The ribosomal RNA of the human protein synthesis machinery comprises numerous chemical modificati... more The ribosomal RNA of the human protein synthesis machinery comprises numerous chemical modifications that are introduced during ribosome biogenesis. We present the 1.9 Å resolution cryo-EM structure of the 80S human ribosome resolving numerous new rRNA modifications and functionally important ions such as Zn2+, K+ and Mg2+ including their associated individual water molecules. 2′-O-methylation, pseudo-uridine and base modifications were confirmed by mass spectrometry resulting in a complete investigation of the > 230 sites many of which could not be addressed previously. They choreograph key interactions within the RNA and at the interface with proteins, including at the ribosomal subunit interfaces of the fully assembled 80S ribosome. Uridine isomerisation turns out to be a key mechanism for U-A base pair stabilisation in RNA in general. The structural environment of chemical modifications & ions is primordial for the RNA architecture of the mature human ribosome, hence providin...
Glossina palipides salivary gland hypertrophy virus (GpSGHV) infects tsetse flies, which are vect... more Glossina palipides salivary gland hypertrophy virus (GpSGHV) infects tsetse flies, which are vectors for African trypanosomosis. This virus represents a major challenge in insect mass rearing and has hampered the implementation of the sterile insect technique programs in the Member States of the International Atomic Energy Agency. GpSGHV virions consist of long rod-shaped particles over 9000 Å in length, but little is known about their detailed structural organization. We show by cryo electron microscopy and cryo electron tomography that the GpSGHV virion has a unique, non-icosahedral helical structure. Its envelope exhibits regularly spaced spikes that protrude from the lipid bilayer and are arranged on a four-start helix. This study provides a detailed insight into the 3D architecture of GpSGHV, which will help to understand the viral life cycle and possibly allow the design of antiviral strategies in the context of tsetse fly infections.
bioRxiv (Cold Spring Harbor Laboratory), Dec 23, 2021
Single molecule localization microscopy (SMLM) with a dichroic image splitter can provide invalua... more Single molecule localization microscopy (SMLM) with a dichroic image splitter can provide invaluable multi-color information regarding colocalization of individual molecules, but it often suffers from technical limitations. So far, demixing algorithms give suboptimal results in terms of localization precision and correction of chromatic aberrations. Here we present an image splitter based multi-color SMLM method (splitSMLM) that offers much improved localization precision & drift correction, compensation of chromatic aberrations, and optimized performance of fluorophores in a specific buffer to equalize their reactivation rates for simultaneous imaging. A novel spectral demixing algorithm, SplitViSu, fully preserves localization precision with essentially no data loss and corrects chromatic aberrations at the nanometer scale. Multi-color performance is further improved by using optimized fluorophore and filter combinations. Applied to three-color imaging of the nuclear pore complex (NPC), this method provides a refined positioning of the individual NPC proteins and reveals that Pom121 clusters act as NPC deposition loci, hence illustrating strength and general applicability of the method. .
Using cryo-electron microscopy (cryo-EM), we determined the structure of the Escherichia coli 70S... more Using cryo-electron microscopy (cryo-EM), we determined the structure of the Escherichia coli 70S ribosome with a global resolution of 2.0 Å. The maps reveal unambiguous positioning of protein and RNA residues, their detailed chemical interactions, and chemical modifications. Notable features include the first examples of isopeptide and thioamide backbone substitutions in ribosomal proteins, the former likely conserved in all domains of life. The maps also reveal extensive solvation of the small (30S) ribosomal subunit, and interactions with A-site and P-site tRNAs, mRNA, and the antibiotic paromomycin. The maps and models of the bacterial ribosome presented here now allow a deeper phylogenetic analysis of ribosomal components including structural conservation to the level of solvation. The high quality of the maps should enable future structural analyses of the chemical basis for translation and aid the development of robust tools for cryo-EM structure modeling and refinement.
Cellular signaling uses two main pathways, the membrane-associated and the nuclear receptors (NRs... more Cellular signaling uses two main pathways, the membrane-associated and the nuclear receptors (NRs). The characteristic of the first is the use of water-soluble ligands which do not cross the membrane. Ligand binding at the cell surface induces, e.g., autophosphorylation of epidermal growth factor (EGF) receptor, starting a cascade of serine- or tyrosine-kinases, finally transducing the signal to the nucleus. NRs work very differently. Located in the nucleus they control the activity of their target genes directly by binding to specific DNA sequences called hormone response elements. They are activated by hydrophobic ligands, like the steroid hormones and retinoids, which reach their receptor in the cytoplasm or the nucleus by crossing the lipid bilayer of the cell membrane. Some of these ligands, such as retinoic acid, need to be metabolically modified, others are completely synthesized in the cell, as Prostaglandins.
The central dogma of molecular biology comprises two fundamental mechanistic steps of gene expres... more The central dogma of molecular biology comprises two fundamental mechanistic steps of gene expression (transcription and translation), which, in bacteria, are coupled. A recent study provides structural insights into a supercomplex between the RNA polymerase and the ribosome, thus highlighting the synergy between two key macromolecular machineries in the cell.
