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In celebration of Pride Month, we asked transgender, genderqueer, and nonbinary scientists to tell us about what fascinates them, their ambitions and achievements, and how their gender identities have shaped their experiences in STEM. We owe a special thanks to 500 Queer Scientists (https://500queerscientists.com/), whose network and efforts at increasing LGBTQ+ scientists' visibility made this article possible.
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Ingeniería , Matemática , Investigadores , Ciencia , Minorías Sexuales y de Género , Tecnología , Personas Transgénero , Femenino , Humanos , MasculinoRESUMEN
Inspired by the period-four oscillation in flash-induced oxygen evolution of photosystem II discovered by Joliot in 1969, Kok performed additional experiments and proposed a five-state kinetic model for photosynthetic oxygen evolution, known as Kok's S-state clock or cycle1,2. The model comprises four (meta)stable intermediates (S0, S1, S2 and S3) and one transient S4 state, which precedes dioxygen formation occurring in a concerted reaction from two water-derived oxygens bound at an oxo-bridged tetra manganese calcium (Mn4CaO5) cluster in the oxygen-evolving complex3-7. This reaction is coupled to the two-step reduction and protonation of the mobile plastoquinone QB at the acceptor side of PSII. Here, using serial femtosecond X-ray crystallography and simultaneous X-ray emission spectroscopy with multi-flash visible laser excitation at room temperature, we visualize all (meta)stable states of Kok's cycle as high-resolution structures (2.04-2.08 Å). In addition, we report structures of two transient states at 150 and 400 µs, revealing notable structural changes including the binding of one additional 'water', Ox, during the S2âS3 state transition. Our results suggest that one water ligand to calcium (W3) is directly involved in substrate delivery. The binding of the additional oxygen Ox in the S3 state between Ca and Mn1 supports O-O bond formation mechanisms involving O5 as one substrate, where Ox is either the other substrate oxygen or is perfectly positioned to refill the O5 position during O2 release. Thus, our results exclude peroxo-bond formation in the S3 state, and the nucleophilic attack of W3 onto W2 is unlikely.
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Oxígeno/metabolismo , Fotosíntesis , Agua/química , Agua/metabolismo , Calcio/metabolismo , Cristalografía por Rayos X , Cianobacterias/química , Rayos Láser , Manganeso/metabolismo , Modelos Moleculares , Oxidación-Reducción , Complejo de Proteína del Fotosistema II/química , Complejo de Proteína del Fotosistema II/metabolismo , Plastoquinona/metabolismoRESUMEN
A major barrier to defining the structural intermediates that arise during the reversible photointerconversion of phytochromes between their biologically inactive and active states has been the lack of crystals that faithfully undergo this transition within the crystal lattice. Here, we describe a crystalline form of the cyclic GMP phosphodiesterases/adenylyl cyclase/FhlA (GAF) domain from the cyanobacteriochrome PixJ in Thermosynechococcus elongatus assembled with phycocyanobilin that permits reversible photoconversion between the blue light-absorbing Pb and green light-absorbing Pg states, as well as thermal reversion of Pg back to Pb. The X-ray crystallographic structure of Pb matches previous models, including autocatalytic conversion of phycocyanobilin to phycoviolobilin upon binding and its tandem thioether linkage to the GAF domain. Cryocrystallography at 150 K, which compared diffraction data from a single crystal as Pb or after irradiation with blue light, detected photoconversion product(s) based on Fobs - Fobs difference maps that were consistent with rotation of the bonds connecting pyrrole rings C and D. Further spectroscopic analyses showed that phycoviolobilin is susceptible to X-ray radiation damage, especially as Pg, during single-crystal X-ray diffraction analyses, which could complicate fine mapping of the various intermediate states. Fortunately, we found that PixJ crystals are amenable to serial femtosecond crystallography (SFX) analyses using X-ray free-electron lasers (XFELs). As proof of principle, we solved by room temperature SFX the GAF domain structure of Pb to 1.55-Å resolution, which was strongly congruent with synchrotron-based models. Analysis of these crystals by SFX should now enable structural characterization of the early events that drive phytochrome photoconversion.
