Recipients of R&D Pilot Awards

Piotr Stępień

DNA NanoNets for improving Cryo-EM imaging of bulky integral membrane proteins 

Membrane proteins are among the essential constituents of living organisms, making up 20-30% of the proteins encoded by all genomes. As membrane proteins govern the transport of materials and information in and out of cells, they are privileged drug targets, with around 60% of small-molecule pharmacophores specifically targeting them. Recently, the study of membrane proteins and drug discovery connected to them was revolutionised by combining nanodiscs and cryo-EM.

The nanodiscs are nanoscale lipid patches into which membrane proteins can be incorporated, effectively rendering them soluble, simplifying their handling and allowing for their facile single-particle analysis using cryo-EM. Despite these advancements, obtaining the highest possible resolution with this combined approach is often challenging because bulky membrane proteins tend to adopt preferred orientations in vitreous ice. This restricts the available “views” of the proteins, resulting in reduced resolution. In my Instruct-ERIC project, I am developing a tool to help overcome this obstacle.

I am incorporating nanodiscs into DNA lattices to position the proteins into underrepresented orientations, creating “DNA NanoNets. " These lattices are designed for easy integration into existing membrane protein preparation and analysis pipelines, enhancing the efficiency of analysis while reducing the number of micrographs which need to be collected. 

Sarah O’Keefe

Towards a movie of nascent protein synthesis in the early secretory pathway 

Our project leverages a photoswitchable translation inhibitor and a light-activated time-resolved cryo-EM approach to develop a new and accurate method to spatiotemporally map the early protein synthesis of any protein of interest, including those that cause human diseases. This method is applicable to capture movie snapshots of how early protein synthesis is accurately co-ordinated in distinct cellular compartments and across cellular life.

First, we established a novel and universal pipeline for programming ribosomes of animal, plant or bacterial origin with stalled nascent chains, their rapid purification and small molecule light-activated ribosomal release. We are now applying these biochemically optimised methods in cryo-EM imaging to reveal new macromolecular insights in protein synthesis. By enabling access to the technologies at the cryo-EM facility at the University of Helsinki, Instruct Centre-FI and the expertise of its staff and users (see photo), this R&D award has also provided invaluable training in instrument usage, sample preparation and image processing for the awardee.

Our results integrated chemical and structural biology in this R&D project have already been used in grant submissions to external funding bodies. 

Sven Klumpe

Shaping focused ion beam in lamella preparation for cryo-electron tomography 

In this project we explore the idea of using probe shaping in focused ion beam (FIB) milling in order to improve the resolution and speed of material removal in lamella preparation for cryo-electron tomography (cryoET). By using the available stigmator as a quasi-cylindrical lens similar to approaches in light sheet microscopy, we stretch the beam profile into a highly elongated shape that we term ‘ion knife'. 

This improves the apparent resolution of spot burns in one direction (knife diameter) at the expense of the perpendicular direction (knife length) compared to a standard, Gaussian-like spot beam profile. In the future direction of the R&D award, we will investigate how specialised hardware could help improve our current approaches to increase the material retention in cryo-lift-out sectioning. Additionally, we will develop open-source software to integrate shaped FIB probes into automated cryo-FIB lamella preparation routines. 

Paola Lanzoni-Mangutchi

Giving GALK1 a stab in the back: the study of GALK1 inactivation through its allosteric site 

Classic galactosaemia (CG) is a rare hereditary disorder of the galactose metabolism, causing a toxic buildup of galactose-1-phosphate (Gal-1P) inside the cells. Galactokinase 1 (GALK1) converts galactose into Gal1P and is a validated therapeutic target against CG. Our group identified GALK1 inhibitors that act via an allosteric site, with a potential for selective drugs with fewer side effects.

This project explores the mechanism of allosteric inhibition as a “stab on GALK1’s back”. We hypothesise that small amino acid modifications at the allosteric site are transmitted across the structure and disrupt the enzymatic activity, causing this mutant protein to self-inhibit in a similar manner as the wild-type would have been by a small molecule inhibitor.

We mutated six candidate residues lining the allosteric site to tryptophan and histidine, due to their chemical similarity to our inhibitors, to test their impact on the enzyme’s overall activity and structure. Initial single mutants show a slight decrease in kinase activity and thermostability. We anticipate that their crystal structures will provide us with an unprecedented understanding of allosteric inhibition.

