My laboratory studies how molecules on the surface of prokaryotic cells mediate cellular interaction with the environment, enabling cellular motility, initiating cellular adhesion to surfaces, and facilitating biofilm formation. For our work, we leverage our expertise in electron cryotomography (cryo-ET) in situ imaging, together with ongoing method development in subtomogram averaging approaches for structure determination of macromolecules in their native context. We combine cryo-EM with FIB milling of specimens and cryo-light microscopy to study molecules on prokaryotic cells.

In sarcomeres, α-actinin crosslinks actin filaments and anchors them to the Z-disk. FATZ proteins interact with α-actinin and five other core Z-disk proteins, contributing to myofibril assembly and maintenance as a protein interaction hub.
Here we report the first structure and its cellular validation of α-actinin-2 in complex with a Z-disk partner, FATZ-1, which is best described as a conformational ensemble. We show that FATZ-1 forms a tight fuzzy complex with α-actinin-2 and propose a molecular interaction mechanism via main molecular recognition elements and secondary binding sites. The obtained integrative model reveals a polar architecture of the complex which, in combination with FATZ-1 multivalent scaffold function, might organise interaction partners and stabilise α-actinin-2 preferential orientation in the Z-disk.
Finally, we uncover FATZ-1 ability to phase-separate and form biomolecular condensates with α-actinin-2, raising the intriguing question whether FATZ proteins can create an interaction hub for Z-disk proteins through membrane-less compartmentalization during myofibrillogenesis.

Bacterial RNA polymerase (RNAP) is an essential multisubunit enzyme performing transcription. Regulation of this process is secured through the stage-dependent interactions of RNAP with different factors (mostly proteins). Here we report the structure-function analysis of the functional complexes between RNAP and a unique helicase-like factor HelD [1] which is present in many Gram-positive bacteria (e.g. Bacillus subtilis and Mycobacterium smegmatis) [2, 3]. HelD forms tightly bound complexes with RNAP. It simultaneously penetrates into RNAP primary and secondary channels which are responsible for nucleic acids binding and substrate delivery. HelD can also interact with the RNAP active site. Structurally, these interactions are incompatible with the binding of DNA to the RNAP core and thus with the elongation stage of transcription. This is in accordance to our functional data showing that HelD is capable of clearing RNAP of nucleic acids and that HelD can dismantle RNAP-DNA complexes. HelD itself is composed of several domains, showing structural changes in solution [2] as well as in complexes with RNAP (three different structural states obtained from the cryo-EM analysis) [3]. Although we were able to link the observed dynamic behaviour with the DNA-clearing role of HelD, the recycling of HelD-bound RNAP and subsequent restart of transcription remains to be explained.

HelD as well as its complexes with RNAP resisted our attempts to crystallize them for many years. In order to get to the structural details we took the advantage of recent developments in the field of single-particle cryo-EM and were able to obtain ~3Å resolution structures. The structure of HelD itself was completely unknown with no homologue in the PDB. We combined X-ray crystallography (structure of one domain) and cryo-EM, together with bioinformatics and homologous modelling and successfully built de novo a complete atomic model of the HelD protein. For the analysis of condition-dependent dynamic behaviour we used small-angle X-ray scattering [2]. Results from our structural studies were supplemented with biochemical and biophysical assays (enzymology, analysis of interactions and stability) and by computational analyses [3].

[1] Wiedermannová, J., Sudzinová, P., Kovaľ, T., Rabatinová, A., Šanderova, H., Ramaniuk, O., Rittich, Š. & Dohnálek, J. (2014). Nucleic Acids Res. 42, 5151-5163.

[2] Kovaľ, T., Sudzinová, P., Perháčová, T., Trundová, M., Skálová, T., Fejfarová, K., Šanderová, H., Krásný, L., Dušková, J. & Dohnálek, J. (2019). FEBS Lett. 593, 996-1005.

[3] Kouba, T., Koval', T., Sudzinová, P., Pospíšil, J., Brezovská, B., Hnilicová, J., Šanderová, H., Janoušková, M., Šiková, M., Halada, P., Sýkora, M., Barvík, I., Nováček, J., Trundová, M., Dušková, J., Skálová, T., Chon, U., Murakami, K.S., Dohnálek, J. & Krásný, L. (2020). Nat Commun.11, 6419.

This work was supported by MEYS (LM2015043 and CZ.1.05/1.1.00/02.0109), CSF (20-12109S and 20-07473S), NIH (grant R35 GM131860), AS CR (86652036), ERDF (CZ.02.1.01/0.0/0.0/16_013/0001776 and CZ.02.1.01/0.0/0.0/15_003/0000447), EMBL (EI3POD) and Marie Skłodowska-Curie grant (664726).

