Project acronym 20SComplexity
Project An integrative approach to uncover the multilevel regulation of 20S proteasome degradation
Researcher (PI) Michal Sharon
Host Institution (HI) WEIZMANN INSTITUTE OF SCIENCE
Call Details Starting Grant (StG), LS1, ERC-2014-STG
Summary For many years, the ubiquitin-26S proteasome degradation pathway was considered the primary route for proteasomal degradation. However, it is now becoming clear that proteins can also be targeted for degradation by a ubiquitin-independent mechanism mediated by the core 20S proteasome itself. Although initially believed to be limited to rare exceptions, degradation by the 20S proteasome is now understood to have a wide range of substrates, many of which are key regulatory proteins. Despite its importance, little is known about the mechanisms that control 20S proteasomal degradation, unlike the extensive knowledge acquired over the years concerning degradation by the 26S proteasome. Our overall aim is to reveal the multiple regulatory levels that coordinate the 20S proteasome degradation route.
To achieve this goal we will carry out a comprehensive research program characterizing three distinct levels of 20S proteasome regulation:
Intra-molecular regulation- Revealing the intrinsic molecular switch that activates the latent 20S proteasome.
Inter-molecular regulation- Identifying novel proteins that bind the 20S proteasome to regulate its activity and characterizing their mechanism of function.
Cellular regulatory networks- Unraveling the cellular cues and multiple pathways that influence 20S proteasome activity using a novel systematic and unbiased screening approach.
Our experimental strategy involves the combination of biochemical approaches with native mass spectrometry, cross-linking and fluorescence measurements, complemented by cell biology analyses and high-throughput screening. Such a multidisciplinary approach, integrating in vitro and in vivo findings, will likely provide the much needed knowledge on the 20S proteasome degradation route. When completed, we anticipate that this work will be part of a new paradigm – no longer perceiving the 20S proteasome mediated degradation as a simple and passive event but rather a tightly regulated and coordinated process.
Summary
For many years, the ubiquitin-26S proteasome degradation pathway was considered the primary route for proteasomal degradation. However, it is now becoming clear that proteins can also be targeted for degradation by a ubiquitin-independent mechanism mediated by the core 20S proteasome itself. Although initially believed to be limited to rare exceptions, degradation by the 20S proteasome is now understood to have a wide range of substrates, many of which are key regulatory proteins. Despite its importance, little is known about the mechanisms that control 20S proteasomal degradation, unlike the extensive knowledge acquired over the years concerning degradation by the 26S proteasome. Our overall aim is to reveal the multiple regulatory levels that coordinate the 20S proteasome degradation route.
To achieve this goal we will carry out a comprehensive research program characterizing three distinct levels of 20S proteasome regulation:
Intra-molecular regulation- Revealing the intrinsic molecular switch that activates the latent 20S proteasome.
Inter-molecular regulation- Identifying novel proteins that bind the 20S proteasome to regulate its activity and characterizing their mechanism of function.
Cellular regulatory networks- Unraveling the cellular cues and multiple pathways that influence 20S proteasome activity using a novel systematic and unbiased screening approach.
Our experimental strategy involves the combination of biochemical approaches with native mass spectrometry, cross-linking and fluorescence measurements, complemented by cell biology analyses and high-throughput screening. Such a multidisciplinary approach, integrating in vitro and in vivo findings, will likely provide the much needed knowledge on the 20S proteasome degradation route. When completed, we anticipate that this work will be part of a new paradigm – no longer perceiving the 20S proteasome mediated degradation as a simple and passive event but rather a tightly regulated and coordinated process.
Max ERC Funding
1 500 000 €
Duration
Start date: 2015-04-01, End date: 2020-03-31
Project acronym 2DNanoSpec
Project Nanoscale Vibrational Spectroscopy of Sensitive 2D Molecular Materials
Researcher (PI) Renato ZENOBI
Host Institution (HI) EIDGENOESSISCHE TECHNISCHE HOCHSCHULE ZUERICH
Call Details Advanced Grant (AdG), PE4, ERC-2016-ADG
Summary I propose to investigate the nanometer scale organization of delicate 2-dimensional molecular materials using nanoscale vibrational spectroscopy. 2D structures are of great scientific and technological importance, for example as novel materials (graphene, MoS2, WS2, etc.), and in the form of biological membranes and synthetic 2D-polymers. Powerful methods for their analysis and imaging with molecular selectivity and sufficient spatial resolution, however, are lacking. Tip-enhanced Raman spectroscopy (TERS) allows label-free spectroscopic identification of molecular species, with ≈10 nm spatial resolution, and with single molecule sensitivity for strong Raman scatterers. So far, however, TERS is not being carried out in liquids, which is the natural environment for membranes, and its application to poor Raman scatterers such as components of 2D polymers, lipids, or other membrane compounds (proteins, sugars) is difficult. TERS has the potential to overcome the restrictions of other optical/spectroscopic methods to study 2D materials, namely (i) insufficient spatial resolution of diffraction-limited optical methods; (ii) the need for labelling for all methods relying on fluorescence; and (iii) the inability of some methods to work in liquids. I propose to address a number of scientific questions associated with the spatial organization, and the occurrence of defects in sensitive 2D molecular materials. The success of these studies will also rely critically on technical innovations of TERS that notably address the problem of energy dissipation. This will for the first time allow its application to study of complex, delicate 2D molecular systems without photochemical damage.
Summary
I propose to investigate the nanometer scale organization of delicate 2-dimensional molecular materials using nanoscale vibrational spectroscopy. 2D structures are of great scientific and technological importance, for example as novel materials (graphene, MoS2, WS2, etc.), and in the form of biological membranes and synthetic 2D-polymers. Powerful methods for their analysis and imaging with molecular selectivity and sufficient spatial resolution, however, are lacking. Tip-enhanced Raman spectroscopy (TERS) allows label-free spectroscopic identification of molecular species, with ≈10 nm spatial resolution, and with single molecule sensitivity for strong Raman scatterers. So far, however, TERS is not being carried out in liquids, which is the natural environment for membranes, and its application to poor Raman scatterers such as components of 2D polymers, lipids, or other membrane compounds (proteins, sugars) is difficult. TERS has the potential to overcome the restrictions of other optical/spectroscopic methods to study 2D materials, namely (i) insufficient spatial resolution of diffraction-limited optical methods; (ii) the need for labelling for all methods relying on fluorescence; and (iii) the inability of some methods to work in liquids. I propose to address a number of scientific questions associated with the spatial organization, and the occurrence of defects in sensitive 2D molecular materials. The success of these studies will also rely critically on technical innovations of TERS that notably address the problem of energy dissipation. This will for the first time allow its application to study of complex, delicate 2D molecular systems without photochemical damage.
Max ERC Funding
2 311 696 €
Duration
Start date: 2017-09-01, End date: 2022-08-31
Project acronym 2D–SYNETRA
Project Two-dimensional colloidal nanostructures - Synthesis and electrical transport
Researcher (PI) Christian Klinke
Host Institution (HI) UNIVERSITAET HAMBURG
Call Details Starting Grant (StG), PE4, ERC-2012-StG_20111012
Summary We propose to develop truly two-dimensional continuous materials and two-dimensional monolayer films composed of individual nanocrystals by the comparatively fast, inexpensive, and scalable colloidal synthesis method. The materials’ properties will be studied in detail, especially regarding their (photo-) electrical transport. This will allow developing new types of device structures, such as Coulomb blockade and field enhancement based transistors.
Recently, we demonstrated the possibility to synthesize in a controlled manner truly two-dimensional colloidal nanostructures. We will investigate their formation mechanism, synthesize further materials as “nanosheets”, develop methodologies to tune their geometrical properties, and study their (photo-) electrical properties.
Furthermore, we will use the Langmuir-Blodgett method to deposit highly ordered monolayers of monodisperse nanoparticles. Such structures show interesting transport properties governed by Coulomb blockade effects known from individual nanoparticles. This leads to semiconductor-like behavior in metal nanoparticle films. The understanding of the electric transport in such “multi-tunnel devices” is still very limited. Thus, we will investigate this concept in detail and take it to its limits. Beside improvement of quality and exchange of material we will tune the nanoparticles’ size and shape in order to gain a deeper understanding of the electrical properties of supercrystallographic assemblies. Furthermore, we will develop device concepts for diode and transistor structures which take into account the novel properties of the low-dimensional assemblies.
