Biology subjects

Meier-Credo, J.

Publications and source records attributed to Meier-Credo, J..

8 recordsLinked to original sources

bioRxiv2025

The vault associates with membranes in situ

The eukaryotic vault particle is a giant ribonucleoprotein complex that assembles into an iconic barrel-like cage. Its cellular function has remained elusive despite extensive characterization. Using cryo-electron tomography of Dictyostelium discoideum cells, we define the distribution, structural states and interaction landscape of vault particles in situ. Surprisingly, we detect a subpopulation of vault particles associated with the endoplasmic reticulum (ER) and nuclear envelope membranes. This association occurs at a defined barrel height of the vault particle and at patches of reduced membrane bilayer thickness and altered curvature. We further find that a substantial fraction of vaults encloses 80S ribosomes in highly ordered orientations. These structural findings are further corroborated by proximity labeling experiments which identify ER-resident proteins and numerous ribosomal components as vault particle interactors. The membrane-bound and ribosome-encapsulating vault populations that we uncover will direct future studies towards revealing vault function.

cell biology↗

bioRxiv2025

Correlative MS Imaging for cellular identification and analysis of in situ cryo-ET

In situ cryo-electron tomography (cryo-ET) offers direct access to protein structures in their native state and environment. A central challenge for this technique is the identification and classification of imaged cells, as sample heterogeneity with respect to cell type and functional state can significantly impact the data. Currently, cells can be classified using subtomogram averaging or fluorescent labeling, which require sufficiently distinguishable macromolecules, genetic modifications, or the availability of suitable labels. In MALDI-MS Imaging (MSI) individual cells can be identified and classified reliably, making use of the distributions of phospholipid species, metabolites and small proteins with characteristic profiles for specific cell types. Here we present an integrated pipeline combining MSI and in situ cryo-ET for single-cell identification and classification. Our approach enables MSI data acquisition directly from EM grids and can be used on any eukaryotic single-cell cryo-ET sample without prior modification in less than 36 h. We apply this workflow to describe cell-to-cell heterogeneity in cultured HeLa cells, to distinguish between distantly related HeLa and Polytomella cells, and between primary cultured rat neurons and glial cells. Finally we present a data integration workflow combining cryo-ET, light microscopy and MSI aligned in the same coordinate system to facilitate efficient data visualization and interpretation. We anticipate that this approach will change the way that structural biologists can approach cellular heterogeneity in cryo-ET, as cells can be identified and classified reliably post acquisition.

biophysics↗

bioRxiv2025

Structural insights into late-stage photosystem II assembly by Psb32

Photosystem II (PSII) assembly is a stepwise process in which intermediate complexes with auxiliary proteins are transiently formed to allow efficient de novo biogenesis or repair of damaged PSII. In particular, the role of extrinsic PSII subunits (PsbO, PsbU, PsbV) and auxiliary proteins such as Psb27 for the formation and photoactivation of the Mn4O5Ca cluster, which catalyzes the unique water splitting reaction in mature PSII, remains unclear. Using cryo-electron microscopy, we have determined the structure of two novel late-stage PSII assembly intermediates from Thermosynechococcus vestitus BP-1. In contrast to previous studies, the resulting monomeric PSII complexes contain both PsbJ and Psb27 and exhibit a fully mature acceptor side, while the oxygen evolving complex (OEC) is still in an immature state. The second complex additionally associates with the late-acting assembly factor Psb32 and the extrinsic subunit PsbV. While Psb32 has received little attention, its proposed role in the complex challenges the previous assumption that all extrinsic subunits associate spontaneously, as well as the notion that PsbO initiates binding and solely drives OEC formation. Our structures of the Psb27-PSII and Psb32-PSII intermediates provide novel insights, how structural changes of C-termini of the D1 and D2 core proteins regulate maturation of the OEC and how the catalytic side is prepared for binding of the Mn4O5Ca cluster. The Psb32-PSII complex potentially represents the final PSII assembly intermediate that precedes the incorporation and photoactivation of the Mn4O5Ca cluster, allowing us to explain the final steps in the PSII biogenesis and assembly pipeline in great detail, as only the two extrinsic subunits PsbO and PsbU are missing.

plant biology↗

bioRxiv2023

P116 from Mycoplasma is a self-sufficient lipid uptake and delivery machinery

Lipid acquisition and transport are fundamental processes in all organisms. Here, we investigate the lipid uptake and delivery mechanism of the minimal model organism Mycoplasma pneumoniae. We show that the essential protein P116 can transport lipids between liposomes independently and without ATP consumption. Our structural data and molecular dynamics simulations reveal the mechanism by which the N-terminal region of P116 perturbs the membrane, the lipid transfer route, and the regulation of membrane binding by the cargo mass within P116s large hydrophobic cavity. When adequately filled with cargo, P116 undergoes a rapid conformational change that modulates membrane binding. Taken together, our results show that Mycoplasma developed one integrated lipid uptake and delivery machinery that simplifies the complex multi-protein pathways used by higher developed organisms.

molecular biology↗

bioRxiv2022

The immunodominant protein P116 is a passive transporter of cholesterol and other essential lipids

