Auswahl der wissenschaftlichen Literatur zum Thema „Time-Resolved cryoEM“

Blotting times for conventional cryoEM specimen preparation complicate time-resolved studies and lead to some specimens adopting preferred orientations or denaturing at the air–water interface. Here, it is shown that solution sprayed onto one side of a holey cryoEM grid can be wicked through the grid by a glass-fiber filter held against the opposite side, often called the `back', of the grid, producing a film suitable for vitrification. This process can be completed in tens of milliseconds. Ultrasonic specimen application and through-grid wicking were combined in a high-speed specimen-preparation device that was named `Back-it-up' or BIU. The high liquid-absorption capacity of the glass fiber compared with self-wicking grids makes the method relatively insensitive to the amount of sample applied. Consequently, through-grid wicking produces large areas of ice that are suitable for cryoEM for both soluble and detergent-solubilized protein complexes. The speed of the device increases the number of views for a specimen that suffers from preferred orientations.

Connexins (Cxs) are a family of integral membrane proteins, which function as both hexameric hemichannels (HCs) and dodecameric gap junction channels (GJCs), behaving as conduits for the electrical and molecular communication between cells and between cells and the extracellular environment, respectively. Their proper functioning is crucial for many processes, including development, physiology, and response to disease and trauma. Abnormal GJC and HC communication can lead to numerous pathological states including inflammation, skin diseases, deafness, nervous system disorders, and cardiac arrhythmias. Over the last 15 years, high-resolution X-ray and electron cryomicroscopy (cryoEM) structures for seven Cx isoforms have revealed conservation in the four-helix transmembrane (TM) bundle of each subunit; an αβ fold in the disulfide-bonded extracellular loops and inter-subunit hydrogen bonding across the extracellular gap that mediates end-to-end docking to form a tight seal between hexamers in the GJC. Tissue injury is associated with cellular Ca2+ overload. Surprisingly, the binding of 12 Ca2+ ions in the Cx26 GJC results in a novel electrostatic gating mechanism that blocks cation permeation. In contrast, acidic pH during tissue injury elicits association of the N-terminal (NT) domains that sterically blocks the pore in a “ball-and-chain” fashion. The NT domains under physiologic conditions display multiple conformational states, stabilized by protein–protein and protein–lipid interactions, which may relate to gating mechanisms. The cryoEM maps also revealed putative lipid densities within the pore, intercalated among transmembrane α-helices and between protomers, the functions of which are unknown. For the future, time-resolved cryoEM of isolated Cx channels as well as cryotomography of GJCs and HCs in cells and tissues will yield a deeper insight into the mechanisms for channel regulation. The cytoplasmic loop (CL) and C-terminal (CT) domains are divergent in sequence and length, are likely involved in channel regulation, but are not visualized in the high-resolution X-ray and cryoEM maps presumably due to conformational flexibility. We expect that the integrated use of synergistic physicochemical, spectroscopic, biophysical, and computational methods will reveal conformational dynamics relevant to functional states. We anticipate that such a wealth of results under different pathologic conditions will accelerate drug discovery related to Cx channel modulation.

Time-resolved carbamazepine crystallization from wet ethanol has been monitored using a combination of cryoTEM and 3D electron diffraction. Carbamazepine is shown to crystallize exclusively as a dihydrate after 180 s. When the timescale was reduced to 30 s, three further polymorphs could be identified. At 20 s, the development of early stage carbamazepine dihydrate was observed through phase separation. This work reveals two possible crystallization pathways present in this active pharmaceutical ingredient.