Nanosheets and monolayers of nanoparticles truly follow the principle of building devices by the bottom-up approach and allow electric transport measurements in a 2D regime. Highly ordered nanomaterial systems possess easy and reliably to manipulate electronic properties what make them interesting for future (inexpensive) electronic devices.
Summary
We propose to develop truly two-dimensional continuous materials and two-dimensional monolayer films composed of individual nanocrystals by the comparatively fast, inexpensive, and scalable colloidal synthesis method. The materials’ properties will be studied in detail, especially regarding their (photo-) electrical transport. This will allow developing new types of device structures, such as Coulomb blockade and field enhancement based transistors.
Recently, we demonstrated the possibility to synthesize in a controlled manner truly two-dimensional colloidal nanostructures. We will investigate their formation mechanism, synthesize further materials as “nanosheets”, develop methodologies to tune their geometrical properties, and study their (photo-) electrical properties.
Furthermore, we will use the Langmuir-Blodgett method to deposit highly ordered monolayers of monodisperse nanoparticles. Such structures show interesting transport properties governed by Coulomb blockade effects known from individual nanoparticles. This leads to semiconductor-like behavior in metal nanoparticle films. The understanding of the electric transport in such “multi-tunnel devices” is still very limited. Thus, we will investigate this concept in detail and take it to its limits. Beside improvement of quality and exchange of material we will tune the nanoparticles’ size and shape in order to gain a deeper understanding of the electrical properties of supercrystallographic assemblies. Furthermore, we will develop device concepts for diode and transistor structures which take into account the novel properties of the low-dimensional assemblies.
Nanosheets and monolayers of nanoparticles truly follow the principle of building devices by the bottom-up approach and allow electric transport measurements in a 2D regime. Highly ordered nanomaterial systems possess easy and reliably to manipulate electronic properties what make them interesting for future (inexpensive) electronic devices.
Max ERC Funding
1 497 200 €
Duration
Start date: 2013-02-01, End date: 2019-01-31
Project acronym 2F4BIODYN
Project Two-Field Nuclear Magnetic Resonance Spectroscopy for the Exploration of Biomolecular Dynamics
Researcher (PI) Fabien Ferrage
Host Institution (HI) CENTRE NATIONAL DE LA RECHERCHE SCIENTIFIQUE CNRS
Call Details Starting Grant (StG), PE4, ERC-2011-StG_20101014
Summary The paradigm of the structure-function relationship in proteins is outdated. Biological macromolecules and supramolecular assemblies are highly dynamic objects. Evidence that their motions are of utmost importance to their functions is regularly identified. The understanding of the physical chemistry of biological processes at an atomic level has to rely not only on the description of structure but also on the characterization of molecular motions.
The investigation of protein motions will be undertaken with a very innovative methodological approach in nuclear magnetic resonance relaxation. In order to widen the ranges of frequencies at which local motions in proteins are probed, we will first use and develop new techniques for a prototype shuttle system for the measurement of relaxation at low fields on a high-field NMR spectrometer. Second, we will develop a novel system: a set of low-field NMR spectrometers designed as accessories for high-field spectrometers. Used in conjunction with the shuttle, this system will offer (i) the sensitivity and resolution (i.e. atomic level information) of a high-field spectrometer (ii) the access to low fields of a relaxometer and (iii) the ability to measure a wide variety of relaxation rates with high accuracy. This system will benefit from the latest technology in homogeneous permanent magnet development to allow a control of spin systems identical to that of a high-resolution probe. This new apparatus will open the way to the use of NMR relaxation at low fields for the refinement of protein motions at an atomic scale.
Applications of this novel approach will focus on the bright side of protein dynamics: (i) the largely unexplored dynamics of intrinsically disordered proteins, and (ii) domain motions in large proteins. In both cases, we will investigate a series of diverse protein systems with implications in development, cancer and immunity.
Summary
The paradigm of the structure-function relationship in proteins is outdated. Biological macromolecules and supramolecular assemblies are highly dynamic objects. Evidence that their motions are of utmost importance to their functions is regularly identified. The understanding of the physical chemistry of biological processes at an atomic level has to rely not only on the description of structure but also on the characterization of molecular motions.
The investigation of protein motions will be undertaken with a very innovative methodological approach in nuclear magnetic resonance relaxation. In order to widen the ranges of frequencies at which local motions in proteins are probed, we will first use and develop new techniques for a prototype shuttle system for the measurement of relaxation at low fields on a high-field NMR spectrometer. Second, we will develop a novel system: a set of low-field NMR spectrometers designed as accessories for high-field spectrometers. Used in conjunction with the shuttle, this system will offer (i) the sensitivity and resolution (i.e. atomic level information) of a high-field spectrometer (ii) the access to low fields of a relaxometer and (iii) the ability to measure a wide variety of relaxation rates with high accuracy. This system will benefit from the latest technology in homogeneous permanent magnet development to allow a control of spin systems identical to that of a high-resolution probe. This new apparatus will open the way to the use of NMR relaxation at low fields for the refinement of protein motions at an atomic scale.
Applications of this novel approach will focus on the bright side of protein dynamics: (i) the largely unexplored dynamics of intrinsically disordered proteins, and (ii) domain motions in large proteins. In both cases, we will investigate a series of diverse protein systems with implications in development, cancer and immunity.
Max ERC Funding
1 462 080 €
Duration
Start date: 2012-01-01, End date: 2017-12-31
Project acronym 3CBIOTECH
Project Cold Carbon Catabolism of Microbial Communities underprinning a Sustainable Bioenergy and Biorefinery Economy
Researcher (PI) Gavin James Collins
Host Institution (HI) NATIONAL UNIVERSITY OF IRELAND GALWAY
Call Details Starting Grant (StG), LS9, ERC-2010-StG_20091118
Summary The applicant will collaborate with Irish, European and U.S.-based colleagues to develop a sustainable biorefinery and bioenergy industry in Ireland and Europe. The focus of this ERC Starting Grant will be the application of classical microbiological, physiological and real-time polymerase chain reaction (PCR)-based assays, to qualitatively and quantitatively characterize microbial communities underpinning novel and innovative, low-temperature, anaerobic waste (and other biomass) conversion technologies, including municipal wastewater treatment and, demonstration- and full-scale biorefinery applications.
Anaerobic digestion (AD) is a naturally-occurring process, which is widely applied for the conversion of waste to methane-containing biogas. Low-temperature (<20 degrees C) AD has been applied by the applicant as a cost-effective alternative to mesophilic (c. 35C) AD for the treatment of several waste categories. However, the microbiology of low-temperature AD is poorly understood. The applicant will work with microbial consortia isolated from anaerobic bioreactors, which have been operated for long-term experiments (>3.5 years), and include organic acid-oxidizing, hydrogen-producing syntrophic microbes and hydrogen-consuming methanogens. A major focus of the project will be the ecophysiology of psychrotolerant and psychrophilic methanogens already identified and cultivated by the applicant. The project will also investigate the role(s) of poorly-understood Crenarchaeota populations and homoacetogenic bacteria, in complex consortia. The host organization is a leading player in the microbiology of waste-to-energy applications. The applicant will train a team of scientists in all aspects of the microbiology and bioengineering of biomass conversion systems.
Summary
The applicant will collaborate with Irish, European and U.S.-based colleagues to develop a sustainable biorefinery and bioenergy industry in Ireland and Europe. The focus of this ERC Starting Grant will be the application of classical microbiological, physiological and real-time polymerase chain reaction (PCR)-based assays, to qualitatively and quantitatively characterize microbial communities underpinning novel and innovative, low-temperature, anaerobic waste (and other biomass) conversion technologies, including municipal wastewater treatment and, demonstration- and full-scale biorefinery applications.