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Ficobilinas/metabolismo , Ficocianina/metabolismo , Fitocromo/química , Fitocromo/efectos de la radiación , Adenilil Ciclasas/química , Adenilil Ciclasas/metabolismo , Cristalografía , Cristalografía por Rayos X , Cianobacterias/química , GMP Cíclico , Luz , Modelos Moleculares , Hidrolasas Diéster Fosfóricas/química , Hidrolasas Diéster Fosfóricas/metabolismo , Células Fotorreceptoras/metabolismo , Ficobilinas/química , Ficocianina/química , Conformación Proteica , Dominios Proteicos , Thermosynechococcus , Transactivadores/químicaRESUMEN
In oxygenic photosynthesis, light-driven oxidation of water to molecular oxygen is carried out by the oxygen-evolving complex (OEC) in photosystem II (PS II). Recently, we reported the room-temperature structures of PS II in the four (semi)stable S-states, S1, S2, S3, and S0, showing that a water molecule is inserted during the S2 â S3 transition, as a new bridging O(H)-ligand between Mn1 and Ca. To understand the sequence of events leading to the formation of this last stable intermediate state before O2 formation, we recorded diffraction and Mn X-ray emission spectroscopy (XES) data at several time points during the S2 â S3 transition. At the electron acceptor site, changes due to the two-electron redox chemistry at the quinones, QA and QB, are observed. At the donor site, tyrosine YZ and His190 H-bonded to it move by 50 µs after the second flash, and Glu189 moves away from Ca. This is followed by Mn1 and Mn4 moving apart, and the insertion of OX(H) at the open coordination site of Mn1. This water, possibly a ligand of Ca, could be supplied via a "water wheel"-like arrangement of five waters next to the OEC that is connected by a large channel to the bulk solvent. XES spectra show that Mn oxidation (τ of â¼350 µs) during the S2 â S3 transition mirrors the appearance of OX electron density. This indicates that the oxidation state change and the insertion of water as a bridging atom between Mn1 and Ca are highly correlated.
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Fotosíntesis , Complejo de Proteína del Fotosistema II/metabolismo , Hidrógeno/metabolismo , Magnesio/metabolismo , Oxidación-Reducción , Oxígeno/metabolismo , Fotones , Complejo de Proteína del Fotosistema II/química , Quinonas/metabolismo , Agua/metabolismoRESUMEN
Light-induced oxidation of water by photosystem II (PS II) in plants, algae and cyanobacteria has generated most of the dioxygen in the atmosphere. PS II, a membrane-bound multi-subunit pigment protein complex, couples the one-electron photochemistry at the reaction centre with the four-electron redox chemistry of water oxidation at the Mn4CaO5 cluster in the oxygen-evolving complex (OEC). Under illumination, the OEC cycles through five intermediate S-states (S0 to S4), in which S1 is the dark-stable state and S3 is the last semi-stable state before O-O bond formation and O2 evolution. A detailed understanding of the O-O bond formation mechanism remains a challenge, and will require elucidation of both the structures of the OEC in the different S-states and the binding of the two substrate waters to the catalytic site. Here we report the use of femtosecond pulses from an X-ray free electron laser (XFEL) to obtain damage-free, room temperature structures of dark-adapted (S1), two-flash illuminated (2F; S3-enriched), and ammonia-bound two-flash illuminated (2F-NH3; S3-enriched) PS II. Although the recent 1.95 Å resolution structure of PS II at cryogenic temperature using an XFEL provided a damage-free view of the S1 state, measurements at room temperature are required to study the structural landscape of proteins under functional conditions, and also for in situ advancement of the S-states. To investigate the water-binding site(s), ammonia, a water analogue, has been used as a marker, as it binds to the Mn4CaO5 cluster in the S2 and S3 states. Since the ammonia-bound OEC is active, the ammonia-binding Mn site is not a substrate water site. This approach, together with a comparison of the native dark and 2F states, is used to discriminate between proposed O-O bond formation mechanisms.