The ribosomal RNA of the human protein synthesis machinery comprises numerous chemical modificati... more The ribosomal RNA of the human protein synthesis machinery comprises numerous chemical modifications that are introduced during ribosome biogenesis. We present the 1.9 Å resolution cryo-EM structure of the 80S human ribosome resolving numerous new rRNA modifications and functionally important ions such as Zn2+, K+ and Mg2+ including their associated individual water molecules. 2′-O-methylation, pseudo-uridine and base modifications were confirmed by mass spectrometry resulting in a complete investigation of the > 230 sites many of which could not be addressed previously. They choreograph key interactions within the RNA and at the interface with proteins, including at the ribosomal subunit interfaces of the fully assembled 80S ribosome. Uridine isomerisation turns out to be a key mechanism for U-A base pair stabilisation in RNA in general. The structural environment of chemical modifications & ions is primordial for the RNA architecture of the mature human ribosome, hence providin...
Glossina palipides salivary gland hypertrophy virus (GpSGHV) infects tsetse flies, which are vect... more Glossina palipides salivary gland hypertrophy virus (GpSGHV) infects tsetse flies, which are vectors for African trypanosomosis. This virus represents a major challenge in insect mass rearing and has hampered the implementation of the sterile insect technique programs in the Member States of the International Atomic Energy Agency. GpSGHV virions consist of long rod-shaped particles over 9000 Å in length, but little is known about their detailed structural organization. We show by cryo electron microscopy and cryo electron tomography that the GpSGHV virion has a unique, non-icosahedral helical structure. Its envelope exhibits regularly spaced spikes that protrude from the lipid bilayer and are arranged on a four-start helix. This study provides a detailed insight into the 3D architecture of GpSGHV, which will help to understand the viral life cycle and possibly allow the design of antiviral strategies in the context of tsetse fly infections.
bioRxiv (Cold Spring Harbor Laboratory), Dec 23, 2021
Single molecule localization microscopy (SMLM) with a dichroic image splitter can provide invalua... more Single molecule localization microscopy (SMLM) with a dichroic image splitter can provide invaluable multi-color information regarding colocalization of individual molecules, but it often suffers from technical limitations. So far, demixing algorithms give suboptimal results in terms of localization precision and correction of chromatic aberrations. Here we present an image splitter based multi-color SMLM method (splitSMLM) that offers much improved localization precision & drift correction, compensation of chromatic aberrations, and optimized performance of fluorophores in a specific buffer to equalize their reactivation rates for simultaneous imaging. A novel spectral demixing algorithm, SplitViSu, fully preserves localization precision with essentially no data loss and corrects chromatic aberrations at the nanometer scale. Multi-color performance is further improved by using optimized fluorophore and filter combinations. Applied to three-color imaging of the nuclear pore complex (NPC), this method provides a refined positioning of the individual NPC proteins and reveals that Pom121 clusters act as NPC deposition loci, hence illustrating strength and general applicability of the method. .
Using cryo-electron microscopy (cryo-EM), we determined the structure of the Escherichia coli 70S... more Using cryo-electron microscopy (cryo-EM), we determined the structure of the Escherichia coli 70S ribosome with a global resolution of 2.0 Å. The maps reveal unambiguous positioning of protein and RNA residues, their detailed chemical interactions, and chemical modifications. Notable features include the first examples of isopeptide and thioamide backbone substitutions in ribosomal proteins, the former likely conserved in all domains of life. The maps also reveal extensive solvation of the small (30S) ribosomal subunit, and interactions with A-site and P-site tRNAs, mRNA, and the antibiotic paromomycin. The maps and models of the bacterial ribosome presented here now allow a deeper phylogenetic analysis of ribosomal components including structural conservation to the level of solvation. The high quality of the maps should enable future structural analyses of the chemical basis for translation and aid the development of robust tools for cryo-EM structure modeling and refinement.
Cellular signaling uses two main pathways, the membrane-associated and the nuclear receptors (NRs... more Cellular signaling uses two main pathways, the membrane-associated and the nuclear receptors (NRs). The characteristic of the first is the use of water-soluble ligands which do not cross the membrane. Ligand binding at the cell surface induces, e.g., autophosphorylation of epidermal growth factor (EGF) receptor, starting a cascade of serine- or tyrosine-kinases, finally transducing the signal to the nucleus. NRs work very differently. Located in the nucleus they control the activity of their target genes directly by binding to specific DNA sequences called hormone response elements. They are activated by hydrophobic ligands, like the steroid hormones and retinoids, which reach their receptor in the cytoplasm or the nucleus by crossing the lipid bilayer of the cell membrane. Some of these ligands, such as retinoic acid, need to be metabolically modified, others are completely synthesized in the cell, as Prostaglandins.
The central dogma of molecular biology comprises two fundamental mechanistic steps of gene expres... more The central dogma of molecular biology comprises two fundamental mechanistic steps of gene expression (transcription and translation), which, in bacteria, are coupled. A recent study provides structural insights into a supercomplex between the RNA polymerase and the ribosome, thus highlighting the synergy between two key macromolecular machineries in the cell.
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Papers by Bruno Klaholz