Protéine | EPFL Graph Search

redresse=1.36|vignette|Représentation d'une protéine, ici deux sous-unités d'une molécule d'hémoglobine. On observe les représentées en couleur, ainsi que deux des quatre molécules d'hème, qui sont les groupes prosthétiques caractéristiques de cette protéine. redresse=1.36|vignette|Liaison peptidique –CO–NH– au sein d'un polypeptide. Le motif constitue le squelette de la protéine, tandis que les groupes liés aux sont les chaînes latérales des résidus d'acides aminés. Les protéines sont des macromolécules biologiques présentes dans toutes les cellules vivantes. Ce sont des polymères, formées d'une ou de plusieurs chaînes polypeptidiques. Chacune de ces chaînes est constituée de l'enchaînement de résidus d'acides aminés liés entre eux par des liaisons peptidiques. Les protéines assurent une multitude de fonctions au sein de la cellule vivante et dans les tissus. Ce sont des protéines enzymatiques (enzymes) qui catalysent les réactions chimiques de synthèse et de dégradation nécess

Overexpression of a mutant form of TGFBI/BIGH3 induces retinal degeneration in transgenic mice

Purpose: Despite ubiquitous expression of the keratoepithelin (KE) protein encoded by the transforming growth factor beta induced/beta induced gene human clone 3 (TGFBI/BIGH3) gene, corneal dystrophies are restricted to the cornea, and no other tissues are affected. We investigated the role of TGFBI/BIGH3 in Groenouw corneal dystrophies by generating transgenic mice overexpressing TGFBI/BIGH3 containing the R555W mutation.

2008

Low-energy Electron Holography Microscope for Imaging of Single Molecules

Sven Alexander Szilagyi

Structure determination techniques are an essential tool to investigate and understand molecules, especially in biology, where functionality crucially depends on structure. The dominant high-resolution methods, such as transmission electron microscopy and X-ray diffraction, rely on ensemble averaging, making them unsuitable for evidencing structural variations of flexible molecules. Common single-molecule imaging techniques like scanning probe microscopy achieve high resolution but lack access to 3D objects. A possible way to overcome these limitations is offered by low-energy electron holography (LEEH). So far, LEEH achieved molecular imaging at resolutions in the range of 7-8 Å on the single-molecule level. A resolution in the range of a few Å, which would give access to atomic details of molecular structures, is in theory attainable. Obtaining a resolution near the theoretical limit requires a fully optimized LEEH setup and workflow, for which in particular sample and emitter preparation are crucial. Here, I present a novel LEEH microscope with a unique experimental workflow, which is capable of resolving molecular substructures with an unprecedented resolution for LEEH experiments. The newly constructed LEEH microscope takes advantage of an efficient vibration isolation system and is remote-controlled to minimize external perturbations. The setup is capable of low-temperature (50 K) measurements, which enhances the stability of the system. A reliable way for preparing highly coherent electron emitters constituted by sharp tungsten tips is presented along with different methods for improving their performance via functionalization and UHV treatments. I show an optimized and reliable substrate preparation protocol resulting in ultra-clean free-standing single-layer graphene (SLG) samples. Native electrospray ion beam deposition (ES-IBD) allows for the transfer of non-volatile molecules from solution into gas phase and onto the substrate. By employing a quadrupole mass selection, highly pure molecular ion beams are obtained, which are soft-landed onto the SLG substrate in a controlled and reproducible manner while retaining an intact structure. This versatile preparation method allows us to study a great variety of molecules by LEEH. Data on proteins with masses ranging from 12-820 kDa reveal molecular shapes consistent with model structures. Locally, structural details as small as 5 Å were identified, thus we are approaching our goal of a resolution in the range of a few Å. The application of our methodology to flexible proteins, exemplified by antibodies, allowed for the imaging of their conformational variability and for determining the influence of the sample preparation on their structure. This gave us the possibility to directly image gas-phase related conformations. Data of compact molecular systems with masses below 3 kDa reveal that our LEEH microscope is able to investigate small molecules, such as porphyrines, at high imaging contrast. The presented results indicate that an optimized LEEH setup and workflow in combination with ES-IBD is a versatile microscopy method, which is filling the gap between high-resolution and single-molecule techniques by elucidating structural details of various molecular species.

EPFL2022

Amplitude and phase reconstruction for Low-Energy Electron Holography of individual proteins

Hannah Julia Ochner

Single-molecule imaging methods are of importance in structural biology, and specifically in the imaging of proteins, since they can elucidate conformational variability and structural changes that might be lost in imaging methods relying on averaging processes. Low-energy electron holography (LEEH) is a promising technique for imaging individual proteins with negligible radiation damage, which can be combined with a sample preparation process by native electrospray ion beam deposition (native ES-IBD) ensuring the creation of chemically pure samples suitable for holographic imaging.The central step in the analysis of data measured by LEEH is the numerical reconstruction of the object from the experimentally acquired holograms.Full information about the imaged object consists of both amplitude and phase information imprinted on the scattered wave during the interaction of the electron beam with the molecule and encoded in the hologram created by the interference of the scattered wave and the transmitted incident wave. A propagation-based algorithm with the goal of reconstructing the wave field in the plane of the object is presented and applied to holograms of individual proteins prepared by ES-IBD and measured with a low-energy electron holography microscope. The information retrieved from the reconstruction of the amplitude distribution in the object plane is discussed by analysing holograms of antibodies, demonstrating that the inherent conformational variability of these molecules can be mapped by LEEH. The influence of the sample preparation process on the surface conformations is tracked by tuning the landing energy of the proteins during deposition.To complement the amplitude data, an iterative phase retrieval algorithm is implemented to reconstruct the phase distribution in the object plane along with the amplitude distribution. The algorithm's performance and robustness is thoroughly evaluated and simulations regarding multiple scattering effects and element-dependent variations in scattering strength are carried out to provide a reference for the interpretation of phase data retrieved from experimentally acquired holograms. The iterative phase retrieval algorithm is then applied to protein data, indicating that both molecular density and charges can be related to features in the phase reconstructions, while the presence of metals does not correlate with specific phase signals at the current resolution obtainable from the experimental data.Since proteins are inherently three-dimensional, approaches towards three-dimensional reconstruction schemes are discussed, which will be the focus of future work.

EPFL2022

Low-energy Electron Holography Microscope for Imaging of Single Molecules

Sven Alexander Szilagyi

EPFL2022

Amplitude and phase reconstruction for Low-Energy Electron Holography of individual proteins

Hannah Julia Ochner

EPFL2022