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Cianobacterias/química , Electrones , Rayos Láser , Complejo de Proteína del Fotosistema II/química , Complejo de Proteína del Fotosistema II/metabolismo , Temperatura , Amoníaco/química , Amoníaco/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Sitios de Unión , Cristalización , Manganeso/metabolismo , Modelos Moleculares , Oxígeno/metabolismo , Especificidad por Sustrato , Agua/metabolismoRESUMEN
Post-transcriptional ribosomal RNA (rRNA) modifications are present in all organisms, but their exact functional roles and positions are yet to be fully characterized. Modified nucleotides have been implicated in the stabilization of RNA structure and regulation of ribosome biogenesis and protein synthesis. In some instances, rRNA modifications can confer antibiotic resistance. High-resolution ribosome structures are thus necessary for precise determination of modified nucleotides' positions, a task that has previously been accomplished by X-ray crystallography. Here, we present a cryo-electron microscopy (cryo-EM) structure of the Escherichia coli 50S subunit at an average resolution of 2.2 Å as an additional approach for mapping modification sites. Our structure confirms known modifications present in 23S rRNA and additionally allows for localization of Mg2+ ions and their coordinated water molecules. Using our cryo-EM structure as a testbed, we developed a program for assessment of cryo-EM map quality. This program can be easily used on any RNA-containing cryo-EM structure, and an associated Coot plugin allows for visualization of validated modifications, making it highly accessible.
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Microscopía por Crioelectrón , Escherichia coli/metabolismo , Escherichia coli/ultraestructura , Nucleótidos/metabolismo , Subunidades Ribosómicas Grandes Bacterianas/ultraestructura , Modelos Moleculares , Péptidos/metabolismo , Peptidil Transferasas/metabolismo , Reproducibilidad de los Resultados , Solventes , Electricidad EstáticaRESUMEN
We report a method for serial X-ray crystallography at X-ray free-electron lasers (XFELs), which allows for full use of the current 120-Hz repetition rate of the Linear Coherent Light Source (LCLS). Using a micropatterned silicon chip in combination with the high-speed Roadrunner goniometer for sample delivery, we were able to determine the crystal structures of the picornavirus bovine enterovirus 2 (BEV2) and the cytoplasmic polyhedrosis virus type 18 polyhedrin, with total data collection times of less than 14 and 10 min, respectively. Our method requires only micrograms of sample and should therefore broaden the applicability of serial femtosecond crystallography to challenging projects for which only limited sample amounts are available. By synchronizing the sample exchange to the XFEL repetition rate, our method allows for most efficient use of the limited beam time available at XFELs and should enable a substantial increase in sample throughput at these facilities.
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Algoritmos , Cristalografía por Rayos X/métodos , Aumento de la Imagen/métodos , Interpretación de Imagen Asistida por Computador/métodos , Imagenología Tridimensional/métodos , Virus/ultraestructura , Reproducibilidad de los Resultados , Tamaño de la Muestra , Sensibilidad y EspecificidadRESUMEN
X-ray crystallography at X-ray free-electron laser sources is a powerful method for studying macromolecules at biologically relevant temperatures. Moreover, when combined with complementary techniques like X-ray emission spectroscopy, both global structures and chemical properties of metalloenzymes can be obtained concurrently, providing insights into the interplay between the protein structure and dynamics and the chemistry at an active site. The implementation of such a multimodal approach can be compromised by conflicting requirements to optimize each individual method. In particular, the method used for sample delivery greatly affects the data quality. We present here a robust way of delivering controlled sample amounts on demand using acoustic droplet ejection coupled with a conveyor belt drive that is optimized for crystallography and spectroscopy measurements of photochemical and chemical reactions over a wide range of time scales. Studies with photosystem II, the phytochrome photoreceptor, and ribonucleotide reductase R2 illustrate the power and versatility of this method.