Transmission electron microscopy of rapidly-frozen, hydrated specimens (cryo-TEM) is a powerful way of examining labile microstructures. This technique avoids some artifacts associated with conventional preparative methods. Use of a controlled environment vitrification system (CEVS) for specimen preparation reduces the risk of unwanted sample changes due to evaporation, and permits the examination of specimens vitrified from a defined temperature. Studies of dynamic processes with time resolution on the order of seconds, in which the process was initiated by changes in sample pH, have been conducted. We now report the development of an optical method for increasing specimen temperature immediately before vitrification. Using our method, processes that are regulated by temperature can be initiated in less than 500 msec on the specimen grid. The ensuing events can then be captured by plunge-freezing within an additional 200 msec.Dimyristoylphosphatidylcholine (DMPC) liposomes, produced by extrusion, were used as test specimens. DMPC undergoes a gel/liquid crystalline transition at 24°C, inducing a change in liposome morphology from polyhedral to spherical. Five-μl aliquots of DMPC dispersions were placed on holey-carbon-filmed copper grids mounted in the CEVS environmental chamber, and maintained at 6-8°C and 80% relative humidity. Immediately before the temperature jump most of the sample was blotted away with filter paper, leaving a thin specimen film on the grid. Upon pressing the trigger, an electronic control circuit generated this timed sequence of events. First, a solenoid-activated shutter was opened to heat the specimen by exposing it for a variable time to the focused beam of a 75W Xenon arc lamp. Simultaneously, a solenoid-activated cryogen shutter in the bottom of the CEVS was opened. Next, the lamp shutter was closed after the desired heating interval. Finally, a solenoid-activated cable release was used to trigger a spring-loaded plunger in the CEVS, propelling the sample into a reservoir of liquid ethane. Vitrified samples were subsequently transferred to a Zeiss EM902 TEM, operated in zero-loss brightfield mode, for examination at −163°C.

As the resolution revolution in CryoEM expands to encompass all manner of macromolecular complexes, an important new frontier is the implementation of cryogenic time resolved EM (cryoTREM). Biological macromolecular complexes are dynamic systems that undergo conformational changes on timescales from microseconds to minutes. Understanding the dynamic nature of biological changes is critical to understanding function. To realize the full potential of CryoEM, time resolved methods will be integral in coupling static structures to dynamic functions. Here, we present an LED-based photo-flash system as a core part of the sample preparation phase in CryoTREM. The plug-and-play system has a wide range of operational parameters, is low cost and ensures uniform irradiation and minimal heating of the sample prior to plunge freezing. The complete design including electronics and optics, manufacturing, control strategies and operating procedures are discussed for the Thermo Scientific™ Vitrobot and Leica™ EM GP2 plunge freezers. Possible adverse heating effects on the biological sample are also addressed through theoretical as well as experimental studies.

We have developed a confocal laser microscope operating in the mid-infrared range for the study of light-sensitive proteins, such as rhodopsins. The microscope features a co-aligned infrared and visible illumination path for the selective excitation and probing of proteins located in the IR focus only. An external-cavity tunable quantum cascade laser provides a wavelength tuning range (5.80–6.35 µm or 1570–1724 cm−1) suitable for studying protein conformational changes as a function of time delay after visible light excitation with a pulsed LED. Using cryogen-free detectors, the relative changes in the infrared absorption of rhodopsin thin films around 10−4 have been observed with a time resolution down to 30 ms. The measured full-width at half maximum of the Airy disk at λ = 6.08 µm in transmission mode with a confocal arrangement of apertures is 6.6 µm or 1.1λ. Dark-adapted sample replacement at the beginning of each photocycle is then enabled by exchanging the illuminated thin-film location with the microscope mapping stage synchronized to data acquisition and LED excitation and by averaging hundreds of time traces acquired in different nearby locations within a homogeneous film area. We demonstrate that this instrument provides crucial advantages for time-resolved IR studies of rhodopsin thin films with a slow photocycle. Time-resolved studies of inhomogeneous samples may also be possible with the presented instrument.

Rosacea is a chronic dermatosis with no cure. Our goal was to evaluate if the combination of flashlamp-pumped Pulsed Dye Laser (PDL) treatment with topical imiquimod could improve therapeutic outcomes. Fourty patients diagnosed with rosacea and aged between 16 and 53 years were assigned for three different types of treatment: i) PDL-only, ii) imiquimod-only, and iii) PDL + imiquimod. The PDL test sites received a single treatment with the VBeam laser (λ = 595 nm; spot size = 7 mm; tp = 1500 msec) at a dosage of 10 J/cm2 with cryogen spurt duration (30 msec) and the delay time (20 msec). For the test sites of PDL + imiquimod and imiquimod-only, the patients applied imiquimod topically to the test sites once a day for 1 month. Patients were followed-up at 1, 3, and 6 months. The primary efficacy was measured with a DermoSpectrometer. Patients were also monitored for adverse effects. Pair-wise analysis showed statistically significant differences between the blanching responses for the PDL + imiquimod and PDL-only and imiquimod-only treatments (p<0.005). Transient hyperpigmentation was noted in 5% (n=2) and 20% (n=8) of patients in the PDL + imiquimod and PDL-only treatment, respectively. Hyperpigmentation resolved spontaneously within 6 months. Permanent hypopigmentation or scarring was not observed. Superior blanching responses were obtained when using PDL + imiquimod than PDL-only or imiquimod-only treatment for rosacea. A larger number of patients are required to support the results of this study.

