Biology subjects

Kyrilis, F. L.

Publications and source records attributed to Kyrilis, F. L..

9 recordsLinked to original sources

bioRxiv2026

A nested shell structure coordinates enzyme communication in pyruvate oxidation

The pyruvate dehydrogenase complex (PDHc)1 links glycolysis to the Krebs cycle by catalyzing the oxidative decarboxylation of pyruvate to acetyl-CoA and CO2, a process essential for life2,3. PDHc is formed by structural proteins (E3-binding protein, E3BP)4-7, enzymatic subunits (E1, E2, E3)4,6, and mobile lipoyl domains (LDs), the latter shuttling intermediates across active sites6,8. Although numerous details regarding pyruvate oxidation steps have been elucidated9, the precise organization of the entire PDHc remains unknown due to its large size and dynamic heterogeneity. Here, we employ in silico, in vitro, and in situ methods to propose a multi-scale model of PDHc that includes approximately one million atoms and to visualize multiple conformational states. This model reveals a [~]40-50 nm nested shell structure, formed by flexible linkers that spatially coordinate the E1 and E3 enzyme complexes around the E2-E3BP core scaffold. This structure acts as a molecular sieve, selectively guiding lipoyl arms while maintaining enzyme positioning with sub-nm precision. During catalysis, the nested shell structure expands and adopts a mechanically reinforced state comparable in magnitude to viral assemblies10. Our findings provide structural context for the textbook "link reaction"11, building on decades of biochemical knowledge, are transferable to functional aspects of other -ketoacid dehydrogenase complexes, and, ultimately, expand our understanding of primary metabolism as a whole. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=195 SRC="FIGDIR/small/724543v1_ufig1.gif" ALT="Figure 1"> View larger version (70K): org.highwire.dtl.DTLVardef@1615612org.highwire.dtl.DTLVardef@159bc8dorg.highwire.dtl.DTLVardef@69f8c1org.highwire.dtl.DTLVardef@14a83a3_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2026

A 37-million-particle dataset from over 250 experiments to accelerate data-driven cryo-EM analysis

Cryogenic Electron Microscopy (cryo-EM) has revolutionized structural biology by enabling near-atomic-resolution structure determination of biological macromolecules. Central to cryo-EM analysis are particles, namely 2D projections of biomolecules extracted from micrographs, which serve as the primary input for 3D reconstruction. While data-driven methods have transformed other scientific domains, their impact on cryo-EM remains limited because existing particle datasets are too small, too narrow in protein diversity, and lack rich per-particle annotations. We introduce cryoPANDA (cryo-EM Particles ANnotated DAtaset), comprising over 37 million annotated particles from 252 experiments spanning a wide range of protein types, more than 10-fold larger than prior collections. Each particle is accompanied by detailed annotations covering acquisition, classification, and re-construction metadata, alongside the corresponding 3D electrostatic potential map, the published EMDB map, and, where available, the PDB model. We validate cryoPANDA in two ways: first, by reconstructing hundreds of distinct high-resolution cryo-EM maps; and second, by training a DINOv2 foundation model and evaluating its learned representations on micrograph segmentation, particle picking, and particle clustering.

bioinformatics↗

bioRxiv2025

Direct evidence of acid-driven protein desolvation

Water and its ability to modulate the protonation states of biomolecules govern the physical chemistry of life, dictating their metabolic functions(1). However, how amino acid protonation alters protein hydration and solubility(2, 3) is an open question since Kuntz and Kauzmann proposed pH-driven protein desolvation in 1974(4). Here, in a series of high-resolution cryo-electron microscopy structures of a protein complex at different pH values (from pH 9.0 to 3.5) we examined thousands of observable hydration sites. Cryo-EM data, in agreement with constant-pH molecular dynamics simulations, show that nearly half of protein-bound waters exchanged with the bulk solvent upon acidification, with [~]100 waters lost per pH unit per molecule. The loss of waters was most significant around the side chains of glutamate and aspartate residues while specific polar residues, mostly asparagine, anchored persistent waters. A positionally conserved hydration layer was observed across all pH conditions, accounting for 40% of resolved waters. Those waters displayed denser packing than less persistent waters, forming a pH-independent solvation shell. Acid-induced water exchange also displaced bound iron, providing a mechanistic link between solvation and metal release. Our findings demonstrate the core principles of acid-driven protein desolvation, resolving a 50-year-old biochemical hypothesis(4).

biochemistry↗

bioRxiv2025

Strategies for Mitigating Radiation Damage and Improving Data Completeness in 3D Electron Diffraction of Protein Crystals

While 3D electron diffraction (3D-ED or microcrystal electron diffraction, MicroED) has emerged as a promising method for protein structure determination, its applicability is hindered by high susceptibility to radiation damage, leading to decreasing signal-to-noise ratio in consecutive diffraction patterns that limits the quality (resolution and redundancy) of the data. In addition, data completeness may be restricted due to the geometrical limitations of current sample holders and stages. In this work, we introduce an approach that addresses these issues using a commonly available 200keV cryo-electron microscope. The multi-position acquisition technique we present here combines (a) multiple data acquisitions from a single crystal over several tilt ranges and (b) merging data from a small number of crystals, each tilted about a different axis. The robustness of this approach is demonstrated by the de novo elucidation of a protein-peptide complex structure from only two orthorhombic microcrystals.