Anaerobic digestion (AD) is a naturally-occurring process, which is widely applied for the conversion of waste to methane-containing biogas. Low-temperature (<20 degrees C) AD has been applied by the applicant as a cost-effective alternative to mesophilic (c. 35C) AD for the treatment of several waste categories. However, the microbiology of low-temperature AD is poorly understood. The applicant will work with microbial consortia isolated from anaerobic bioreactors, which have been operated for long-term experiments (>3.5 years), and include organic acid-oxidizing, hydrogen-producing syntrophic microbes and hydrogen-consuming methanogens. A major focus of the project will be the ecophysiology of psychrotolerant and psychrophilic methanogens already identified and cultivated by the applicant. The project will also investigate the role(s) of poorly-understood Crenarchaeota populations and homoacetogenic bacteria, in complex consortia. The host organization is a leading player in the microbiology of waste-to-energy applications. The applicant will train a team of scientists in all aspects of the microbiology and bioengineering of biomass conversion systems.
Max ERC Funding
1 499 797 €
Duration
Start date: 2011-05-01, End date: 2016-04-30
Project acronym 3DBIOLUNG
Project Bioengineering lung tissue using extracellular matrix based 3D bioprinting
Researcher (PI) Darcy WAGNER
Host Institution (HI) LUNDS UNIVERSITET
Call Details Starting Grant (StG), LS9, ERC-2018-STG
Summary Chronic lung diseases are increasing in prevalence with over 65 million patients worldwide. Lung transplantation remains the only potential option at end-stage disease. Around 4000 patients receive lung transplants annually with more awaiting transplantation, including 1000 patients in Europe. New options to increase available tissue for lung transplantation are desperately needed.
An exciting new research area focuses on generating lung tissue ex vivo using bioengineering approaches. Scaffolds can be generated from synthetic or biologically-derived (acellular) materials, seeded with cells and grown in a bioreactor prior to transplantation. Ideally, scaffolds would be seeded with cells derived from the transplant recipient, thus obviating the need for long-term immunosuppression. However, functional regeneration has yet to be achieved. New advances in 3D printing and 3D bioprinting (when cells are printed) indicate that this once thought of science-fiction concept might finally be mature enough for complex tissues, including lung. 3D bioprinting addresses a number of concerns identified in previous approaches, such as a) patient heterogeneity in acellular human scaffolds, b) anatomical differences in xenogeneic sources, c) lack of biological cues on synthetic materials and d) difficulty in manufacturing the complex lung architecture. 3D bioprinting could be a reproducible, scalable, and controllable approach for generating functional lung tissue.
The aim of this proposal is to use custom 3D bioprinters to generate constructs mimicking lung tissue using an innovative approach combining primary cells, the engineering reproducibility of synthetic materials, and the biologically conductive properties of acellular lung (hybrid). We will 3D bioprint hybrid murine and human lung tissue models and test gas exchange, angiogenesis and in vivo immune responses. This proposal will be a critical first step in demonstrating feasibility of 3D bioprinting lung tissue.
Summary
Chronic lung diseases are increasing in prevalence with over 65 million patients worldwide. Lung transplantation remains the only potential option at end-stage disease. Around 4000 patients receive lung transplants annually with more awaiting transplantation, including 1000 patients in Europe. New options to increase available tissue for lung transplantation are desperately needed.
An exciting new research area focuses on generating lung tissue ex vivo using bioengineering approaches. Scaffolds can be generated from synthetic or biologically-derived (acellular) materials, seeded with cells and grown in a bioreactor prior to transplantation. Ideally, scaffolds would be seeded with cells derived from the transplant recipient, thus obviating the need for long-term immunosuppression. However, functional regeneration has yet to be achieved. New advances in 3D printing and 3D bioprinting (when cells are printed) indicate that this once thought of science-fiction concept might finally be mature enough for complex tissues, including lung. 3D bioprinting addresses a number of concerns identified in previous approaches, such as a) patient heterogeneity in acellular human scaffolds, b) anatomical differences in xenogeneic sources, c) lack of biological cues on synthetic materials and d) difficulty in manufacturing the complex lung architecture. 3D bioprinting could be a reproducible, scalable, and controllable approach for generating functional lung tissue.
The aim of this proposal is to use custom 3D bioprinters to generate constructs mimicking lung tissue using an innovative approach combining primary cells, the engineering reproducibility of synthetic materials, and the biologically conductive properties of acellular lung (hybrid). We will 3D bioprint hybrid murine and human lung tissue models and test gas exchange, angiogenesis and in vivo immune responses. This proposal will be a critical first step in demonstrating feasibility of 3D bioprinting lung tissue.
Max ERC Funding
1 499 975 €
Duration
Start date: 2019-01-01, End date: 2023-12-31
Project acronym 3DCellPhase-
Project In situ Structural Analysis of Molecular Crowding and Phase Separation
Researcher (PI) Julia MAHAMID
Host Institution (HI) EUROPEAN MOLECULAR BIOLOGY LABORATORY
Call Details Starting Grant (StG), LS1, ERC-2017-STG
Summary This proposal brings together two fields in biology, namely the emerging field of phase-separated assemblies in cell biology and state-of-the-art cellular cryo-electron tomography, to advance our understanding on a fundamental, yet illusive, question: the molecular organization of the cytoplasm.
Eukaryotes organize their biochemical reactions into functionally distinct compartments. Intriguingly, many, if not most, cellular compartments are not membrane enclosed. Rather, they assemble dynamically by phase separation, typically triggered upon a specific event. Despite significant progress on reconstituting such liquid-like assemblies in vitro, we lack information as to whether these compartments in vivo are indeed amorphous liquids, or whether they exhibit structural features such as gels or fibers. My recent work on sample preparation of cells for cryo-electron tomography, including cryo-focused ion beam thinning, guided by 3D correlative fluorescence microscopy, shows that we can now prepare site-specific ‘electron-transparent windows’ in suitable eukaryotic systems, which allow direct examination of structural features of cellular compartments in their cellular context. Here, we will use these techniques to elucidate the structural principles and cytoplasmic environment driving the dynamic assembly of two phase-separated compartments: Stress granules, which are RNA bodies that form rapidly in the cytoplasm upon cellular stress, and centrosomes, which are sites of microtubule nucleation. We will combine these studies with a quantitative description of the crowded nature of cytoplasm and of its local variations, to provide a direct readout of the impact of excluded volume on molecular assembly in living cells. Taken together, these studies will provide fundamental insights into the structural basis by which cells form biochemical compartments.
Summary
This proposal brings together two fields in biology, namely the emerging field of phase-separated assemblies in cell biology and state-of-the-art cellular cryo-electron tomography, to advance our understanding on a fundamental, yet illusive, question: the molecular organization of the cytoplasm.
Eukaryotes organize their biochemical reactions into functionally distinct compartments. Intriguingly, many, if not most, cellular compartments are not membrane enclosed. Rather, they assemble dynamically by phase separation, typically triggered upon a specific event. Despite significant progress on reconstituting such liquid-like assemblies in vitro, we lack information as to whether these compartments in vivo are indeed amorphous liquids, or whether they exhibit structural features such as gels or fibers. My recent work on sample preparation of cells for cryo-electron tomography, including cryo-focused ion beam thinning, guided by 3D correlative fluorescence microscopy, shows that we can now prepare site-specific ‘electron-transparent windows’ in suitable eukaryotic systems, which allow direct examination of structural features of cellular compartments in their cellular context. Here, we will use these techniques to elucidate the structural principles and cytoplasmic environment driving the dynamic assembly of two phase-separated compartments: Stress granules, which are RNA bodies that form rapidly in the cytoplasm upon cellular stress, and centrosomes, which are sites of microtubule nucleation. We will combine these studies with a quantitative description of the crowded nature of cytoplasm and of its local variations, to provide a direct readout of the impact of excluded volume on molecular assembly in living cells. Taken together, these studies will provide fundamental insights into the structural basis by which cells form biochemical compartments.