Selon l’organisation mondiale de la santé, la résistance aux antibiotiques est un problème majeur pour l’humanité en raison de l’émergence de bactérie multi-résistantes. L’émergence de ces résistances chez les bactéries provient du fait qu’elles sont capables de mettre en place de nombreuses stratégies pour empêcher les antibiotiques de fonctionner. En particulier, la première ligne de défense de ces bactéries est la surexpression de transporteurs ABC (ATP-Binding Cassette) qui expulsent les antibiotiques en dehors de la cellule bactérienne, diminuant leurs concentrations sous leurs seuils de cytotoxicité. Plus de 50 ans d’étude sur ces transporteurs ont permis à la communauté scientifique d’établir un mécanisme global, notamment grâce à l’acquisition de structures 3D de plus en plus nombreuses. Ceci a été intimement lié par l’évolution technologique et méthodologique de la biologie structurale ces dernières années avec notamment l’émergence de la cryoEM. Plus les connaissances avancent plus les questions deviennent précises, et il demeure toujours de nombreuses questions sur la compréhension de leur fonctionnement. Dans le cadre de mon projet, j’ai étudié BmrA, l’un de ces transporteur ABC exprimé chez Bacillus subtilis qui lui confère une résistance à la cervimycine C, un antibiotique sécrété par Streptomyces tendae son compétiteur naturel dans le même biotope. De plus, ce transporteur est capable de fixer et de transporter une grande variété de molécules dont de nombreux antibiotiques en adoptant à la fois une conformation prenant en charge le ligand (IF, conformation ouverte vers l’intérieur de la cellule), et une conformation ouverte vers l’extérieur de la cellule (OF) pour le relarguer. Cette capacité de manipuler plusieurs molécules reste une question très discutée, surtout dans la compréhension du mécanisme de transport au niveau moléculaire. Au cours de ma thèse, j’ai participé à une étude d’enzymologie structurale sur un mutant inactif E504A en présence de ligands (Rhodamine 6G, Hœchst 33342) afin d’améliorer les connaissances sur ce mécanisme. Ces ligands jouent un rôle d’effecteur allostérique sur la fixation d’ATP de BmrA, impactant la transition entre les conformations IF et OF. La résolution de plusieurs structures 3D par cryoEM a été réalisé en variant la concentration d’ATP. Une analyse de la flexibilité de chacune de ces conformations a mis en lumière les réarrangements moléculaires que BmrA peut adopter pour assurer sa poly-spécificité. De plus, j’ai apporté de nombreuses informations fonctionnelles en ce qui concerne le couplage entre le transport du ligand et l’activité ATPasique de ce transporteur. La deuxième partie de mes travaux repose sur l’étude de la transition conformationnelle se produisant chez BmrA après la fixation d’ATP à l’aide de techniques dites « en temps résolu ». L’objectif a été de suivre ces changements conformationnels au cours du temps grâce à la fluorescence intrinsèque de BmrA couplée à la cryoEM. J’ai développé et optimisé les conditions expérimentales pour réaliser cette étude, et notamment acquis des informations cinétiques et dynamiques sur des mutants ainsi que la protéine sauvage.Enfin, la dernière partie du manuscrit a consisté à reconstituer BmrA dans un environnement amphipathique plus natif que les détergents afin d’acquérir sa structure 3D par cryoEM. J’ai optimisé ce protocole de reconstitution pour obtenir le meilleur échantillon possible à déposer sur grille. Au cours de ce processus, j’ai caractérisé la formation du protéoliposome à chaque étape du protocole en l’observant par cryoEM. Grâce à cette étude, j’ai pu obtenir les premières classes 2D de BmrA en bicouche lipidique. En conclusion, cette thèse offre une nouvelle façon d’étudier la relation structure-fonction des protéines en développant des outils et une méthodologie d’enzymologie structurale pour visualiser la dynamique de ce transporteur ABC, ainsi qu’une première approche pour l’étudier en liposome
According to the World Health Organization, antibiotic resistance is a major problem for humanity due to the emergence of multiresistant bacteria. The emergence of these resistances in bacteria is due to their ability to implement numerous strategies to prevent antibiotics from working. In particular, the first line of defense of these bacteria is the overexpression of ABC (ATP-Binding Cassette) transporters, which expel antibiotics out of the bacterial cell, reducing their concentrations below their cytotoxic thresholds. Over 50 years of study on these transporters have enabled the scientific community to establish a global mechanism, particularly thanks to the increasing acquisition of 3D structures. This has been closely linked to the technological and methodological evolution of structural biology in recent years, especially with the emergence of cryoEM. As knowledge advances, the questions become more precise, and many questions remain about understanding their functioning. As part of my project, I studied BmrA, one of these ABC transporters expressed in Bacillus subtilis, which confers resistance to cervimycin C, an antibiotic secreted by Streptomyces tendae, its natural competitor in the same biotope. Additionally, this transporter is capable of binding and transporting a wide variety of molecules, including many antibiotics, by adopting both a conformation that takes up the ligand (IF, inward-facing conformation) and an outward-facing conformation (OF) to release it. This ability to handle multiple molecules remains a highly debated question, especially in understanding the transport mechanism at the molecular level. During my Ph.D., I participated in a structural enzymology study on an inactive E504A mutant in the presence of ligands (Rhodamine 6G, Hoechst 33342) to improve knowledge of this mechanism. These ligands act as allosteric effectors on the ATP binding of BmrA, impacting the transition between IF and OF conformations. The resolution of several 3D structures by cryoEM was achieved by varying the concentration of ATP. An analysis of the flexibility of each of these conformations highlighted the molecular rearrangements that BmrA can adopt to ensure its polyspecificity. Moreover, I provided numerous functional insights regarding the coupling between ligand transport and the ATPase activity of this transporter. The second part of my work focused on studying the conformational transition occurring in BmrA after ATP binding using so-called "time-resolved" techniques. The objective was to monitor these conformational changes over time using the intrinsic fluorescence of BmrA coupled with cryoEM. I developed and optimized the experimental conditions to conduct this study, particularly acquiring kinetic and dynamic information on mutants as well as the wild-type protein. Finally, the last part of the manuscript involved reconstituting BmrA in a more native amphipathic environment than detergents to obtain its 3D structure by cryoEM. I optimized this reconstitution protocol to obtain the best possible sample for grid deposition. During this process, I characterized the formation of the proteoliposome at each stage of the protocol by observing it with cryoEM. Thanks to this study, I was able to obtain the first 2D classes of BmrA in a lipid bilayer. In conclusion, this thesis offers a new way to study the structure-function relationship of proteins by developing structural enzymology tools and methodology to visualize the dynamics of this ABC transporter, as well as a first approach to studying it in liposomes