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Cristalografía por Rayos X/métodos , Rayos Láser , Acústica , Complejo de Proteína del Fotosistema II/química , Fitocromo/química , Ribonucleótido Reductasas/química , Espectrometría por Rayos X/métodosRESUMEN
We describe a concentric-flow electrokinetic injector for efficiently delivering microcrystals for serial femtosecond X-ray crystallography analysis that enables studies of challenging biological systems in their unadulterated mother liquor. We used the injector to analyze microcrystals of Geobacillus stearothermophilus thermolysin (2.2-Å structure), Thermosynechococcus elongatus photosystem II (<3-Å diffraction) and Thermus thermophilus small ribosomal subunit bound to the antibiotic paromomycin at ambient temperature (3.4-Å structure).
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Cristalografía/métodos , Complejo de Proteína del Fotosistema II/metabolismo , Ribosomas/metabolismo , Modelos MolecularesRESUMEN
In nature, an oxo-bridged Mn4 CaO5 cluster embedded in photosystem II (PSII), a membrane-bound multi-subunit pigment protein complex, catalyzes the water oxidation reaction that is driven by light-induced charge separations in the reaction center of PSII. The Mn4 CaO5 cluster accumulates four oxidizing equivalents to enable the four-electron four-proton catalysis of two water molecules to one dioxygen molecule and cycles through five intermediate S-states, S0 - S4 in the Kok cycle. One important question related to the catalytic mechanism of the oxygen-evolving complex (OEC) that remains is, whether structural isomers are present in some of the intermediate S-states and if such equilibria are essential for the mechanism of the O-O bond formation. Here we compare results from electron paramagnetic resonance (EPR) and X-ray absorption spectroscopy (XAS) obtained at cryogenic temperatures for the S2 state of PSII with structural data collected of the S1 , S2 and S3 states by serial crystallography at neutral pH (â¼6.5) using an X-ray free electron laser at room temperature. While the cryogenic data show the presence of at least two structural forms of the S2 state, the room temperature crystallography data can be well-described by just one S2 structure. We discuss the deviating results and outline experimental strategies for clarifying this mechanistically important question.
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Complejo de Proteína del Fotosistema II/metabolismo , Cristalografía , Espectroscopía de Resonancia por Spin del Electrón , Temperatura , Espectroscopía de Absorción de Rayos XRESUMEN
Chitin is an abundant biopolymer and pathogen-associated molecular pattern that stimulates a host innate immune response. Mammals express chitin-binding and chitin-degrading proteins to remove chitin from the body. One of these proteins, Acidic Mammalian Chitinase (AMCase), is an enzyme known for its ability to function under acidic conditions in the stomach but is also active in tissues with more neutral pHs, such as the lung. Here, we used a combination of biochemical, structural, and computational modeling approaches to examine how the mouse homolog (mAMCase) can act in both acidic and neutral environments. We measured kinetic properties of mAMCase activity across a broad pH range, quantifying its unusual dual activity optima at pH 2 and 7. We also solved high resolution crystal structures of mAMCase in complex with oligomeric GlcNAcn, the building block of chitin, where we identified extensive conformational ligand heterogeneity. Leveraging these data, we conducted molecular dynamics simulations that suggest how a key catalytic residue could be protonated via distinct mechanisms in each of the two environmental pH ranges. These results integrate structural, biochemical, and computational approaches to deliver a more complete understanding of the catalytic mechanism governing mAMCase activity at different pH. Engineering proteins with tunable pH optima may provide new opportunities to develop improved enzyme variants, including AMCase, for therapeutic purposes in chitin degradation.
RESUMEN
Chitin is an abundant biopolymer and pathogen-associated molecular pattern that stimulates a host innate immune response. Mammals express chitin-binding and chitin-degrading proteins to remove chitin from the body. One of these proteins, Acidic Mammalian Chitinase (AMCase), is an enzyme known for its ability to function under acidic conditions in the stomach but is also active in tissues with more neutral pHs, such as the lung. Here, we used a combination of biochemical, structural, and computational modeling approaches to examine how the mouse homolog (mAMCase) can act in both acidic and neutral environments. We measured kinetic properties of mAMCase activity across a broad pH range, quantifying its unusual dual activity optima at pH 2 and 7. We also solved high-resolution crystal structures of mAMCase in complex with oligomeric GlcNAcn, the building block of chitin, where we identified extensive conformational ligand heterogeneity. Leveraging these data, we conducted molecular dynamics simulations that suggest how a key catalytic residue could be protonated via distinct mechanisms in each of the two environmental pH ranges. These results integrate structural, biochemical, and computational approaches to deliver a more complete understanding of the catalytic mechanism governing mAMCase activity at different pH. Engineering proteins with tunable pH optima may provide new opportunities to develop improved enzyme variants, including AMCase, for therapeutic purposes in chitin degradation.
