Biology subjects

von Loeffelholz, O.

Publications and source records attributed to von Loeffelholz, O..

5 recordsLinked to original sources

bioRxiv2025

EXPLICIT CORRECTION OF SEVERELY NON-UNIFORM DISTRIBUTIONS OF CRYO-EM VIEWS

The quality of three-dimensional macromolecular image reconstructions by cryo electron microscopy strongly depends on the number and quality of respective two-dimensional projections and on their angular distribution in space. Distributions with one or a few strongly preferred particle orientations may result in maps deformed in certain directions. A simple removal of overrepresented views may improve the quality of the reconstructed maps when the level of noise in the two-dimensional projections is low and the dataset-size can afford this removal but is counterproductive or insufficient otherwise. Giving complementarily an increased weight to underrepresented views, or taking multiple copies of them during the reconstruction, may improve the results, naturally, depending on how much the views distribution is non-uniform. This work describes the results of three-dimensional reconstructions using an explicit correction of the number of over- and underrepresented projections for non-uniformly distributed sets. SynopsisAn explicit numerical leveling of non-uniformly distributed sets of 2D projections with the program VUE improves the 3D reconstructions and illustrate sources of image distortions.

molecular biology↗

bioRxiv2025

Translation initiation by the Kozak mRNA sequence is based on a conformational readout on the ribosome

The recognition mechanism of Kozak mRNA, typically comprising purines in the - 3 and +4 positions flanking the AUG start codon, has remained enigmatic for decades. To address this fundamental function in eukaryotes during translation initiation, we analysed the cryo-EM structures of human 48S preinitiation complexes with mRNA point mutations. They reveal a fan-like intercalation of the pre-codon triplet into the 18S ribosomal RNA (rRNA), while the -3 purine as opposed to the pyrimidine in the non-Kozak context favours stabilization of the ternary complex between initiation factor eIF2 and initiator tRNA. Specificity towards the +4 purine is achieved beyond a single residue recognition by mutual conformational adaptations of eIF1A, mRNA and rRNA and involves the insertion of a reading head in which decoding residue A1825 (rRNA) stacks with the A-site codon to stabilize the fully accommodated state. Hence, instead of relying on base pairing as in bacteria, the specific recognition of the Kozak sequence on eukaryotic ribosomes is based on an induced-fit mechanism that triggers a conformational readout of the mRNA.

biochemistry↗

bioRxiv2023

Structure of the human 80S ribosome at 1.9 A resolution - the molecular role of chemical modifications and ions in RNA

The ribosomal RNA of the human protein synthesis machinery comprises numerous chemical modifications that are introduced during ribosome biogenesis. We present the 1.9 [A] 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 providing a structural framework to address their role in healthy context and human diseases.

biochemistry↗

bioRxiv2020

Cryo-EM of human Arp2/3 complexes provides structural insights into actin nucleation modulation by ARPC5 isoforms

The Arp2/3 complex regulates many cellular processes by stimulating formation of branched actin filament networks. Because three of its seven subunits exist as two different isoforms, mammals produce a family of Arp2/3 complexes with different properties that may be suited to different physiological contexts. To shed light on how isoform diversification affects Arp2/3 function, we determined a 4.2 [A] resolution cryo-EM structure of the most active human Arp2/3 complex containing ARPC1B and ARPC5L, and compared it with the structure of the least active ARPC1A-ARPC5-containing complex. The architecture of each isoform-specified Arp2/3 is the same. Strikingly, however, the N-terminal half of ARPC5L is partially disordered compared to ARPC5, suggesting that this region of ARPC5/ARPC5L is an important determinant of complex activity. Confirming this idea, the nucleation activity of Arp2/3 complexes containing hybrid ARPC5/ARPC5L subunits is higher when the ARPC5L N-terminus is present, thereby explaining activity differences between the different Arp2/3 complexes.

biophysics↗

bioRxiv2018

Near-atomic cryo-EM structure of yeast kinesin 5-microtubule complex reveals a distinct binding footprint

Kinesin-5s are essential members of the superfamily of microtubule-dependent motors that undertake conserved roles in cell division. We investigated coevolution of the motor-microtubule interface using cryo-electron microscopy to determine the near-atomic structure of the motor domain of Cut7, the fission yeast kinesin-5, bound to fission yeast microtubules. AMPPNP-bound Cut7 adopts a kinesin-conserved ATP-like conformation, with a closed nucleotide binding pocket and docked neck linker that supports cover neck bundle formation. Compared to mammalian tubulin microtubules, Cut7s footprint on S. pombe microtubule surface is subtly different because of their different architecture. However, the core motor-microtubule interaction that stimulates motor ATPase is tightly conserved, reflected in similar Cut7 ATPase activities on each microtubule type. The S. pombe microtubules were bound by the drug epothilone, which is visible in the taxane binding pocket. Stabilization of S. pombe microtubules is mediated by drug binding at this conserved site despite their noncanonical architecture and mechanochemistry.\n\nHighlightsO_LIS. pombe Cut7 has a distinct binding footprint on S. pombe microtubules\nC_LIO_LIThe core interface driving microtubule activation of motor ATPase is conserved\nC_LIO_LIThe neck linker is docked in AMPPNP-bound Cut7 and the cover neck bundle is formed\nC_LIO_LIEpothilone binds at the taxane binding site to stabilize S. pombe microtubules\nC_LI\n\neTOC textTo investigate coevolution of the motor-microtubule interface, we used cryo-electron microscopy to determine the near-atomic structure of the motor domain of Cut7, the fission yeast kinesin-5, bound to microtubules polymerized from natively purified fission yeast tubulin and stabilised by the drug epothilone.

biophysics↗