Max ERC Funding
1 228 125 €
Duration
Start date: 2018-02-01, End date: 2023-01-31
Project acronym 3DIMAGE
Project 3D Imaging Across Lengthscales: From Atoms to Grains
Researcher (PI) Paul Anthony Midgley
Host Institution (HI) THE CHANCELLOR MASTERS AND SCHOLARS OF THE UNIVERSITY OF CAMBRIDGE
Call Details Advanced Grant (AdG), PE4, ERC-2011-ADG_20110209
Summary "Understanding structure-property relationships across lengthscales is key to the design of functional and structural materials and devices. Moreover, the complexity of modern devices extends to three dimensions and as such 3D characterization is required across those lengthscales to provide a complete understanding and enable improvement in the material’s physical and chemical behaviour. 3D imaging and analysis from the atomic scale through to granular microstructure is proposed through the development of electron tomography using (S)TEM, and ‘dual beam’ SEM-FIB, techniques offering complementary approaches to 3D imaging across lengthscales stretching over 5 orders of magnitude.
We propose to extend tomography to include novel methods to determine atom positions in 3D with approaches incorporating new reconstruction algorithms, image processing and complementary nano-diffraction techniques. At the nanoscale, true 3D nano-metrology of morphology and composition is a key objective of the project, minimizing reconstruction and visualization artefacts. Mapping strain and optical properties in 3D are ambitious and exciting challenges that will yield new information at the nanoscale. Using the SEM-FIB, 3D ‘mesoscale’ structures will be revealed: morphology, crystallography and composition can be mapped simultaneously, with ~5nm resolution and over volumes too large to tackle by (S)TEM and too small for most x-ray techniques. In parallel, we will apply 3D imaging to a wide variety of key materials including heterogeneous catalysts, aerospace alloys, biomaterials, photovoltaic materials, and novel semiconductors.
We will collaborate with many departments in Cambridge and institutes worldwide. The personnel on the proposal will cover all aspects of the tomography proposed using high-end TEMs, including an aberration-corrected Titan, and a Helios dual beam. Importantly, a postdoc is dedicated to developing new algorithms for reconstruction, image and spectral processing."
Summary
"Understanding structure-property relationships across lengthscales is key to the design of functional and structural materials and devices. Moreover, the complexity of modern devices extends to three dimensions and as such 3D characterization is required across those lengthscales to provide a complete understanding and enable improvement in the material’s physical and chemical behaviour. 3D imaging and analysis from the atomic scale through to granular microstructure is proposed through the development of electron tomography using (S)TEM, and ‘dual beam’ SEM-FIB, techniques offering complementary approaches to 3D imaging across lengthscales stretching over 5 orders of magnitude.
We propose to extend tomography to include novel methods to determine atom positions in 3D with approaches incorporating new reconstruction algorithms, image processing and complementary nano-diffraction techniques. At the nanoscale, true 3D nano-metrology of morphology and composition is a key objective of the project, minimizing reconstruction and visualization artefacts. Mapping strain and optical properties in 3D are ambitious and exciting challenges that will yield new information at the nanoscale. Using the SEM-FIB, 3D ‘mesoscale’ structures will be revealed: morphology, crystallography and composition can be mapped simultaneously, with ~5nm resolution and over volumes too large to tackle by (S)TEM and too small for most x-ray techniques. In parallel, we will apply 3D imaging to a wide variety of key materials including heterogeneous catalysts, aerospace alloys, biomaterials, photovoltaic materials, and novel semiconductors.
We will collaborate with many departments in Cambridge and institutes worldwide. The personnel on the proposal will cover all aspects of the tomography proposed using high-end TEMs, including an aberration-corrected Titan, and a Helios dual beam. Importantly, a postdoc is dedicated to developing new algorithms for reconstruction, image and spectral processing."
Max ERC Funding
2 337 330 €
Duration
Start date: 2012-01-01, End date: 2017-12-31
Project acronym 3DNANOMECH
Project Three-dimensional molecular resolution mapping of soft matter-liquid interfaces
Researcher (PI) Ricardo Garcia
Host Institution (HI) AGENCIA ESTATAL CONSEJO SUPERIOR DEINVESTIGACIONES CIENTIFICAS
Call Details Advanced Grant (AdG), PE4, ERC-2013-ADG
Summary Optical, electron and probe microscopes are enabling tools for discoveries and knowledge generation in nanoscale sicence and technology. High resolution –nanoscale or molecular-, noninvasive and label-free imaging of three-dimensional soft matter-liquid interfaces has not been achieved by any microscopy method.
Force microscopy (AFM) is considered the second most relevant advance in materials science since 1960. Despite its impressive range of applications, the technique has some key limitations. Force microscopy has not three dimensional depth. What lies above or in the subsurface is not readily characterized.
3DNanoMech proposes to design, build and operate a high speed force-based method for the three-dimensional characterization soft matter-liquid interfaces (3D AFM). The microscope will combine a detection method based on force perturbations, adaptive algorithms, high speed piezo actuators and quantitative-oriented multifrequency approaches. The development of the microscope cannot be separated from its applications: imaging the error-free DNA repair and to understand the relationship existing between the nanomechanical properties and the malignancy of cancer cells. Those problems encompass the different spatial –molecular-nano-mesoscopic- and time –milli to seconds- scales of the instrument.
In short, 3DNanoMech aims to image, map and measure with picoNewton, millisecond and angstrom resolution soft matter surfaces and interfaces in liquid. The long-term vision of 3DNanoMech is to replace models or computer animations of bimolecular-liquid interfaces by real time, molecular resolution maps of properties and processes.
Summary
Optical, electron and probe microscopes are enabling tools for discoveries and knowledge generation in nanoscale sicence and technology. High resolution –nanoscale or molecular-, noninvasive and label-free imaging of three-dimensional soft matter-liquid interfaces has not been achieved by any microscopy method.
Force microscopy (AFM) is considered the second most relevant advance in materials science since 1960. Despite its impressive range of applications, the technique has some key limitations. Force microscopy has not three dimensional depth. What lies above or in the subsurface is not readily characterized.
3DNanoMech proposes to design, build and operate a high speed force-based method for the three-dimensional characterization soft matter-liquid interfaces (3D AFM). The microscope will combine a detection method based on force perturbations, adaptive algorithms, high speed piezo actuators and quantitative-oriented multifrequency approaches. The development of the microscope cannot be separated from its applications: imaging the error-free DNA repair and to understand the relationship existing between the nanomechanical properties and the malignancy of cancer cells. Those problems encompass the different spatial –molecular-nano-mesoscopic- and time –milli to seconds- scales of the instrument.
In short, 3DNanoMech aims to image, map and measure with picoNewton, millisecond and angstrom resolution soft matter surfaces and interfaces in liquid. The long-term vision of 3DNanoMech is to replace models or computer animations of bimolecular-liquid interfaces by real time, molecular resolution maps of properties and processes.
Max ERC Funding
2 499 928 €
Duration
Start date: 2014-02-01, End date: 2019-01-31
Project acronym 3DPROTEINPUZZLES
Project Shape-directed protein assembly design
Researcher (PI) Lars Ingemar ANDRÉ
Host Institution (HI) LUNDS UNIVERSITET
Call Details Consolidator Grant (CoG), LS9, ERC-2017-COG
Summary Large protein complexes carry out some of the most complex functions in biology. Such structures are often assembled spontaneously from individual components through the process of self-assembly. If self-assembled protein complexes could be engineered from first principle it would enable a wide range of applications in biomedicine, nanotechnology and materials science. Recently, approaches to rationally design proteins to self-assembly into predefined structures have emerged. The highlight of this work is the design of protein cages that may be engineered into protein containers. However, current approaches for self-assembly design does not result in the assemblies with the required structural complexity to encode many of the sophisticated functions found in nature. To move forward, we have to learn how to engineer protein subunits with more than one designed interface that can assemble into tightly interacting complexes. In this proposal we propose a new protein design paradigm, shape directed protein design, in order to address shortcomings of the current methodology. The proposed method combines geometric shape matching and computational protein design. Using this approach we will de novo design assemblies with a wide variety of structural states, including protein complexes with cyclic and dihedral symmetry as well as icosahedral protein capsids built from novel protein building blocks. To enable these two design challenges we also develop a high-throughput assay to measure assembly stability in vivo that builds on a three-color fluorescent assay. This method will not only facilitate the screening of orders of magnitude more design constructs, but also enable the application of directed evolution to experimentally improve stable and assembly properties of designed containers as well as other designed assemblies.
