Biology subjects

Pachota, M.

Publications and source records attributed to Pachota, M..

8 recordsLinked to original sources

bioRxiv2026

Injector-based pump-probe serial synchrotron crystallography of CrPhotLOV1 at the MicroMAX beamline

Time-resolved serial synchrotron crystallography (TR-SSX) has become a powerful approach for investigating structural dynamics in proteins under near-physiological conditions. Its successful application, however, depends on robust sample delivery, reliable pump-probe synchronization, and reproducible data-processing strategies. Here, an injector-based pump-probe TR-SSX workflow at the MicroMAX beamline is applied to investigate the structural dynamics of the LOV1 domain of the blue-light photoreceptor phototropin from Chlamydomonas reinhardtii (CrPhotLOV1). Room-temperature serial datasets were collected for an independently measured laser-off dark state together with four light-activated pump-probe time windows (0-350, 500-850, 1000-1350 and 1500-1850 {micro}s after a microsecond laser pulse). All four time windows and an interleaved pump-probe dark reference were recorded in a single run with the JUNGFRAU detector at 2 kHz. Because thioether adduct formation in CrPhotLOV1 is complete within about 20 {micro}s and the adduct persists for hundreds of seconds, the four delays probe the same photoadduct state and serve as independent replicates. The workflow was evaluated in terms of data quality, light contamination in the pump-probe dark reference, difference Fourier analysis, extrapolated electron-density maps and refinement of light-state models. Consistent activation levels were obtained across all time points, enabling reliable structural interpretation of the light-activated state. Difference Fourier maps and refined extrapolated models reproducibly revealed formation of the characteristic FMN-Cys57 thioether adduct together with localized rearrangements within the chromophore-binding pocket. The refined light-state structures agree closely with previously reported CrPhotLOV1 models. These results establish a robust and reproducible workflow for injector-based pump-probe TR-SSX at the MicroMAX beamline, suitable for routine time-resolved studies of light-responsive proteins.

biophysics↗

bioRxiv2026

Room-temperature light-state structure of the AsLOV2-Rac1 optogenetic actuator by sheet-on-sheet serial femtosecond crystallography

Light-oxygen-voltage (LOV) domains are blue-light sensors that make fused effector proteins optically controllable. The AsLOV2-Rac1 fusion (PA-Rac1) is one of the most widely used of these tools, yet the structural basis of its activation has been inferred largely from cryogenic structures and from solution studies, mostly of the isolated AsLOV2 domain. Here, room-temperature dark and light-state structures of AsLOV2-Rac1 at 1.8 [A] resolution are reported, determined by serial femtosecond crystallography at the SwissFEL Cristallina beamline with sheet-on-sheet (SOS) chip sample delivery. Repeated 450 nm pump-laser excitation during chip rastering accumulated the FMN-cysteine adduct in about 80 % of the molecules, giving strong Fourier difference maps. To our knowledge, this is the first light-state structure of PA-Rac1 or of any AsLOV2 fusion and the first AsLOV2 light state in which the thioether adduct is not compromised by X-ray radiolysis. Apart from the N-terminal residue and one marginal Rac1 loop residue, C displacements above 0.3 [A] between dark and light models recorded at the same temperature are confined to the chromophore environment, the D helix and the G{beta}-H{beta} and H{beta}-I{beta} loops of AsLOV2. These changes, including the Ile445 rotamer switch, are also seen in light-state structures of the isolated AsLOV2 domain, but the Asn414 rotation reported there is absent in the fusion. Comparison with the cryogenic dark-state structure also shows temperature-dependent differences that exaggerate the apparent changes. In the crystal, the GTP, the Mg2+ site and the switch regions of Rac1 show no detectable light-induced change and the J helix does not unfold, although transient or low-population states cannot be excluded. This modest response is consistent with the small difference in effector affinity between dark- and lit-state mutants in solution, with lattice contacts and an interdomain Ca2+ ion as possible further contributions.

biophysics↗

bioRxiv2026

Photoactivatable Rac1: a photosensor tilt by time-resolved crystallography and a partially open switch I when GDP-bound

The LOV2 domain of Avena sativa phototropin 1 is widely used to control proteins with light, but how its signal reaches a fused effector has not been visualized. We followed photoactivatable Rac1 by time-resolved serial crystallography at room temperature. Within 350 {micro}s, a fifth of the photosensors form the flavin-cysteine adduct and tilt as a rigid body on Rac1 about an axis through the domain interface, while the J helix stays docked and Rac1 keeps GTP and dark-state switches up to 1.85 ms. Dark crystals contain a variable GDP-bound sub-population, so light data must be referenced to interleaved dark frames. In this sub-population, switch I is only partially open, held at a Ca2+ ion that bridges LOV2 and Rac1, a conformation that can persist in simulations without the lattice. The photosensor thus first acts on Rac1 at the interface that also holds switch I in the GDP-bound state.