Steffen Klein

Optimising labels for accurately localizing macromolecules in cryo-electron tomography of intact cells 

In this project, we develop a correlative method using fluorescent and structurally defined tags that can be directly localized in cryo-ET data to allow identification and pinpointing the location of new targets in human cells for structure determination by subtomogram averaging. Based on our previously developed tags, we aim to optimise the method by generating a simplified one-component labeling system and improving its efficiency. By coupling different inducible dimerisation modules directly to genetically encoded multimeric particles (GEMs), we reduce the complexity and size of the tags while allowing tight temporal control of target labeling. The reduced distance between the GEM tag and the target protein will increase the localisation accuracy of the target position, potentially improving automated particle picking during data analysis, which is essential for structure determination. 

Heikki Saari

In-line Raman detector for flow analytics of macromolecular biocomplexes 

Our project is focused on establishing a novel inline Raman detector to be used for preparative or analytic methods using liquid flow. Usually, Raman spectroscopy is applied for dried samples, as the Raman scattering of photons is rare, and the intensity of an acquired spectra is dependent on sample concentration. A special focus is using it during extracellular vesicle (EV) isolation to monitor the purification process. As Raman spectroscopy is a non-destructive analysis method that provides information regarding chemical functional groups in a given sample, it can be used to identify and assess several types of biomolecules, including proteins and lipids.

For our first sub-project, we have been applying the inline Raman detector in conjunction with an anion exchange chromatography purification scheme for platelet derived EVs. Since anion exchange is simultaneously able to concentrate and purify EVs, we found that the EVs are quite specifically enriched into the eluate and most of the impurities are discarded in the flowthrough. These phases can be identified with the inline Raman detector, and changes such as additional impurities or increased sample load can also be distinguished from a standard sample based on the Raman spectra. 

 Alexandre Ourjoumtsev

New tools and concepts for efficient real-space refinement and validation of macromolecular atomic models versus inhomogeneous cryo electron microscopy or crystallographic maps  

A new type of modelling based on a specially designed function allows a new representation of information about cryo-EM and crystallographic maps and about atomic models refined versus these maps. This can lead to new refinement procedures. To numerically compare model maps with the experimentally obtained ones, the former, mimicking major imperfections of the latter, are calculated as a sum of atomic images expressed by analytic functions of atomic position, values of the displacement parameter (B), occupancy and a local resolution (D). This resolution is associated now with atoms contributing to the given map point.

The proposed method requires no Fourier transform and allows the generation of variable-resolution maps in a single run. Inversely, developed analytic expressions for finite-resolution atomic images, together with analytic expressions for the target functions comparing two maps, allow the estimation and simultaneous refinement of the values of all atomic parameters. The algorithms already implemented recover B and D values assuming that atomic positions are known. In particular, several cases of such values, previously incorrectly assigned by conventional software, have been identified and fixed. The values found with the new method made the calculated maps much more similar to the respective experimental ones. Development of full-parameters refinement software is under development. 

Francesca Paoletti 

Illuminating the role of the intrinsically disordered regions of pro-neurotrophins and their interacting cartography with ligands and receptors by integrative structural biology with hybrid methods. 

Nerve growth factor (NGF), the prototypical neurotrophin (NT), is involved in the maintenance and growth of neuronal populations, whereas its precursor, proNGF, is involved in neuronal apoptosis. Binding of NGF or proNGF to TrkA, p75NTR, and VP10p receptors triggers complex intracellular signalling pathways that can be modulated by Small Endogenous Ligands (SELs). Due to the wide spectrum of diseases involving NTs (e.g. Alzheimer’s Disease, corneal pathologies, chronic pain, etc.), there is a wide pharmacological interest in targeting NTs with SELs. The role of SELs in these processes remains poorly understood because of the lack of architectural details on the pro-NTs pro-peptide, predicted to be intrinsically unstructured.

This proposal aims at widening the molecular understanding of proNGF biological responses, through integrative structural biology. The NMR spectral assignment of proNGF represents a fundamental milestone for interaction biophysical studies (HDXMS, AUC, ITC, GCI…) aimed to unravel the molecular determinants of SELs in the modulation of receptors’ binding. The optimisation of a workflow for proNGF would allow to extend it to different SELs, as well as to other pro-NTs. The project foresees to overcome resilience in unravelling how proNTs structure affect their function and to foster advances in their diagnostic and pharmacological applications.