Translation is the process by which the cell produces new proteins on the ribosome, as directed by genetic instructions, in all living organisms. Structural studies of the ribosome have shed considerable lights on its mechanism and regulation. Cryogenic electron microscopy (cryo-EM) and single-particle reconstruction technique is one of the major approaches to studying ribosome structure. In this thesis, I report the use of cryo-EM and related new techniques to study the structure of ribosome complexes. This work is divided into three parts. First, in Chapter 3, I describe the development of a computational method in the classification of cryo-EM data. Recently developed classification methods have enabled resolving multiple structures/conformations of the molecules from cryo-EM data obtained on a heterogeneous biological sample. However, the classification methods all involve various amounts of arbitrary decisions made by researchers, which can limit the use of these methods by inexperienced users. As a step toward fully automated classification, I worked with colleagues to develop a "jumper analysis" to determine the number of distinguishable classes of 3D reconstruction, based on the statistics of cryo-EM particles. Second, in Chapter 4, I document the cryo-EM study of EttA-70S ribosome complex, which provided structural insights into the mechanism of EttA in translation regulation. Energy-dependent translation throttle A (EttA, previously named YjjK in Escherichia coli) is the most prevalent member of ATP-binding cassette F family proteins in eubacteria. Through a collaboration among the Hunt, Frank, and Gonzalez labs, we combined crystallography, biochemical, cryo-EM and single-molecule fluorescence energy transfer techniques to elucidate the function and mechanism of EttA. We demonstrated that EttA gates ribosome entry into the translation elongation cycle through a nucleotide-dependent interaction sensitive to ATP/ADP ratio. We also showed that the ATP-bound form of EttA binds to the ribosomal tRNA-exit site, and restricts the ribosome and tRNA dynamics required for translation. Thirdly, in Chapter 5, I discuss the improvements to a new technique, time-resolved cryo-EM by mixing-spraying, and its application to ribosome studies. The mixing-spraying method can study processes involving two big biological molecules that are in the sub-second time scale. I worked with colleagues to apply this method to studying ribosome subunit association. By mixing the subunits and reacting for 60 ms and 140 ms, we were able to capture the association reaction in a pre-equilibrium state. We detected three 70S ribosome conformations in the system. Quantification of the proportions of particles assuming these conformations suggested that the 70S ribosome can undergo fast conformational changes upon formation, and reaches equilibrium among these conformations earlier than 60 ms. In addition, I present preliminary results of studying translation decoding using the mixing-spraying method. This study, performed before improving the mixing-spraying method, was inconclusive mainly due to the limited size of cryo-EM data. Now that we have demonstrated the capability of the mixing-spraying method to visualize multiple states of molecules in a sub-second reaction, the translation decoding process can be revisited and many other processes, such as translation initiation, can be studied.