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Quitina , Quitinasas , Simulación de Dinámica Molecular , Quitinasas/metabolismo , Quitinasas/química , Animales , Concentración de Iones de Hidrógeno , Ratones , Quitina/metabolismo , Quitina/química , Conformación Proteica , Cristalografía por Rayos X , Unión Proteica , Ligandos , Cinética , Acetilglucosamina/metabolismo , Acetilglucosamina/química , Modelos MolecularesRESUMEN
Enzymes populate ensembles of structures necessary for catalysis that are difficult to experimentally characterize. We use time-resolved mix-and-inject serial crystallography at an x-ray free electron laser to observe catalysis in a designed mutant isocyanide hydratase (ICH) enzyme that enhances sampling of important minor conformations. The active site exists in a mixture of conformations, and formation of the thioimidate intermediate selects for catalytically competent substates. The influence of cysteine ionization on the ICH ensemble is validated by determining structures of the enzyme at multiple pH values. Large molecular dynamics simulations in crystallo and time-resolved electron density maps show that Asp17 ionizes during catalysis and causes conformational changes that propagate across the dimer, permitting water to enter the active site for intermediate hydrolysis. ICH exhibits a tight coupling between ionization of active site residues and catalysis-activated protein motions, exemplifying a mechanism of electrostatic control of enzyme dynamics.
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Simulación de Dinámica Molecular , Proteínas , Cristalografía por Rayos X , Proteínas/química , Catálisis , Conformación Proteica , HidrolasasRESUMEN
This chapter discusses the use of diffraction simulators to improve experimental outcomes in macromolecular crystallography, in particular for future experiments aimed at diffuse scattering. Consequential decisions for upcoming data collection include the selection of either a synchrotron or free electron laser X-ray source, rotation geometry or serial crystallography, and fiber-coupled area detector technology vs. pixel-array detectors. The hope is that simulators will provide insights to make these choices with greater confidence. Simulation software, especially those packages focused on physics-based calculation of the diffraction, can help to predict the location, size, shape, and profile of Bragg spots and diffuse patterns in terms of an underlying physical model, including assumptions about the crystal's mosaic structure, and therefore can point to potential issues with data analysis in the early planning stages. Also, once the data are collected, simulation may offer a pathway to improve the measurement of diffraction, especially with weak data, and might help to treat problematic cases such as overlapping patterns.
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Análisis de Datos , Programas Informáticos , Simulación por Computador , Cristalografía , Sustancias MacromolecularesRESUMEN
Understanding and controlling protein motion at atomic resolution is a hallmark challenge for structural biologists and protein engineers because conformational dynamics are essential for complex functions such as enzyme catalysis and allosteric regulation. Time-resolved crystallography offers a window into protein motions, yet without a universal perturbation to initiate conformational changes the method has been limited in scope. Here we couple a solvent-based temperature jump with time-resolved crystallography to visualize structural motions in lysozyme, a dynamic enzyme. We observed widespread atomic vibrations on the nanosecond timescale, which evolve on the submillisecond timescale into localized structural fluctuations that are coupled to the active site. An orthogonal perturbation to the enzyme, inhibitor binding, altered these dynamics by blocking key motions that allow energy to dissipate from vibrations into functional movements linked to the catalytic cycle. Because temperature jump is a universal method for perturbing molecular motion, the method demonstrated here is broadly applicable for studying protein dynamics.
