These scripts demonstrate the use of IMP, MODELLER, FoXS, and AllosMod, and Minimal Ensemble Sear... more These scripts demonstrate the use of IMP, MODELLER, FoXS, and AllosMod, and Minimal Ensemble Search in the modeling of the Nup133 protein in the <em>S. cerevisiae</em> Nuclear Pore Complex (NPC). First, MODELLER is used to generate an initial comparative model of Nup133 guided by FoXS fits to SAXS data. Then, the model was subjected to conformational sampling with AllosMod, and finally Minimal Ensemble Search was used to identify four models that together reproduced both the SAXS data and a set of electron microscopy class averages. The final model was also validated against a set of chemical cross-links, that were not used in the modeling. <strong>For more information</strong> about how to reproduce this modeling, see the Sali lab website or the README file.
The presence of amyloid fibrils is a hallmark of several neurodegenerative diseases. Some amyloid... more The presence of amyloid fibrils is a hallmark of several neurodegenerative diseases. Some amyloidogenic proteins, such as α-synuclein and amyloid β, can interact with lipids, and this interaction can strongly favor the formation of amyloid fibrils. In particular the primary nucleation step, i.e. the de novo formation of amyloid fibrils, has been shown to be accelerated by lipids. However, the exact mechanism of this acceleration is still mostly unclear. Here we use a range of scattering methods, such as dynamic light scattering (DLS) and small angle X-ray and neutron scattering (SAXS and SANS) to obtain structural information on the binding of α-synuclein to vesicles formed from negatively charged lipids and their co-assembly into amyloid fibrils. We find that the lipid vesicles do not simply act as a surface that catalyses the nucleation reaction, but that lipid molecules take an active role in the reaction. The binding of α-synuclein to the lipid vesicles immediately induces a maj...
Pentameric ligand-gated ion channels (pLGICs) perform electrochemical signal transduction in orga... more Pentameric ligand-gated ion channels (pLGICs) perform electrochemical signal transduction in organisms ranging from bacteria to humans. Among the prokaryotic pLGICs there is architectural diversity involving N-terminal domains (NTDs) not found in eukaryotic relatives, exemplified by the calcium-sensitive channel (DeCLIC) from a Desulfofustis deltaproteobacterium, which has an NTD in addition to the canonical pLGIC structure. Here we have characterized the structure and dynamics of DeCLIC through cryo-electron microscopy (cryo-EM), small-angle neutron scattering (SANS), and molecular dynamics (MD) simulations. In the presence and absence of calcium, cryo-EM yielded structures with alternative conformations of the calcium binding site. SANS profiles further revealed conformational diversity at room temperature beyond that observed in static structures, shown through MD to be largely attributable to rigid body motions of the NTD relative to the protein core, with expanded and asymmetri...
The dense accumulation of α-Synuclein fibrils in neurons is considered to be strongly associated ... more The dense accumulation of α-Synuclein fibrils in neurons is considered to be strongly associated with Parkinson’s disease. These intracellular inclusions, called Lewy bodies, also contain significant amounts of lipids. To better understand such accumulations, it should be important to study α-Synuclein fibril formation under conditions where the fibrils lump together, mimicking what is observed in Lewy bodies. In the present study, we have therefore investigated the overall structural arrangements of α-synuclein fibrils, formed under mildly acidic conditions, pH = 5.5, in pure buffer or in the presence of various model membrane systems, by means of small-angle neutron scattering (SANS). At this pH, α-synuclein fibrils are colloidally unstable and aggregate further into dense clusters. SANS intensities show a power law dependence on the scattering vector,q, indicating that the clusters can be described as mass fractal aggregates. The experimentally observed fractal dimension wasd= 2....
ABSTRACTThe myelin sheath is an essential, multilayered membrane structure that insulates axons, ... more ABSTRACTThe myelin sheath is an essential, multilayered membrane structure that insulates axons, enabling the rapid transmission of nerve impulses. The tetraspan myelin proteolipid protein (PLP) is the most abundant protein of compact myelin in the central nervous system (CNS). The integral membrane protein PLP adheres myelin membranes together and enhances the compaction of myelin, having a fundamental role in myelin stability and axonal support. PLP is linked to severe CNS neuropathies, including inherited Pelizaeus-Merzbacher disease and spastic paraplegia type 2, as well as multiple sclerosis. Nevertheless, the structure, lipid interaction properties, and membrane organization mechanisms of PLP have remained unidentified. We expressed, purified, and structurally characterized human PLP and its shorter isoform DM20. Synchrotron radiation circular dichroism spectroscopy and small-angle X-ray and neutron scattering revealed a dimeric, α-helical conformation for both PLP and DM20 in...