biochemistry↗

bioRxiv2024

Mechanism of SHP2 activation by bis-Tyr-phosphorylated Gab1

The non-receptor tyrosine phosphatase SHP2 (PTPN11) is a regulator of diverse cellular functions including mitogenic activation and cell migration. SHP2 consists of two tandem SH2 domains followed by the catalytic domain, and is autoinhibited by the N-terminal SH2 domain that blocks access to the active site. Mutations that influence auto-inhibition have been implicated in cancer and other diseases, and allosteric inhibitors have been developed that stabilise the inactive state. The mechanism of SHP2 activation remains unclear, however. Here, we show that the intrinsically disordered bis-phosphorylated SHP2-activating peptide pY627pY659-Gab1 binds to both SH2 domains, undergoing a partial disorder-to-order transition in the process. In addition to eliciting changes in SH2 domain dynamics, the peptide reorganises their relative orientations to provide a new SH2-SH2 interface. Our data suggest an active conformation for SHP2 that is also applicable to the hematopoietic cell-specific SHP1 (PTPN6), shedding light on the activation mechanism of both enzymes and paving the way for the development of novel compounds that modulate SHP2 activity.

biochemistry↗

bioRxiv2022

AI-guided cryo-EM probes a thermophilic cell-free system with succinyl-coA manufacturing capability

Cell-free systems display tremendous potential for biotechnological applications, complementing in vitro reconstituted enzymatic processes and traditional expression systems. However, they often represent "black boxes" without much insight into their components. Here, we characterize a thermophilic cell-free system that produces succinyl-CoA and discern its intrinsic, non-stochastic organization. By employing biochemical, biophysical, and bioinformatic methods we resolve its molecular composition, 3D architecture and molecular function at atomic resolution. We further report the high-resolution cryo-EM structure of the reactions main component, the oxoglutarate dehydrogenase complex core (E2o), which displays various structural adaptations. These include hydrogen bonding patterns confining interactions of participating enzymes (E1o-E2o-E3), electrostatic tunneling that drives inter-communication between subunits, and the presence of a flexible subunit, the E3BPo connecting E2o and E3. This multi-scale analysis of a cell-free system provides a blueprint for structure-function studies of complex mixtures of biotechnological value.

biochemistry↗

bioRxiv2022

Delineating organizational principles of the endogenous L-A virus by cryo-EM and computational analysis of native cell extracts

The high abundance of most viruses in infected host cells benefits their structural characterization; endogenous viruses are present in low copy numbers, however, and are therefore challenging to investigate. Here, we retrieve cell extracts enriched with an endogenous virus, the yeast L-A virus. The determined cryo-EM structure discloses capsid-stabilizing cation-{pi} stacking and an interplay of non-covalent interactions from ten distinct capsomere interfaces. The capsid-embedded mRNA decapping active site trench is supported by a constricting movement of two opposite-facing loops. tRNA-loaded polysomes and other biomacromolecules, presumably mRNA, are found in virus proximity while stacked dsRNA bundles and the sub-stoichiometric polymerase localize underneath the capsid surface. Mature viruses participate in larger viral communities resembling their rare in-cell equivalents in terms of size, composition, and inter-virus distances. Our results collectively describe a 3D-architecture of a viral milieu, opening the door to cellextract-based high-resolution structural virology.

biochemistry↗

bioRxiv2022

Cryo-EM structure of the Rhodobaca bogoriensis RC-LH1-PufX dimeric complex at 2.9 angstrom

The reaction centre-light harvesting 1 (RC-LH1) complex is essential for converting light into proton motive force in photosynthetic bacteria. RC-LH1 is a monomer in most purple bacteria, but in Rhodobacter species, it is a dimer. Its assembly depends on an accessory polypeptide (PufX) and, ultimately, on photosynthetic growth. To date, knowledge on the RC-LH1-PufX structure, where the dimer has two incomplete C-shaped antenna rings surrounding an RC, is mainly limited to the model organism Rhodobacter sphaeroides. Here we present a cryo-electron microscopy structure at 2.9 [A] from Rhodobaca bogorensis strain LBB1. RCs are surrounded by 30 antennas and incorporate protein Y and PufX. RCs are stably connected by PufX, which self-interacts, electrostatically attracts cytochrome c2 (cyt c2) and forms extensive networks with co-factors. This structure underlines coordinated energy transfer in a combinatorial manner, providing a basis to describe bacterial photosynthesis within a dimeric photosynthetic apparatus.

biophysics↗

bioRxiv2019

2.7 A cryo-EM structure of vitrified M. musculus H-chain apoferritin from 200 keV \"screening microscope\"

Here we present the structure of mouse H-chain apoferritin at 2.7 [A] (FSC=0.143) solved by single particle cryogenic electron microscopy (cryo-EM) using a 200 kV device. Data were collected using a compact, two-lens illumination system with a constant power objective lens, without the use of energy filters or aberration correctors. Coulomb potential maps reveal clear densities for main chain carbonyl oxygens, residue side chains (including alternative conformations) and bound solvent molecules. We argue that the advantages offered by (a) the high electronic and mechanical stability of the microscope, (b) the high emission stability and low beam energy spread of the high brightness Field Emission Gun (x-FEG), (c) direct electron detection technology and (d) particle-based Contrast Transfer Function (CTF) refinement have contributed to achieving resolution close to the Rayleigh limit. Overall, we show that basic electron optical settings for automated cryo-electron microscopy imaging, widely thought of as a "screening cryo-microscope", can be used to determine structures approaching atomic resolution. HighlightsO_LIThe 2.7 [A] structure of mouse apoferritin was solved using a 200 keV screening cryo-microscope C_LIO_LIThe apoferritin reconstruction was resolved without an energy filter, aberration correctors, or constant-power condenser lenses C_LIO_LIComparison to available crystallographic and cryo-EM structures from high-end cryo-microscopes demonstrates consistency in resolved water molecules, metals and side chain orientations C_LIO_LIAlthough radiation damage is more prominent at 200 keV compared to 300 keV, this type of instrumentation is more accessible to research laboratories due to its compactness and simplicity C_LI

biophysics↗