Also, 3D classification of sub-volumes could reveals important rearrangement of a virus.
Electron Tomography
Correlation of structural data and physical properties at the single particle level
The structural data obtained by 3D-cryo-EM from the different experimental systems under study in our group are currently combined with the study of the nano-mechanical properties of individual viral particles using atomic force microscopy and spectroscopy. We are also exploring the application of optical tweezers to test small forces involved in specific capsid transformations. The study of the deformation behaviour and material properties at the level of individual viral particles opens the possibility to perform systematic studies to correlate molecular structure, nanoscopic behaviour, and macroscopic properties of these macromolecular viral containers.
Whole cell cartography by cryo-soft X-ray tomography (cryo-SXT)
Cryo Soft X-ray tomography (cryo-SXT) is an emerging X-ray microscopy technique for three-dimensional visualization of cryo-preserved, whole, unstained cells at a spatial resolution of 40 nm (half-pitch). The penetration power of soft X-rays in the so-called water window spectral range, between the carbon and oxygen absorption edges (Figure 4), allows penetrating water layers up to 10 μm thickness while carbon-rich structures, strongly absorbent, are visualized with good contrast. Thus, frozen-hydrated specimens can be imaged close to their native state providing significant complementary information to the today existing biological imaging techniques.
Synthetic Biology
Physical characterization of the packaging machinery
Based on our previous structural knowledge on the packaging machinery of two viral model systems (T7 and phi29), we are developing a synthetic biology approach to insert phage connectors into nano-engineered lipidic bilayers, in collaboration with the group of Francisco Monroy (UCM). We are exploring the possibility of building up the entire packaging motor on these inserted connectors which will allow us to obtain efficient lipidic nanocontainers capable to carrying large amounts of DNA. On the other hand, we are also improving our optical tweezers set up to study, at the single molecule level, the physical properties of the membrane-inserted connectors and the molecular basis of their function.​