Summary
Large protein complexes carry out some of the most complex functions in biology. Such structures are often assembled spontaneously from individual components through the process of self-assembly. If self-assembled protein complexes could be engineered from first principle it would enable a wide range of applications in biomedicine, nanotechnology and materials science. Recently, approaches to rationally design proteins to self-assembly into predefined structures have emerged. The highlight of this work is the design of protein cages that may be engineered into protein containers. However, current approaches for self-assembly design does not result in the assemblies with the required structural complexity to encode many of the sophisticated functions found in nature. To move forward, we have to learn how to engineer protein subunits with more than one designed interface that can assemble into tightly interacting complexes. In this proposal we propose a new protein design paradigm, shape directed protein design, in order to address shortcomings of the current methodology. The proposed method combines geometric shape matching and computational protein design. Using this approach we will de novo design assemblies with a wide variety of structural states, including protein complexes with cyclic and dihedral symmetry as well as icosahedral protein capsids built from novel protein building blocks. To enable these two design challenges we also develop a high-throughput assay to measure assembly stability in vivo that builds on a three-color fluorescent assay. This method will not only facilitate the screening of orders of magnitude more design constructs, but also enable the application of directed evolution to experimentally improve stable and assembly properties of designed containers as well as other designed assemblies.
Max ERC Funding
2 325 292 €
Duration
Start date: 2018-06-01, End date: 2023-05-31
Project acronym 3MC
Project 3D Model Catalysts to explore new routes to sustainable fuels
Researcher (PI) Petra Elisabeth De jongh
Host Institution (HI) UNIVERSITEIT UTRECHT
Call Details Consolidator Grant (CoG), PE4, ERC-2014-CoG
Summary Currently fuels, plastics, and drugs are predominantly manufactured from oil. A transition towards renewable resources critically depends on new catalysts, for instance to convert small molecules (such as solar or biomass derived hydrogen, carbon monoxide, water and carbon dioxide) into more complex ones (such as oxygenates, containing oxygen atoms in their structure). Catalyst development now often depends on trial and error rather than rational design, as the heterogeneity of these composite systems hampers detailed understanding of the role of each of the components.
I propose 3D model catalysts as a novel enabling tool to overcome this problem. Their well-defined nature allows unprecedented precision in the variation of structural parameters (morphology, spatial distribution) of the individual components, while at the same time they mimic real catalysts closely enough to allow testing under industrially relevant conditions. Using this approach I will address fundamental questions, such as:
* What are the mechanisms (structural, electronic, chemical) by which non-metal promoters influence the functionality of copper-based catalysts?
* Which nanoalloys can be formed, how does their composition influence the surface active sites and catalytic functionality under reaction conditions?
* Which size and interface effects occur, and how can we use them to tune the actitivity and selectivity towards desired products?
Our 3D model catalysts will be assembled from ordered mesoporous silica and carbon support materials and Cu-based promoted and bimetallic nanoparticles. The combination with high resolution characterization and testing under realistic conditions allows detailed insight into the role of the different components; critical for the rational design of novel catalysts for a future more sustainable production of chemicals and fuels from renewable resources.
Summary
Currently fuels, plastics, and drugs are predominantly manufactured from oil. A transition towards renewable resources critically depends on new catalysts, for instance to convert small molecules (such as solar or biomass derived hydrogen, carbon monoxide, water and carbon dioxide) into more complex ones (such as oxygenates, containing oxygen atoms in their structure). Catalyst development now often depends on trial and error rather than rational design, as the heterogeneity of these composite systems hampers detailed understanding of the role of each of the components.
I propose 3D model catalysts as a novel enabling tool to overcome this problem. Their well-defined nature allows unprecedented precision in the variation of structural parameters (morphology, spatial distribution) of the individual components, while at the same time they mimic real catalysts closely enough to allow testing under industrially relevant conditions. Using this approach I will address fundamental questions, such as:
* What are the mechanisms (structural, electronic, chemical) by which non-metal promoters influence the functionality of copper-based catalysts?
* Which nanoalloys can be formed, how does their composition influence the surface active sites and catalytic functionality under reaction conditions?
* Which size and interface effects occur, and how can we use them to tune the actitivity and selectivity towards desired products?
Our 3D model catalysts will be assembled from ordered mesoporous silica and carbon support materials and Cu-based promoted and bimetallic nanoparticles. The combination with high resolution characterization and testing under realistic conditions allows detailed insight into the role of the different components; critical for the rational design of novel catalysts for a future more sustainable production of chemicals and fuels from renewable resources.
Max ERC Funding
1 999 625 €
Duration
Start date: 2015-09-01, End date: 2020-08-31
Project acronym 3S-BTMUC
Project Soft, Slimy, Sliding Interfaces: Biotribological Properties of Mucins and Mucus gels
Researcher (PI) Seunghwan Lee
Host Institution (HI) DANMARKS TEKNISKE UNIVERSITET
Call Details Starting Grant (StG), LS9, ERC-2010-StG_20091118
Summary Mucins are a family of high-molecular-weight glycoproteins and a major macromolecular constituent in slimy mucus gels that are covering the surface of internal biological tissues. A primary role of mucus gels in biological systems is known to be the protection and lubrication of underlying epithelial cell surfaces. This is intuitively well appreciated by both science community and the public, and yet detailed lubrication properties of mucins and mucus gels have remained largely unexplored to date. Detailed and systematic understanding of the lubrication mechanism of mucus gels is significant from many angles; firstly, lubricity of mucus gels is closely related with fundamental functions of various human organs, such as eye blinking, mastication in oral cavity, swallowing through esophagus, digestion in stomach, breathing through air way and respiratory organs, and thus often indicates the health state of those organs. Furthermore, for the application of various tissue-contacting devices or personal care products, e.g. catheters, endoscopes, and contact lenses, mucus gel layer is the first counter surface that comes into the mechanical and tribological contacts with them. Finally, remarkable lubricating performance by mucins and mucus gels in biological systems may provide many useful and possibly innovative hints in utilizing water as base lubricant for man-made engineering systems. This project thus proposes to carry out a 5 year research program focusing on exploring the lubricity of mucins and mucus gels by combining a broad range of experimental approaches in biology and tribology.
Summary
Mucins are a family of high-molecular-weight glycoproteins and a major macromolecular constituent in slimy mucus gels that are covering the surface of internal biological tissues. A primary role of mucus gels in biological systems is known to be the protection and lubrication of underlying epithelial cell surfaces. This is intuitively well appreciated by both science community and the public, and yet detailed lubrication properties of mucins and mucus gels have remained largely unexplored to date. Detailed and systematic understanding of the lubrication mechanism of mucus gels is significant from many angles; firstly, lubricity of mucus gels is closely related with fundamental functions of various human organs, such as eye blinking, mastication in oral cavity, swallowing through esophagus, digestion in stomach, breathing through air way and respiratory organs, and thus often indicates the health state of those organs. Furthermore, for the application of various tissue-contacting devices or personal care products, e.g. catheters, endoscopes, and contact lenses, mucus gel layer is the first counter surface that comes into the mechanical and tribological contacts with them. Finally, remarkable lubricating performance by mucins and mucus gels in biological systems may provide many useful and possibly innovative hints in utilizing water as base lubricant for man-made engineering systems. This project thus proposes to carry out a 5 year research program focusing on exploring the lubricity of mucins and mucus gels by combining a broad range of experimental approaches in biology and tribology.
Max ERC Funding
1 432 920 €
Duration
Start date: 2011-04-01, End date: 2016-03-31
Project acronym a SMILE
Project analyse Soluble + Membrane complexes with Improved LILBID Experiments
Researcher (PI) Nina Morgner
Host Institution (HI) JOHANN WOLFGANG GOETHE-UNIVERSITATFRANKFURT AM MAIN
Call Details Starting Grant (StG), PE4, ERC-2013-StG
Summary Crucial processes within cells depend on specific non-covalent interactions which mediate the assembly of proteins and other biomolecules. Deriving structural information to understand the function of these complex systems is the primary goal of Structural Biology.
In this application, the recently developed LILBID method (Laser Induced Liquid Bead Ion Desorption) will be optimized for investigation of macromolecular complexes with a mass accuracy two orders of magnitude better than in 1st generation spectrometers.