biophysics↗

bioRxiv2026

Covalent bond formation caught in a LOV photoreceptor

Light-oxygen-voltage (LOV) domains are blue-light photoreceptors of plants, algae and fungi, and among the most widely used tools in optogenetics. They switch on by forming a covalent thioether bond between a conserved cysteine and their flavin chromophore, in a reaction that needs a proton to cross from the cysteine to the flavin through a pocket containing essentially no water. Its mechanism has been debated for two decades1, and because the chemistry is over within a microsecond its elementary steps have stayed hidden. Here we combine 10 time-resolved serial femtosecond crystallography snapshots and infrared spectroscopy with QM/MM calculations to resolve the entire sequence of events at 1.4 [A] resolution: from excited-state distortion of the flavin ring (10-100 ps), through hydration of a surface channel (10 ns) and a single ordered water reaching the active site as the reactive cysteine shifts between its conformations (100-500 ns), to the thioether bond itself, caught half-formed at 1 {micro}s (half the molecules reacted, half still poised) and complete at 10-100 {micro}s. That water bridges the cysteine and the flavin and shuttles the proton, lowering the barrier from [~]35 to [~]15 kcal/mol and accelerating the reaction by roughly fourteen orders of magnitude (without it, the half-life would be [~]237,000 years), then departs before the bond forms. Proteins can therefore hydrate a dehydrated active site transiently and on demand to overcome otherwise prohibitive reaction barriers, a catalytic strategy that reaches well beyond photoreceptors.

biophysics↗

bioRxiv2023

Fixed-target time-resolved crystallography at XFELs: the scourge of light contamination but reduced sample consumption

X-ray free electron laser (XFEL) light sources have allowed for the rapid growth of time-resolved structural experiments, which provide crucial information on the function of biological machines and their mechanisms. We set out to commission the SwissMX fixed-target sample delivery system at the SwissFEL Cristallina experimental station using the PSI developed MISP-chip for pump-probe time-resolved experiments. To characterise the system, we used the light-sensitive protein crystals of the Light-Oxygen-Voltage domain 1 (LOV1) from Chlamydomonas reinhardtii. Using different experimental settings, the adjacent-well light contamination was carefully assessed, indicating that it is crucial to control the light scattering from solid supports otherwise significant contamination can occur. However, our results show that, after the initial experiments and parameter refinement, the opaque MISP-chips are suitable for pump-probing a light-sensitive protein. This crystallographic experiment also probed the sub-millisecond structural dynamics of the LOV1 and indicated that at {Delta}t=10 s the covalent thioether bond is already established between the reactive Cys57 and FMN cofactor. This experiment validated the crystals to be suitable for in-depth follow up studies of the still poorly understood signal transduction mechanism. Importantly, the fixed-target delivery system also permitted a tenfold reduction in protein sample consumption compared to the most successful system used at XFEL, the high-viscosity extruder. This development creates the prospect of an exciting increase in XFEL project throughput for the field.

biophysics↗

bioRxiv2022

Refolding of lid subdomain of SARS-CoV-2 nsp14 upon nsp10 interaction releases exonuclease activity.

During the RNA replication, coronaviruses require proofreading to maintain the integrity of their large genomes. Nsp14 associates with viral polymerase complex to excise the mismatched nucleotides. Aside from the exonuclease activity, nsp14 methyltransferase domain mediates cap methylation, facilitating translation initiation and protecting viral RNA from recognition by the innate immune sensors. The nsp14 exonuclease activity is modulated by a protein co-factor nsp10. While the nsp10/nsp14 complex structure is available, the mechanistic basis for nsp10 mediated modulation remains unclear in the absence of nsp14 structure. Here we provide a crystal structure of nsp14 in an apo-form. Comparative analysis of the apo- and nsp10 bound structures explain the modulatory role of the co-factor protein. Further, the structure presented in this study rationalizes the recently proposed idea of nsp14/nsp10/nsp16 ternary complex.

microbiology↗

bioRxiv2022

Despite the odds: formation of the SARS-CoV-2 methylation complex.

Coronaviruses protect their single-stranded RNA genome with a methylated cap during replication. The capping process is initiated by several nonstructural proteins (nsp) encoded in the viral genome. The methylation is performed by two methyltransferases, nsp14 and nsp16 where nsp10 acts as a co-factor to both. Aditionally, nsp14 carries an exonuclease domain, which operates in the proofreading system during RNA replication of the viral genome. Both nsp14 and nsp16 were reported to independently bind nsp10, but the available structural information suggests that the concomitant interaction between these three proteins should be impossible due to steric clashes. Here, we show that nsp14, nsp10, and nsp16 can form a heterotrimer complex. This interaction is expected to encourage formation of mature capped viral mRNA, modulating the nsp14s exonuclease activity, and protecting the viral RNA. Our findings show that nsp14 is amenable to allosteric regulation and may serve as a novel target for therapeutic approaches.

microbiology↗

bioRxiv2021

Acriflavine, a clinically aproved drug, inhibits SARS-CoV-2 and other betacoronaviruses

SO_SCPLOWUMMARYC_SCPLOWThe COVID-19 pandemic caused by SARS-CoV-2 has been socially and economically devastating. Despite an unprecedented research effort, effective therapeutics are still missing to limit severe disease and mortality. Using high-throughput screening, we identified acriflavine as a potent papain-like protease (PLpro) inhibitor. NMR titrations and a co-crystal structure confirm that acriflavine blocks the PLpro catalytic pocket in an unexpected binding mode. We show that the drug inhibits viral replication at nanomolar concentration in cellular models, in vivo in mice and ex vivo in human airway epithelia, with broad range activity against SARS-CoV-2 and other betacoronaviruses. Considering that acriflavine is an inexpensive drug approved in some countries, it may be immediately tested in clinical trials and play an important role during the current pandemic and future outbreaks.

microbiology↗