Single-particle reconstruction technique is one of the major approaches to studying ribosome structure and membrane proteins. In this thesis, I report the use of time-resolved cryo-EM technique to study the structure of short-lived ribosome complexes and conventional cryo-EM technique to study the structure of ribosome complexes and membrane proteins. The thesis consists three parts. The first part is the development of time-resolved cryo-EM technique. I document the protocol for how to capture short-lived states of the molecules with time-resolved cryo-EM technique using microfluidic chip. Working closely with Dr. Lin’s lab at Columbia University Engineering Department, I designed and tested a well-controlled and effective microspraying-plunging method to prepare cryo-grids. I demonstrated the performance of this device by a 3-Å reconstruction from about 4000 particles collected on grids sprayed with apoferritin suspension. The second part is the application of time-resolved cryo-EM technique for studying short-lived ribosome complexes in bacteria translation processes on the time-scale of 10-1000 ms. I document three applications on bacterial translation processes. The initiation project is collaborated with Dr. Gonzalez’s lab at Chemistry Department, Columbia University. The termination and recycling projects are collaborated with Dr. Ehrenberg’s lab at Department of Cell and Molecular Biology, Uppsala University. I captured and solved short-lived ribosome intermediates complexes in these processes. The results demonstrate the power of time-resolved cryo-EM to determine how a time-ordered series of conformational changes contribute to the mechanism and regulation of one of the most fundamental processes in biology. The last part is the application of conventional cryo-EM technique to study ribosome complexes and membrane proteins. This part includes five collaboration projects. Human GABA(B) receptor project is the collaboration with Dr. Fan at Department of Pharmacology, Columbia University. Cyclic nucleotide-gated (CNG) channels project is the collaboration with Dr. Yang at Department of Biological Sciences, Columbia University. The cryo-EM study of Ybit-70S ribosome complex and Cystic fibrosis transmembrane conductance regulator (CFTR) project are the collaboration with Dr. Hunt at Department of Biological Sciences, Columbia University. The cryo-EM study of native lipid bilayer in membrane protein transporter is the collaboration with Dr. Hendrickson at Department of Biochemistry and Molecular Biophysics, Columbia University and Dr. Guo at Department of Medicinal Chemistry, Virginia Commonwealth University.

Abstract During the early 1980s, Dubochet and his colleagues developed a technique for preparing and rapidly freezing thin aqueous films suspended on an electron microscope grid. This technique has encouraged an enormous expansion in structural analysis by electron microscopy of ice-embedded macromolecules, macromolecular assemblies such as viruses and ribosomes, and tubular or two-dimensional crystals of membrane proteins. The critical step of rapid freezing, which had been adapted from earlier procedures to achieve verification of aqueous solutions and to explore fast biological processes, simply consists of plunging a thin (10-100 nm) aqueous film into a cryogen such as liquid ethane at a temperature near its freezing point (~90 K). The cooling rate attained at the surface of the aqueous film is thought to be, in excess of 10 Ks-1 so that the time to cool from room temperature to near liquid nitrogen temperature is a small fraction of a millisecond.