Ferritin, a spherically shaped protein complex, is responsible for iron storage in bacteria, plan... more Ferritin, a spherically shaped protein complex, is responsible for iron storage in bacteria, plants, animals, and humans. Various ferritin iron core compositions in organisms are associated with specific living requirements, health state, and different biochemical roles of ferritin isomers. Magnetoferritin, a synthetic ferritin derivative, serves as an artificial model system of unusual iron phase structures found in humans. We present the results of a complex structural study of magnetoferritins prepared by controlled in vitro synthesis. Using various complementary methods, it was observed that manipulation of the synthesis technology can improve the physicochemical parameters of the system, which is useful in applications. Thus, a higher synthesis temperature leads to an increase in magnetization due to the formation of the magnetite phase. An increase in the iron loading factor has a more pronounced impact on the protein shell structure in comparison with the pH of the aqueous me...
Small-angle neutron scattering (SANS), combined with macromolecular deuteration and solvent contr... more Small-angle neutron scattering (SANS), combined with macromolecular deuteration and solvent contrast variation (H2O/D2O exchange) allows focussing selectively on the signal of specific proteins in multi-protein complexes or mixtures of isolated proteins. We illustrate this unique capacity by the example of a functional protein-degradation system in solution, the PAN-20S proteasome complex in the presence of a protein substrate, ssrA-tagged GFP. By comparing experimental SANS data with synthetic SAXS (small-angle X-ray scattering) data, predicted for the same system under identical conditions, we show that SANS, when combined with macromolecular deuteration and solvent contrast variation, can specifically focus on the conformation of the PAN unfoldase, even in the presence of very large GFP aggregates. Likewise, structural information of native GFP states can be visualized in detail, even in the presence of the much larger PAN-20S unfoldase-protease oligomers, which would dominate th...
Exploiting small-angle X-ray and neutron scattering (SAXS/SANS) on the same sample volume at the ... more Exploiting small-angle X-ray and neutron scattering (SAXS/SANS) on the same sample volume at the same time provides complementary nanoscale structural information in two different contrast situations. Unlike an independent experimental approach, the truly combined SAXS/SANS experimental approach ensures the exactness of the probed samples, particularly for in situ studies. Here, an advanced portable SAXS system that is dimensionally suitable for installation in the D22 zone of ILL is introduced. The SAXS apparatus is based on a Rigaku switchable copper/molybdenum microfocus rotating-anode X-ray generator and a DECTRIS detector with a changeable sample-to-detector distance of up to 1.6 m in a vacuum chamber. A case study is presented to demonstrate the uniqueness of the newly established method. Temporal structural rearrangements of both the organic stabilizing agent and organically capped gold colloidal particles during gold nanoparticle growth are simultaneously probed, enabling th...
By using wide-angle X-ray scattering (WAXS), small-angle neutron scattering, and theoretical scat... more By using wide-angle X-ray scattering (WAXS), small-angle neutron scattering, and theoretical scattering function simulation, we have clarified the effect of glycerol on both the thermal structure transition and the hydration-shell of myoglobin. At the glycerol concentration, ≤ ∼40 % v/v, the decreasing tendency in the maximum dimension and the radius of gyration was observed by X-ray scattering. The neutron scattering result using the inverse contrast variation method directly shows the preservation of the hydration-shell density at the concentration ≤ ∼40 % v/v. This phenomenon is reasonably explained by the preferential exclusion of glycerol from the protein surface to preserve the hydration shell, as suggested by the previous studies. While, at the concentration, ≥ 50 % v/v, the opposite tendency was observed. It suggests the preferential solvation (partial preferential penetration or replacement of glycerol into or with hydration-shell water surrounding the protein surface) occu...
Enabling control over macromolecular ordering and the spatial distribution of structures formed v... more Enabling control over macromolecular ordering and the spatial distribution of structures formed via the mechanisms of molecular self-assembly is a challenge that could yield a range of new functional materials. In particular, using the self-assembly of minimalist peptides, to drive the incorporation of large complex molecules will allow a functionalization strategy for the next generation of biomaterial engineering. Here, for the first time, we show that co-assembly with increasing concentrations of a highly charged polysaccharide, fucoidan, the microscale ordering of Fmoc-FRGDF peptide fibrils and subsequent mechanical properties of the resultant hydrogel can be easily and effectively manipulated without disruption to the nanofibrillar structure of the assembly.