Controlled disassembly of the multiprotein complexes in the mass spectrometric analysis while keeping the 3D structure intact, will allow for the determination of complex stoichiometry and connectivity of the constituting proteins. Methods for such controlled disassembly will be developed in two separate units of the proposed LILBID spectrometer, in a collision chamber and in a laser dissociation chamber, enabling gas phase dissociation of protein complexes and removal of excess water/buffer molecules. As a third unit, a chamber allowing determination of ion mobility (IM) will be integrated to determine collisional cross sections (CCS). From CCS, unique information regarding the spatial arrangement of proteins in complexes or subcomplexes will then be obtainable from LILBID.
The proposed design of the new spectrometer will offer fundamentally new possibilities for the investigation of non-covalent RNA, soluble and membrane protein complexes, as well as broadening the applicability of non-covalent MS towards supercomplexes.
Summary
Crucial processes within cells depend on specific non-covalent interactions which mediate the assembly of proteins and other biomolecules. Deriving structural information to understand the function of these complex systems is the primary goal of Structural Biology.
In this application, the recently developed LILBID method (Laser Induced Liquid Bead Ion Desorption) will be optimized for investigation of macromolecular complexes with a mass accuracy two orders of magnitude better than in 1st generation spectrometers.
Controlled disassembly of the multiprotein complexes in the mass spectrometric analysis while keeping the 3D structure intact, will allow for the determination of complex stoichiometry and connectivity of the constituting proteins. Methods for such controlled disassembly will be developed in two separate units of the proposed LILBID spectrometer, in a collision chamber and in a laser dissociation chamber, enabling gas phase dissociation of protein complexes and removal of excess water/buffer molecules. As a third unit, a chamber allowing determination of ion mobility (IM) will be integrated to determine collisional cross sections (CCS). From CCS, unique information regarding the spatial arrangement of proteins in complexes or subcomplexes will then be obtainable from LILBID.
The proposed design of the new spectrometer will offer fundamentally new possibilities for the investigation of non-covalent RNA, soluble and membrane protein complexes, as well as broadening the applicability of non-covalent MS towards supercomplexes.
Max ERC Funding
1 264 477 €
Duration
Start date: 2014-02-01, End date: 2019-01-31
Project acronym A-LIFE
Project The asymmetry of life: towards a unified view of the emergence of biological homochirality
Researcher (PI) Cornelia MEINERT
Host Institution (HI) CENTRE NATIONAL DE LA RECHERCHE SCIENTIFIQUE CNRS
Call Details Starting Grant (StG), PE4, ERC-2018-STG
Summary What is responsible for the emergence of homochirality, the almost exclusive use of one enantiomer over its mirror image? And what led to the evolution of life’s homochiral biopolymers, DNA/RNA, proteins and lipids, where all the constituent monomers exhibit the same handedness?
Based on in-situ observations and laboratory studies, we propose that this handedness occurs when chiral biomolecules are synthesized asymmetrically through interaction with circularly polarized photons in interstellar space. The ultimate goal of this project will be to demonstrate how the diverse set of heterogeneous enantioenriched molecules, available from meteoritic impact, assembles into homochiral pre-biopolymers, by simulating the evolutionary stages on early Earth. My recent research has shown that the central chiral unit of RNA, ribose, forms readily under simulated comet conditions and this has provided valuable new insights into the accessibility of precursors of genetic material in interstellar environments. The significance of this project arises due to the current lack of experimental demonstration that amino acids, sugars and lipids can simultaneously and asymmetrically be synthesized by a universal physical selection process.
A synergistic methodology will be developed to build a unified theory for the origin of all chiral biological building blocks and their assembly into homochiral supramolecular entities. For the first time, advanced analyses of astrophysical-relevant samples, asymmetric photochemistry triggered by circularly polarized synchrotron and laser sources, and chiral amplification due to polymerization processes will be combined. Intermediates and autocatalytic reaction kinetics will be monitored and supported by quantum calculations to understand the underlying processes. A unified theory on the asymmetric formation and self-assembly of life’s biopolymers is groundbreaking and will impact the whole conceptual foundation of the origin of life.
Summary
What is responsible for the emergence of homochirality, the almost exclusive use of one enantiomer over its mirror image? And what led to the evolution of life’s homochiral biopolymers, DNA/RNA, proteins and lipids, where all the constituent monomers exhibit the same handedness?
Based on in-situ observations and laboratory studies, we propose that this handedness occurs when chiral biomolecules are synthesized asymmetrically through interaction with circularly polarized photons in interstellar space. The ultimate goal of this project will be to demonstrate how the diverse set of heterogeneous enantioenriched molecules, available from meteoritic impact, assembles into homochiral pre-biopolymers, by simulating the evolutionary stages on early Earth. My recent research has shown that the central chiral unit of RNA, ribose, forms readily under simulated comet conditions and this has provided valuable new insights into the accessibility of precursors of genetic material in interstellar environments. The significance of this project arises due to the current lack of experimental demonstration that amino acids, sugars and lipids can simultaneously and asymmetrically be synthesized by a universal physical selection process.
A synergistic methodology will be developed to build a unified theory for the origin of all chiral biological building blocks and their assembly into homochiral supramolecular entities. For the first time, advanced analyses of astrophysical-relevant samples, asymmetric photochemistry triggered by circularly polarized synchrotron and laser sources, and chiral amplification due to polymerization processes will be combined. Intermediates and autocatalytic reaction kinetics will be monitored and supported by quantum calculations to understand the underlying processes. A unified theory on the asymmetric formation and self-assembly of life’s biopolymers is groundbreaking and will impact the whole conceptual foundation of the origin of life.
Max ERC Funding
1 500 000 €
Duration
Start date: 2019-04-01, End date: 2024-03-31
Project acronym AAMDDR
Project DNA damage response and genome stability: The role of ATM, ATR and the Mre11 complex
Researcher (PI) Vincenzo Costanzo
Host Institution (HI) CANCER RESEARCH UK LBG
Call Details Starting Grant (StG), LS1, ERC-2007-StG
Summary Chromosomal DNA is continuously subjected to exogenous and endogenous damaging insults. In the presence of DNA damage cells activate a multi-faceted checkpoint response that delays cell cycle progression and promotes DNA repair. Failures in this response lead to genomic instability, the main feature of cancer cells. Several cancer-prone human syndromes including the Ataxia teleangiectasia (A-T), the A-T Like Disorder (ATLD) and the Seckel Syndrome reflect defects in the specific genes of the DNA damage response such as ATM, MRE11 and ATR. DNA damage response pathways are poorly understood at biochemical level in vertebrate organisms. We have established a cell-free system based on Xenopus laevis egg extract to study molecular events underlying DNA damage response. This is the first in vitro system that recapitulates different aspects of the DNA damage response in vertebrates. Using this system we propose to study the biochemistry of the ATM, ATR and the Mre11 complex dependent DNA damage response. In particular we will: 1) Dissect the signal transduction pathway that senses DNA damage and promotes cell cycle arrest and DNA damage repair; 2) Analyze at molecular level the role of ATM, ATR, Mre11 in chromosomal DNA replication and mitosis during normal and stressful conditions; 3) Identify substrates of the ATM and ATR dependent DNA damage response using an innovative screening procedure.
Summary
Chromosomal DNA is continuously subjected to exogenous and endogenous damaging insults. In the presence of DNA damage cells activate a multi-faceted checkpoint response that delays cell cycle progression and promotes DNA repair. Failures in this response lead to genomic instability, the main feature of cancer cells. Several cancer-prone human syndromes including the Ataxia teleangiectasia (A-T), the A-T Like Disorder (ATLD) and the Seckel Syndrome reflect defects in the specific genes of the DNA damage response such as ATM, MRE11 and ATR. DNA damage response pathways are poorly understood at biochemical level in vertebrate organisms. We have established a cell-free system based on Xenopus laevis egg extract to study molecular events underlying DNA damage response. This is the first in vitro system that recapitulates different aspects of the DNA damage response in vertebrates. Using this system we propose to study the biochemistry of the ATM, ATR and the Mre11 complex dependent DNA damage response. In particular we will: 1) Dissect the signal transduction pathway that senses DNA damage and promotes cell cycle arrest and DNA damage repair; 2) Analyze at molecular level the role of ATM, ATR, Mre11 in chromosomal DNA replication and mitosis during normal and stressful conditions; 3) Identify substrates of the ATM and ATR dependent DNA damage response using an innovative screening procedure.