Many human pathogens need to extract lipids from their environment for survival and proliferation. How this is accomplished on a molecular level is largely unknown1. Here, we report a comprehensive structural and functional analysis of the previously uncharacterized protein P116 (MPN_213) from Mycoplasma pneumoniae, a human pathogen responsible for approximately 30% of community-acquired human pneumonia2. Single-particle cryo-electron microscopy of P116 at 3.3 [A] resolution reveals a homodimer with a core domain presenting a previously unseen fold. This fold creates a large cavity of [~]18,000 [A]3 with a hydrophobic internal surface that is accessible to solvent. Within the cavity ligands with a length of 10-19 [A] and a width of 4 [A] could be observed. These ligands were identified as the essential lipids phosphatidylcholine, sphingomyelin and cholesterol using mass spectrometry. When the cavity is emptied, the protein undergoes an extensive conformational change that can no longer accommodate lipids. When emptied P116 is incubated with high-density lipoproteins (HDLs) a net transfer of cholesterol is demonstrated by a radioactivity experiment and cryo-electron microscopy resolves a complex between P116 and HDL. Taken together, our results reveal the mechanism by which P116 extracts essential lipids from the host environment and possibly then delivers them into the membrane by a wringing movement. This mechanism may be precedential for other cholesterol-auxotrophic bacteria.

microbiology↗

bioRxiv2022

Isolation of a novel heterodimeric PSII complex via strep-tagged PsbO

The multi-subunit membrane protein complex Photosystem II (PSII) catalyzes the light-driven oxidation of water and with this the initial step of photosynthetic electron transport in plants, algae, and cyanobacteria. Its biogenesis is coordinated by a network of auxiliary proteins that facilitate the stepwise assembly of individual subunits and cofactors, forming various intermediate complexes until fully functional mature PSII is present at the end of the process. In the current study, we purified PSII complexes from a mutant line of the thermophilic cyanobacterium Thermosynechococcus vestitus BP-1 in which the extrinsic subunit PsbO, characteristic for active PSII, was fused with an N-terminal Twin-Strep-tag. Three distinct PSII complexes were separated by ion-exchange chromatography after the initial affinity purification. Two complexes differ in their oligomeric state (monomeric and dimeric) but share the typical subunit composition of mature PSII. They are characterized by the very high oxygen-evolving activity of approx. 6,000 {micro}mol O2{middle dot} (mg Chl{middle dot}h)-1. Analysis of the third (heterodimeric) PSII complex revealed lower oxygen-evolving activity of approx. 3,000 {micro}mol O2{middle dot} (mg Chl{middle dot}h)-1 and manganese content of 2.7 ({+/-} 0.2) per reaction center compared to 3.7 ({+/-} 0.2) of fully active PSII. Mass spectrometry and time-resolved fluorescence spectroscopy further indicated that PsbO is partially replaced by Psb27 in this PSII fraction, thus implying a role in the repair of the complex.

plant biology↗

bioRxiv2022

Conformational changes in mitochondrial complex I from the thermophilic eukaryote Chaetomium thermophilum

Mitochondrial complex I is a redox-driven proton pump that generates proton-motive force across the inner mitochondrial membrane, powering oxidative phosphorylation and ATP synthesis in eukaryotes. We report the structure of complex I from the thermophilic fungus Chaetomium thermophilum, determined by cryoEM up to 2.4 [A] resolution. We show that the complex undergoes a transition between two conformations, which we refer to as form 1 and 2. The conformational switch is manifest in a twisting movement of the peripheral arm relative to the membrane arm, but most notably in substantial rearrangements of the Q-binding cavity and the E-channel, resulting in a continuous aqueous passage from the E-channel to subunit ND5 at the far end of the membrane arm. The conformational changes in the complex interior resemble those reported for mammalian complex I, suggesting a highly conserved, universal mechanism of coupling electron transport to proton pumping.

biochemistry↗

bioRxiv2020

How to build a water-splitting machine: structural insights into photosystem II assembly

Biogenesis of photosystem II (PSII), natures water splitting catalyst, is assisted by auxiliary proteins that form transient complexes with PSII components to facilitate stepwise assembly events. Using cryo-electron microscopy, we solved the structure of such a PSII assembly intermediate with 2.94 [A] resolution. It contains three assembly factors (Psb27, Psb28, Psb34) and provides detailed insights into their molecular function. Binding of Psb28 induces large conformational changes at the PSII acceptor side, which distort the binding pocket of the mobile quinone (QB) and replace bicarbonate with glutamate as a ligand of the non-heme iron, a structural motif found in reaction centers of non-oxygenic photosynthetic bacteria. These results reveal novel mechanisms that protect PSII from damage during biogenesis until water splitting is activated. Our structure further demonstrates how the PSII active site is prepared for the incorporation of the Mn4CaO5 cluster, which performs the unique water splitting reaction. One Sentence HighlightThe high-resolution Cryo-EM structure of the photosystem II assembly intermediate PSII-I reveals how natures water splitting catalyst is assembled, protected and prepared for photoactivation by help of the three assembly factors Psb27, Psb28 and Psb34.

plant biology↗