These scripts demonstrate the use of IMP, MODELLER, FoXS, and AllosMod, and Minimal Ensemble Sear... more These scripts demonstrate the use of IMP, MODELLER, FoXS, and AllosMod, and Minimal Ensemble Search in the modeling of the Nup133 protein in the <em>S. cerevisiae</em> Nuclear Pore Complex (NPC). First, MODELLER is used to generate an initial comparative model of Nup133 guided by FoXS fits to SAXS data. Then, the model was subjected to conformational sampling with AllosMod, and finally Minimal Ensemble Search was used to identify four models that together reproduced both the SAXS data and a set of electron microscopy class averages. The final model was also validated against a set of chemical cross-links, that were not used in the modeling. <strong>For more information</strong> about how to reproduce this modeling, see the Sali lab website or the README file.
The presence of amyloid fibrils is a hallmark of several neurodegenerative diseases. Some amyloid... more The presence of amyloid fibrils is a hallmark of several neurodegenerative diseases. Some amyloidogenic proteins, such as α-synuclein and amyloid β, can interact with lipids, and this interaction can strongly favor the formation of amyloid fibrils. In particular the primary nucleation step, i.e. the de novo formation of amyloid fibrils, has been shown to be accelerated by lipids. However, the exact mechanism of this acceleration is still mostly unclear. Here we use a range of scattering methods, such as dynamic light scattering (DLS) and small angle X-ray and neutron scattering (SAXS and SANS) to obtain structural information on the binding of α-synuclein to vesicles formed from negatively charged lipids and their co-assembly into amyloid fibrils. We find that the lipid vesicles do not simply act as a surface that catalyses the nucleation reaction, but that lipid molecules take an active role in the reaction. The binding of α-synuclein to the lipid vesicles immediately induces a maj...
Pentameric ligand-gated ion channels (pLGICs) perform electrochemical signal transduction in orga... more Pentameric ligand-gated ion channels (pLGICs) perform electrochemical signal transduction in organisms ranging from bacteria to humans. Among the prokaryotic pLGICs there is architectural diversity involving N-terminal domains (NTDs) not found in eukaryotic relatives, exemplified by the calcium-sensitive channel (DeCLIC) from a Desulfofustis deltaproteobacterium, which has an NTD in addition to the canonical pLGIC structure. Here we have characterized the structure and dynamics of DeCLIC through cryo-electron microscopy (cryo-EM), small-angle neutron scattering (SANS), and molecular dynamics (MD) simulations. In the presence and absence of calcium, cryo-EM yielded structures with alternative conformations of the calcium binding site. SANS profiles further revealed conformational diversity at room temperature beyond that observed in static structures, shown through MD to be largely attributable to rigid body motions of the NTD relative to the protein core, with expanded and asymmetri...
The dense accumulation of α-Synuclein fibrils in neurons is considered to be strongly associated ... more The dense accumulation of α-Synuclein fibrils in neurons is considered to be strongly associated with Parkinson’s disease. These intracellular inclusions, called Lewy bodies, also contain significant amounts of lipids. To better understand such accumulations, it should be important to study α-Synuclein fibril formation under conditions where the fibrils lump together, mimicking what is observed in Lewy bodies. In the present study, we have therefore investigated the overall structural arrangements of α-synuclein fibrils, formed under mildly acidic conditions, pH = 5.5, in pure buffer or in the presence of various model membrane systems, by means of small-angle neutron scattering (SANS). At this pH, α-synuclein fibrils are colloidally unstable and aggregate further into dense clusters. SANS intensities show a power law dependence on the scattering vector,q, indicating that the clusters can be described as mass fractal aggregates. The experimentally observed fractal dimension wasd= 2....
ABSTRACTThe myelin sheath is an essential, multilayered membrane structure that insulates axons, ... more ABSTRACTThe myelin sheath is an essential, multilayered membrane structure that insulates axons, enabling the rapid transmission of nerve impulses. The tetraspan myelin proteolipid protein (PLP) is the most abundant protein of compact myelin in the central nervous system (CNS). The integral membrane protein PLP adheres myelin membranes together and enhances the compaction of myelin, having a fundamental role in myelin stability and axonal support. PLP is linked to severe CNS neuropathies, including inherited Pelizaeus-Merzbacher disease and spastic paraplegia type 2, as well as multiple sclerosis. Nevertheless, the structure, lipid interaction properties, and membrane organization mechanisms of PLP have remained unidentified. We expressed, purified, and structurally characterized human PLP and its shorter isoform DM20. Synchrotron radiation circular dichroism spectroscopy and small-angle X-ray and neutron scattering revealed a dimeric, α-helical conformation for both PLP and DM20 in...