Max ERC Funding
1 000 000 €
Duration
Start date: 2008-07-01, End date: 2013-06-30
Project acronym ABACUS
Project Ab-initio adiabatic-connection curves for density-functional analysis and construction
Researcher (PI) Trygve Ulf Helgaker
Host Institution (HI) UNIVERSITETET I OSLO
Call Details Advanced Grant (AdG), PE4, ERC-2010-AdG_20100224
Summary Quantum chemistry provides two approaches to molecular electronic-structure calculations: the systematically refinable but expensive many-body wave-function methods and the inexpensive but not systematically refinable Kohn Sham method of density-functional theory (DFT). The accuracy of Kohn Sham calculations is determined by the quality of the exchange correlation functional, from which the effects of exchange and correlation among the electrons are extracted using the density rather than the wave function. However, the exact exchange correlation functional is unknown—instead, many approximate forms have been developed, by fitting to experimental data or by satisfying exact relations. Here, a new approach to density-functional analysis and construction is proposed: the Lieb variation principle, usually regarded as conceptually important but impracticable. By invoking the Lieb principle, it becomes possible to approach the development of approximate functionals in a novel manner, being directly guided by the behaviour of exact functional, accurately calculated for a wide variety of chemical systems. In particular, this principle will be used to calculate ab-initio adiabatic connection curves, studying the exchange correlation functional for a fixed density as the electronic interactions are turned on from zero to one. Pilot calculations have indicated the feasibility of this approach in simple cases—here, a comprehensive set of adiabatic-connection curves will be generated and utilized for calibration, construction, and analysis of density functionals, the objective being to produce improved functionals for Kohn Sham calculations by modelling or fitting such curves. The ABACUS approach will be particularly important in cases where little experimental information is available—for example, for understanding and modelling the behaviour of the exchange correlation functional in electromagnetic fields.
Summary
Quantum chemistry provides two approaches to molecular electronic-structure calculations: the systematically refinable but expensive many-body wave-function methods and the inexpensive but not systematically refinable Kohn Sham method of density-functional theory (DFT). The accuracy of Kohn Sham calculations is determined by the quality of the exchange correlation functional, from which the effects of exchange and correlation among the electrons are extracted using the density rather than the wave function. However, the exact exchange correlation functional is unknown—instead, many approximate forms have been developed, by fitting to experimental data or by satisfying exact relations. Here, a new approach to density-functional analysis and construction is proposed: the Lieb variation principle, usually regarded as conceptually important but impracticable. By invoking the Lieb principle, it becomes possible to approach the development of approximate functionals in a novel manner, being directly guided by the behaviour of exact functional, accurately calculated for a wide variety of chemical systems. In particular, this principle will be used to calculate ab-initio adiabatic connection curves, studying the exchange correlation functional for a fixed density as the electronic interactions are turned on from zero to one. Pilot calculations have indicated the feasibility of this approach in simple cases—here, a comprehensive set of adiabatic-connection curves will be generated and utilized for calibration, construction, and analysis of density functionals, the objective being to produce improved functionals for Kohn Sham calculations by modelling or fitting such curves. The ABACUS approach will be particularly important in cases where little experimental information is available—for example, for understanding and modelling the behaviour of the exchange correlation functional in electromagnetic fields.
Max ERC Funding
2 017 932 €
Duration
Start date: 2011-03-01, End date: 2016-02-29
Project acronym ABCTRANSPORT
Project Minimalist multipurpose ATP-binding cassette transporters
Researcher (PI) Dirk Jan Slotboom
Host Institution (HI) RIJKSUNIVERSITEIT GRONINGEN
Call Details Starting Grant (StG), LS1, ERC-2011-StG_20101109
Summary Many Gram-positive (pathogenic) bacteria are dependent on the uptake of vitamins from the environment or from the infected host. We have recently discovered the long-elusive family of membrane protein complexes catalyzing such transport. The vitamin transporters have an unprecedented modular architecture consisting of a single multipurpose energizing module (the Energy Coupling Factor, ECF) and multiple exchangeable membrane proteins responsible for substrate recognition (S-components). The S-components have characteristics of ion-gradient driven transporters (secondary active transporters), whereas the energizing modules are related to ATP-binding cassette (ABC) transporters (primary active transporters).
The aim of the proposal is threefold: First, we will address the question how properties of primary and secondary transporters are combined in ECF transporters to obtain a novel transport mechanism. Second, we will study the fundamental and unresolved question how protein-protein recognition takes place in the hydrophobic environment of the lipid bilayer. The modular nature of the ECF proteins offers a natural system to study the driving forces used for membrane protein interaction. Third, we will assess whether the ECF transport systems could become targets for antibacterial drugs. ECF transporters are found exclusively in prokaryotes, and their activity is often essential for viability of Gram-positive pathogens. Thus they could turn out to be an Achilles’ heel for the organisms.
Structural and mechanistic studies (X-ray crystallography, microscopy, spectroscopy and biochemistry) will reveal how the different transport modes are combined in a single protein complex, how transport is energized and catalyzed, and how protein-protein recognition takes place. Microbiological screens will be developed to search for compounds that inhibit prokaryote-specific steps of the mechanism of ECF transporters.
Summary
Many Gram-positive (pathogenic) bacteria are dependent on the uptake of vitamins from the environment or from the infected host. We have recently discovered the long-elusive family of membrane protein complexes catalyzing such transport. The vitamin transporters have an unprecedented modular architecture consisting of a single multipurpose energizing module (the Energy Coupling Factor, ECF) and multiple exchangeable membrane proteins responsible for substrate recognition (S-components). The S-components have characteristics of ion-gradient driven transporters (secondary active transporters), whereas the energizing modules are related to ATP-binding cassette (ABC) transporters (primary active transporters).
The aim of the proposal is threefold: First, we will address the question how properties of primary and secondary transporters are combined in ECF transporters to obtain a novel transport mechanism. Second, we will study the fundamental and unresolved question how protein-protein recognition takes place in the hydrophobic environment of the lipid bilayer. The modular nature of the ECF proteins offers a natural system to study the driving forces used for membrane protein interaction. Third, we will assess whether the ECF transport systems could become targets for antibacterial drugs. ECF transporters are found exclusively in prokaryotes, and their activity is often essential for viability of Gram-positive pathogens. Thus they could turn out to be an Achilles’ heel for the organisms.
Structural and mechanistic studies (X-ray crystallography, microscopy, spectroscopy and biochemistry) will reveal how the different transport modes are combined in a single protein complex, how transport is energized and catalyzed, and how protein-protein recognition takes place. Microbiological screens will be developed to search for compounds that inhibit prokaryote-specific steps of the mechanism of ECF transporters.
Max ERC Funding
1 500 000 €
Duration
Start date: 2012-01-01, End date: 2017-12-31
Project acronym ABCvolume
Project The ABC of Cell Volume Regulation
Researcher (PI) Berend Poolman
Host Institution (HI) RIJKSUNIVERSITEIT GRONINGEN
Call Details Advanced Grant (AdG), LS1, ERC-2014-ADG
Summary Cell volume regulation is crucial for any living cell because changes in volume determine the metabolic activity through e.g. changes in ionic strength, pH, macromolecular crowding and membrane tension. These physical chemical parameters influence interaction rates and affinities of biomolecules, folding rates, and fold stabilities in vivo. Understanding of the underlying volume regulatory mechanisms has immediate application in biotechnology and health, yet these factors are generally ignored in systems analyses of cellular functions.
My team has uncovered a number of mechanisms and insights of cell volume regulation. The next step forward is to elucidate how the components of a cell volume regulatory circuit work together and control the physicochemical conditions of the cell.