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Proteínas , Cristalografía por Rayos X , Modelos Moleculares , Temperatura , Proteínas/química , Conformación Molecular , Conformación ProteicaRESUMEN
Enzymes populate ensembles of structures with intrinsically different catalytic proficiencies that are difficult to experimentally characterize. We use time-resolved mix-and-inject serial crystallography (MISC) at an X-ray free electron laser (XFEL) to observe catalysis in a designed mutant (G150T) isocyanide hydratase (ICH) enzyme that enhances sampling of important minor conformations. The active site exists in a mixture of conformations and formation of the thioimidate catalytic intermediate selects for catalytically competent substates. A prior proposal for active site cysteine charge-coupled conformational changes in ICH is validated by determining structures of the enzyme over a range of pH values. A combination of large molecular dynamics simulations of the enzyme in crystallo and time-resolved electron density maps shows that ionization of the general acid Asp17 during catalysis causes additional conformational changes that propagate across the dimer interface, connecting the two active sites. These ionization-linked changes in the ICH conformational ensemble permit water to enter the active site in a location that is poised for intermediate hydrolysis. ICH exhibits a tight coupling between ionization of active site residues and catalysis-activated protein motions, exemplifying a mechanism of electrostatic control of enzyme dynamics.
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Alteration of antibiotic binding sites through modification of ribosomal RNA (rRNA) is a common form of resistance to ribosome-targeting antibiotics. The rRNA-modifying enzyme Cfr methylates an adenosine nucleotide within the peptidyl transferase center, resulting in the C-8 methylation of A2503 (m8A2503). Acquisition of cfr results in resistance to eight classes of ribosome-targeting antibiotics. Despite the prevalence of this resistance mechanism, it is poorly understood whether and how bacteria modulate Cfr methylation to adapt to antibiotic pressure. Moreover, direct evidence for how m8A2503 alters antibiotic binding sites within the ribosome is lacking. In this study, we performed directed evolution of Cfr under antibiotic selection to generate Cfr variants that confer increased resistance by enhancing methylation of A2503 in cells. Increased rRNA methylation is achieved by improved expression and stability of Cfr through transcriptional and post-transcriptional mechanisms, which may be exploited by pathogens under antibiotic stress as suggested by natural isolates. Using a variant that achieves near-stoichiometric methylation of rRNA, we determined a 2.2 Å cryo-electron microscopy structure of the Cfr-modified ribosome. Our structure reveals the molecular basis for broad resistance to antibiotics and will inform the design of new antibiotics that overcome resistance mediated by Cfr.
Antibiotics treat or prevent infections by killing bacteria or slowing down their growth. A large proportion of these drugs do this by disrupting an essential piece of cellular machinery called the ribosome which the bacteria need to make proteins. However, over the course of the treatment, some bacteria may gain genetic alterations that allow them to resist the effects of the antibiotic. Antibiotic resistance is a major threat to global health, and understanding how it emerges and spreads is an important area of research. Recent studies have discovered populations of resistant bacteria carrying a gene for a protein named chloramphenicol-florfenicol resistance, or Cfr for short. Cfr inserts a small modification in to the ribosome that prevents antibiotics from inhibiting the production of proteins, making them ineffective against the infection. To date, Cfr has been found to cause resistance to eight different classes of antibiotics. Identifying which mutations enhance its activity and protect bacteria is vital for designing strategies that fight antibiotic resistance. To investigate how the gene for Cfr could mutate and make bacteria more resistant, Tsai et al. performed a laboratory technique called directed evolution, a cyclic process which mimics natural selection. Genetic changes were randomly introduced in the gene for the Cfr protein and bacteria carrying these mutations were treated with tiamulin, an antibiotic rendered ineffective by the modification Cfr introduces into the ribosome. Bacteria that survived were then selected and had more mutations inserted. By repeating this process several times, Tsai et al. identified 'super' variants of the Cfr protein that lead to greater resistance. The experiments showed that these variants boosted resistance by increasing the proportion of ribosomes that contained the protective modification. This process was facilitated by mutations that enabled higher levels of Cfr protein to accumulate in the cell. In addition, the current study allowed, for the first time, direct visualization of how the Cfr modification disrupts the effect antibiotics have on the ribosome. These findings will make it easier for clinics to look out for bacteria that carry these 'super' resistant mutations. They could also help researchers design a new generation of antibiotics that can overcome resistance caused by the Cfr protein.