Ferritin, a spherically shaped protein complex, is responsible for iron storage in bacteria, plan... more Ferritin, a spherically shaped protein complex, is responsible for iron storage in bacteria, plants, animals, and humans. Various ferritin iron core compositions in organisms are associated with specific living requirements, health state, and different biochemical roles of ferritin isomers. Magnetoferritin, a synthetic ferritin derivative, serves as an artificial model system of unusual iron phase structures found in humans. We present the results of a complex structural study of magnetoferritins prepared by controlled in vitro synthesis. Using various complementary methods, it was observed that manipulation of the synthesis technology can improve the physicochemical parameters of the system, which is useful in applications. Thus, a higher synthesis temperature leads to an increase in magnetization due to the formation of the magnetite phase. An increase in the iron loading factor has a more pronounced impact on the protein shell structure in comparison with the pH of the aqueous me...
Small-angle neutron scattering (SANS), combined with macromolecular deuteration and solvent contr... more Small-angle neutron scattering (SANS), combined with macromolecular deuteration and solvent contrast variation (H2O/D2O exchange) allows focussing selectively on the signal of specific proteins in multi-protein complexes or mixtures of isolated proteins. We illustrate this unique capacity by the example of a functional protein-degradation system in solution, the PAN-20S proteasome complex in the presence of a protein substrate, ssrA-tagged GFP. By comparing experimental SANS data with synthetic SAXS (small-angle X-ray scattering) data, predicted for the same system under identical conditions, we show that SANS, when combined with macromolecular deuteration and solvent contrast variation, can specifically focus on the conformation of the PAN unfoldase, even in the presence of very large GFP aggregates. Likewise, structural information of native GFP states can be visualized in detail, even in the presence of the much larger PAN-20S unfoldase-protease oligomers, which would dominate th...
Exploiting small-angle X-ray and neutron scattering (SAXS/SANS) on the same sample volume at the ... more Exploiting small-angle X-ray and neutron scattering (SAXS/SANS) on the same sample volume at the same time provides complementary nanoscale structural information in two different contrast situations. Unlike an independent experimental approach, the truly combined SAXS/SANS experimental approach ensures the exactness of the probed samples, particularly for in situ studies. Here, an advanced portable SAXS system that is dimensionally suitable for installation in the D22 zone of ILL is introduced. The SAXS apparatus is based on a Rigaku switchable copper/molybdenum microfocus rotating-anode X-ray generator and a DECTRIS detector with a changeable sample-to-detector distance of up to 1.6 m in a vacuum chamber. A case study is presented to demonstrate the uniqueness of the newly established method. Temporal structural rearrangements of both the organic stabilizing agent and organically capped gold colloidal particles during gold nanoparticle growth are simultaneously probed, enabling th...
By using wide-angle X-ray scattering (WAXS), small-angle neutron scattering, and theoretical scat... more By using wide-angle X-ray scattering (WAXS), small-angle neutron scattering, and theoretical scattering function simulation, we have clarified the effect of glycerol on both the thermal structure transition and the hydration-shell of myoglobin. At the glycerol concentration, ≤ ∼40 % v/v, the decreasing tendency in the maximum dimension and the radius of gyration was observed by X-ray scattering. The neutron scattering result using the inverse contrast variation method directly shows the preservation of the hydration-shell density at the concentration ≤ ∼40 % v/v. This phenomenon is reasonably explained by the preferential exclusion of glycerol from the protein surface to preserve the hydration shell, as suggested by the previous studies. While, at the concentration, ≥ 50 % v/v, the opposite tendency was observed. It suggests the preferential solvation (partial preferential penetration or replacement of glycerol into or with hydration-shell water surrounding the protein surface) occu...
Enabling control over macromolecular ordering and the spatial distribution of structures formed v... more Enabling control over macromolecular ordering and the spatial distribution of structures formed via the mechanisms of molecular self-assembly is a challenge that could yield a range of new functional materials. In particular, using the self-assembly of minimalist peptides, to drive the incorporation of large complex molecules will allow a functionalization strategy for the next generation of biomaterial engineering. Here, for the first time, we show that co-assembly with increasing concentrations of a highly charged polysaccharide, fucoidan, the microscale ordering of Fmoc-FRGDF peptide fibrils and subsequent mechanical properties of the resultant hydrogel can be easily and effectively manipulated without disruption to the nanofibrillar structure of the assembly.
Uploads