I propose construction of a synthetic cell in which an osmoregulatory transporter and mechanosensitive channel form a minimal volume regulatory network. My group has developed the technology to reconstitute membrane proteins into lipid vesicles (synthetic cells). One of the challenges is to incorporate into the vesicles an efficient pathway for ATP production and maintain energy homeostasis while the load on the system varies. We aim to control the transmembrane flux of osmolytes, which requires elucidation of the molecular mechanism of gating of the osmoregulatory transporter. We will focus on the glycine betaine ABC importer, which is one of the most complex transporters known to date with ten distinct protein domains, transiently interacting with each other.
The proposed synthetic metabolic circuit constitutes a fascinating out-of-equilibrium system, allowing us to understand cell volume regulatory mechanisms in a context and at a level of complexity minimally needed for life. Analysis of this circuit will address many outstanding questions and eventually allow us to design more sophisticated vesicular systems with applications, for example as compartmentalized reaction networks.
Summary
Cell volume regulation is crucial for any living cell because changes in volume determine the metabolic activity through e.g. changes in ionic strength, pH, macromolecular crowding and membrane tension. These physical chemical parameters influence interaction rates and affinities of biomolecules, folding rates, and fold stabilities in vivo. Understanding of the underlying volume regulatory mechanisms has immediate application in biotechnology and health, yet these factors are generally ignored in systems analyses of cellular functions.
My team has uncovered a number of mechanisms and insights of cell volume regulation. The next step forward is to elucidate how the components of a cell volume regulatory circuit work together and control the physicochemical conditions of the cell.
I propose construction of a synthetic cell in which an osmoregulatory transporter and mechanosensitive channel form a minimal volume regulatory network. My group has developed the technology to reconstitute membrane proteins into lipid vesicles (synthetic cells). One of the challenges is to incorporate into the vesicles an efficient pathway for ATP production and maintain energy homeostasis while the load on the system varies. We aim to control the transmembrane flux of osmolytes, which requires elucidation of the molecular mechanism of gating of the osmoregulatory transporter. We will focus on the glycine betaine ABC importer, which is one of the most complex transporters known to date with ten distinct protein domains, transiently interacting with each other.
The proposed synthetic metabolic circuit constitutes a fascinating out-of-equilibrium system, allowing us to understand cell volume regulatory mechanisms in a context and at a level of complexity minimally needed for life. Analysis of this circuit will address many outstanding questions and eventually allow us to design more sophisticated vesicular systems with applications, for example as compartmentalized reaction networks.
Max ERC Funding
2 247 231 €
Duration
Start date: 2015-07-01, End date: 2020-06-30
Project acronym ABDESIGN
Project Computational design of novel protein function in antibodies
Researcher (PI) Sarel-Jacob Fleishman
Host Institution (HI) WEIZMANN INSTITUTE OF SCIENCE
Call Details Starting Grant (StG), LS1, ERC-2013-StG
Summary We propose to elucidate the structural design principles of naturally occurring antibody complementarity-determining regions (CDRs) and to computationally design novel antibody functions. Antibodies represent the most versatile known system for molecular recognition. Research has yielded many insights into antibody design principles and promising biotechnological and pharmaceutical applications. Still, our understanding of how CDRs encode specific loop conformations lags far behind our understanding of structure-function relationships in non-immunological scaffolds. Thus, design of antibodies from first principles has not been demonstrated. We propose a computational-experimental strategy to address this challenge. We will: (a) characterize the design principles and sequence elements that rigidify antibody CDRs. Natural antibody loops will be subjected to computational modeling, crystallography, and a combined in vitro evolution and deep-sequencing approach to isolate sequence features that rigidify loop backbones; (b) develop a novel computational-design strategy, which uses the >1000 solved structures of antibodies deposited in structure databases to realistically model CDRs and design them to recognize proteins that have not been co-crystallized with antibodies. For example, we will design novel antibodies targeting insulin, for which clinically useful diagnostics are needed. By accessing much larger sequence/structure spaces than are available to natural immune-system repertoires and experimental methods, computational antibody design could produce higher-specificity and higher-affinity binders, even to challenging targets; and (c) develop new strategies to program conformational change in CDRs, generating, e.g., the first allosteric antibodies. These will allow targeting, in principle, of any molecule, potentially revolutionizing how antibodies are generated for research and medicine, providing new insights on the design principles of protein functional sites.
Summary
We propose to elucidate the structural design principles of naturally occurring antibody complementarity-determining regions (CDRs) and to computationally design novel antibody functions. Antibodies represent the most versatile known system for molecular recognition. Research has yielded many insights into antibody design principles and promising biotechnological and pharmaceutical applications. Still, our understanding of how CDRs encode specific loop conformations lags far behind our understanding of structure-function relationships in non-immunological scaffolds. Thus, design of antibodies from first principles has not been demonstrated. We propose a computational-experimental strategy to address this challenge. We will: (a) characterize the design principles and sequence elements that rigidify antibody CDRs. Natural antibody loops will be subjected to computational modeling, crystallography, and a combined in vitro evolution and deep-sequencing approach to isolate sequence features that rigidify loop backbones; (b) develop a novel computational-design strategy, which uses the >1000 solved structures of antibodies deposited in structure databases to realistically model CDRs and design them to recognize proteins that have not been co-crystallized with antibodies. For example, we will design novel antibodies targeting insulin, for which clinically useful diagnostics are needed. By accessing much larger sequence/structure spaces than are available to natural immune-system repertoires and experimental methods, computational antibody design could produce higher-specificity and higher-affinity binders, even to challenging targets; and (c) develop new strategies to program conformational change in CDRs, generating, e.g., the first allosteric antibodies. These will allow targeting, in principle, of any molecule, potentially revolutionizing how antibodies are generated for research and medicine, providing new insights on the design principles of protein functional sites.
Max ERC Funding
1 499 930 €
Duration
Start date: 2013-09-01, End date: 2018-08-31
Project acronym ABEL
Project "Alpha-helical Barrels: Exploring, Understanding and Exploiting a New Class of Protein Structure"
Researcher (PI) Derek Neil Woolfson
Host Institution (HI) UNIVERSITY OF BRISTOL
Call Details Advanced Grant (AdG), LS9, ERC-2013-ADG
Summary "Recently through de novo peptide design, we have discovered and presented a new protein structure. This is an all-parallel, 6-helix bundle with a continuous central channel of 0.5 – 0.6 nm diameter. We posit that this is one of a broader class of protein structures that we call the alpha-helical barrels. Here, in three Work Packages, we propose to explore these structures and to develop protein functions within them. First, through a combination of computer-aided design, peptide synthesis and thorough biophysical characterization, we will examine the extents and limits of the alpha-helical-barrel structures. Whilst this is curiosity driven research, it also has practical consequences for the studies that will follow; that is, alpha-helical barrels made from increasing numbers of helices have channels or pores that increase in a predictable way. Second, we will use rational and empirical design approaches to engineer a range of functions within these cavities, including binding capabilities and enzyme-like activities. Finally, and taking the programme into another ambitious area, we will use the alpha-helical barrels to template other folds that are otherwise difficult to design and engineer, notably beta-barrels that insert into membranes to render ion-channel and sensor functions."
Summary
"Recently through de novo peptide design, we have discovered and presented a new protein structure. This is an all-parallel, 6-helix bundle with a continuous central channel of 0.5 – 0.6 nm diameter. We posit that this is one of a broader class of protein structures that we call the alpha-helical barrels. Here, in three Work Packages, we propose to explore these structures and to develop protein functions within them. First, through a combination of computer-aided design, peptide synthesis and thorough biophysical characterization, we will examine the extents and limits of the alpha-helical-barrel structures. Whilst this is curiosity driven research, it also has practical consequences for the studies that will follow; that is, alpha-helical barrels made from increasing numbers of helices have channels or pores that increase in a predictable way. Second, we will use rational and empirical design approaches to engineer a range of functions within these cavities, including binding capabilities and enzyme-like activities. Finally, and taking the programme into another ambitious area, we will use the alpha-helical barrels to template other folds that are otherwise difficult to design and engineer, notably beta-barrels that insert into membranes to render ion-channel and sensor functions."
Max ERC Funding
2 467 844 €
Duration
Start date: 2014-02-01, End date: 2019-01-31