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Evolución Molecular Dirigida/métodos , Farmacorresistencia Microbiana/genética , Proteínas de Escherichia coli/genética , Escherichia coli/genética , Metiltransferasas/genética , ARN Ribosómico/genética , Adenosina/metabolismo , Antibacterianos/farmacología , Sitios de Unión , Escherichia coli/efectos de los fármacos , MetilaciónRESUMEN
The antibiotic linezolid, the first clinically approved member of the oxazolidinone class, inhibits translation of bacterial ribosomes by binding to the peptidyl transferase center. Recent work has demonstrated that linezolid does not inhibit peptide bond formation at all sequences but rather acts in a context-specific manner, namely when alanine occupies the penultimate position of the nascent chain. However, the molecular basis for context-specificity has not been elucidated. Here we show that the second-generation oxazolidinone radezolid also induces stalling with a penultimate alanine, and we determine high-resolution cryo-EM structures of linezolid- and radezolid-stalled ribosome complexes to explain their mechanism of action. These structures reveal that the alanine side chain fits within a small hydrophobic crevice created by oxazolidinone, resulting in improved ribosome binding. Modification of the ribosome by the antibiotic resistance enzyme Cfr disrupts stalling due to repositioning of the modified nucleotide. Together, our findings provide molecular understanding for the context-specificity of oxazolidinones.
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Antibacterianos/química , Antibacterianos/farmacología , Oxazolidinonas/química , Oxazolidinonas/farmacología , Biosíntesis de Proteínas/efectos de los fármacos , Alanina/química , Sitios de Unión , Microscopía por Crioelectrón , Linezolid/química , Linezolid/farmacología , Modelos Moleculares , Peptidil Transferasas/metabolismo , ARN Ribosómico/química , ARN Ribosómico/metabolismo , ARN de Transferencia/química , ARN de Transferencia/metabolismo , Ribosomas/efectos de los fármacos , Ribosomas/metabolismo , Ribosomas/ultraestructuraRESUMEN
Cry11Aa and Cry11Ba are the two most potent toxins produced by mosquitocidal Bacillus thuringiensis subsp. israelensis and jegathesan, respectively. The toxins naturally crystallize within the host; however, the crystals are too small for structure determination at synchrotron sources. Therefore, we applied serial femtosecond crystallography at X-ray free electron lasers to in vivo-grown nanocrystals of these toxins. The structure of Cry11Aa was determined de novo using the single-wavelength anomalous dispersion method, which in turn enabled the determination of the Cry11Ba structure by molecular replacement. The two structures reveal a new pattern for in vivo crystallization of Cry toxins, whereby each of their three domains packs with a symmetrically identical domain, and a cleavable crystal packing motif is located within the protoxin rather than at the termini. The diversity of in vivo crystallization patterns suggests explanations for their varied levels of toxicity and rational approaches to improve these toxins for mosquito control.
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Bacillus thuringiensis , Nanopartículas , Animales , Proteínas Bacterianas/toxicidad , Endotoxinas , Proteínas Hemolisinas/toxicidad , Larva , Control de MosquitosRESUMEN
Light-driven oxidation of water to molecular oxygen is catalyzed by the oxygen-evolving complex (OEC) in Photosystem II (PS II). This multi-electron, multi-proton catalysis requires the transport of two water molecules to and four protons from the OEC. A high-resolution 1.89 Å structure obtained by averaging all the S states and refining the data of various time points during the S2 to S3 transition has provided better visualization of the potential pathways for substrate water insertion and proton release. Our results indicate that the O1 channel is the likely water intake pathway, and the Cl1 channel is the likely proton release pathway based on the structural rearrangements of water molecules and amino acid side chains along these channels. In particular in the Cl1 channel, we suggest that residue D1-E65 serves as a gate for proton transport by minimizing the back reaction. The results show that the water oxidation reaction at the OEC is well coordinated with the amino acid side chains and the H-bonding network over the entire length of the channels, which is essential in shuttling substrate waters and protons.