Anomalous small-angle X-ray scattering (ASAXS) utilizes the changes of the scattering patterns emerging due to the variation of the scattering amplitude of a particular atom type upon changing the X-ray wavelength in the vicinity of the absorption edge of the atom. ASAXS on biological macromolecules is challenging due to the weak anomalous scattering effect. Biological macromolecules are also prone to radiation damage and often only available in small quantities, which further complicates the ASAXS measurements. First biological ASAXS experiments were done in the early 80-s at the European Molecular Biology Laboratory (EMBL) beamlines of the synchrotron DESY in Hamburg on metallo-proteins [1] but overall, ASAXS was not widely used in biological studies.

Recent progress in synchrotron instrumentation and dramatic increase of the brilliance of modern synchrotron sources revitalized the interest to biological ASAXS. The biological SAXS beamline P12 operated by EMBL at PETRA III storage ring (DESY, Hamburg) is dedicated to study macromolecular solutions [2] and allows for ASAXS on macromolecular solutions. The beamline was adapted to accommodate the needs for ASAXS by implementing careful data collection and reduction procedures and we report the recent developments at P12 allowing to conduct ASAXS on different macromolecular systems. Examples of utilizing ASAXS on various systems including, in particular, surfactants, nanoparticles, polymers and metal-loaded proteins are presented. The beamline control, data acquisition and data reduction pipeline developed for ASAXS on P12 are now available as standard tools for the biological SAXS community.

[1] H. B. Stuhrmann, Q. Rev. Biophys. 14, 433 (1981).

[2] C. E. Blanchet et al., J. Appl. Crystallogr. 48, 431 (2015).

Bacterial flagella are self-assembling nanomachines that enable cell motility by connecting a rotary motor to a long filament. Members of the Spirochaetes phylum, which includes important pathogens, swim using body undulations powered by the rotation of supercoiled flagella that remain confined within the periplasmic space and wrap around the cell body [1]. This behaviour diverges from that of other bacteria, where flagella function as extracellular propellers [2]. Spirochetal filaments are correspondingly distinct in composition and organization, but their molecular structure has remained elusive, obscuring the underlying mechanism for locomotion.

Here we show that, unlike all other known bacterial flagella, a highly asymmetric sheath layer coats the flagellar filament of the Leptospira spirochete and enforces filament supercoiling [3]. We solved 3D structures of wild-type and mutant flagellar filaments from L. biflexa, by integrating customized cryo-electron tomographic averaging methods with crystallographic analyses of sheath components FcpA and FcpB (Fig. 1). A central core made by FlaB (flagellin-like) protein units, delimits a central ~2nm pore.

Surrounding the core, FcpA and FcpB proteins colocalize exclusively on the outer, convex side of the filament, as an asymmetric sheath. Distinct sheath components, likely corresponding to FlaA isoforms, instead localize to the concave, inner side of the appendage. Sheath proteins were shown to produce filament supercoiling, an essential feature for flagellar-driven motility [1]. Such radial asymmetry represents a new paradigm of bacterial flagellar architecture, promoting filament supercoiling and ultimately enabling periplasmic flagella to exert their function in Leptospira translational motility. Given the large conservation of flagellar proteins across the Phylum, this asymmetric filament paradigm might prove valid for other spirochetes.

Candida albicans is the most common commensal fungus colonizing humans, and normally it does not impact the human health. However under certain conditions, it can rapidly outgrow bacterial flora causing mucocutaneous or systemic (and potentially fatal) infections. In most (mild) cases the treatment with topical and oral medications works well, however the resistant strains of C. albicans appear at the alarming pace, requiring the prompt development of new medications targeting this pathogen. One of the most promising routes to fight pathogens is to interfere with their protein synthesis machinery, therefore the structural information on ribosomes from pathogenic organisms is essential.

In this research we used an integrative structural biology approach based on the combination of single-particle cryo-Electron microscopy and macromolecular X-ray crystallography to resolve the structure of C. albicans ribosome.

We obtained 2.4 Å resolution structure of the 80S ribosome from C. albicans with the bound antibiotic and 4.2 Å resolution structure of the vacant C. albicans ribosome by single particle cryo-EM and X-ray crystallography. The comparison with other available eukaryotic ribosomes revealed unique features of C. albicans. These results can be used as a structural basis to decipher the mechanisms of antifungal resistance in C. albicans and to design novel inhibitors.