The Ribosome—Structure and Functional Ligand-Binding Experiments Using Cryo-electron Microscopy

1998 ◽  
Vol 124 (2-3) ◽  
pp. 142-150 ◽  
Author(s):  
Joachim Frank
Keyword(s):  
Electron Microscopy ◽  
Ligand Binding ◽  
Ribosome Structure ◽  
Cryo Electron Microscopy

scholarly journals Architecture and subunit arrangement of native AMPA receptors elucidated by cryo-EM

Science ◽  
2019 ◽  
Vol 364 (6438) ◽  
pp. 355-362 ◽  
Author(s):  
Yan Zhao ◽  
Shanshuang Chen ◽  
Adam C. Swensen ◽  
Wei-Jun Qian ◽  
Eric Gouaux
Keyword(s):  
Molecular Structure ◽  
Electron Microscopy ◽  
Signal Transduction ◽  
Ligand Binding ◽  
Ampa Receptor ◽  
Ampa Receptors ◽  
Mammalian Brain ◽  
Receptor Complexes ◽  
Cryo Electron Microscopy ◽  
Chemical Synapses

Glutamate-gated AMPA receptors mediate the fast component of excitatory signal transduction at chemical synapses throughout all regions of the mammalian brain. AMPA receptors are tetrameric assemblies composed of four subunits, GluA1–GluA4. Despite decades of study, the subunit composition, subunit arrangement, and molecular structure of native AMPA receptors remain unknown. Here we elucidate the structures of 10 distinct native AMPA receptor complexes by single-particle cryo–electron microscopy (cryo-EM). We find that receptor subunits are arranged nonstochastically, with the GluA2 subunit preferentially occupying the B and D positions of the tetramer and with triheteromeric assemblies comprising a major population of native AMPA receptors. Cryo-EM maps define the structure for S2-M4 linkers between the ligand-binding and transmembrane domains, suggesting how neurotransmitter binding is coupled to ion channel gating.

scholarly journals Cryo-EM structure of a 40 kDa SAM-IV riboswitch RNA at 3.7 Å resolution

2019 ◽  
Vol 10 (1) ◽  
Author(s):  
Kaiming Zhang ◽  
Shanshan Li ◽  
Kalli Kappel ◽  
Grigore Pintilie ◽  
Zhaoming Su ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
High Resolution ◽  
Ligand Binding ◽  
Small Rnas ◽  
Sulfur Metabolism ◽  
Cryo Electron Microscopy ◽  
Binding Core ◽  
New Antibiotics ◽  
Binding Pockets ◽  
High Flexibility

AbstractSpecimens below 50 kDa have generally been considered too small to be analyzed by single-particle cryo-electron microscopy (cryo-EM). The high flexibility of pure RNAs makes it difficult to obtain high-resolution structures by cryo-EM. In bacteria, riboswitches regulate sulfur metabolism through binding to the S-adenosylmethionine (SAM) ligand and offer compelling targets for new antibiotics. SAM-I, SAM-I/IV, and SAM-IV are the three most commonly found SAM riboswitches, but the structure of SAM-IV is still unknown. Here, we report the structures of apo and SAM-bound SAM-IV riboswitches (119-nt, ~40 kDa) to 3.7 Å and 4.1 Å resolution, respectively, using cryo-EM. The structures illustrate homologies in the ligand-binding core but distinct peripheral tertiary contacts in SAM-IV compared to SAM-I and SAM-I/IV. Our results demonstrate the feasibility of resolving small RNAs with enough detail to enable detection of their ligand-binding pockets and suggest that cryo-EM could play a role in structure-assisted drug design for RNA.

scholarly journals Cryo-electron microscopy structures and progress toward a dynamic understanding of KATP channels

2018 ◽  
Vol 150 (5) ◽  
pp. 653-669 ◽  
Author(s):  
Michael C. Puljung
Keyword(s):  
Electron Microscopy ◽  
Ligand Binding ◽  
Adenine Nucleotide ◽  
Adenine Nucleotides ◽  
Cell Metabolism ◽  
Katp Channels ◽  
K Channel ◽  
Sulfonylurea Receptor ◽  
Binding Domains ◽  
Cryo Electron Microscopy

Adenosine triphosphate (ATP)–sensitive K+ (KATP) channels are molecular sensors of cell metabolism. These hetero-octameric channels, comprising four inward rectifier K+ channel subunits (Kir6.1 or Kir6.2) and four sulfonylurea receptor (SUR1 or SUR2A/B) subunits, detect metabolic changes via three classes of intracellular adenine nucleotide (ATP/ADP) binding site. One site, located on the Kir subunit, causes inhibition of the channel when ATP or ADP is bound. The other two sites, located on the SUR subunit, excite the channel when bound to Mg nucleotides. In pancreatic β cells, an increase in extracellular glucose causes a change in oxidative metabolism and thus turnover of adenine nucleotides in the cytoplasm. This leads to the closure of KATP channels, which depolarizes the plasma membrane and permits Ca2+ influx and insulin secretion. Many of the molecular details regarding the assembly of the KATP complex, and how changes in nucleotide concentrations affect gating, have recently been uncovered by several single-particle cryo-electron microscopy structures of the pancreatic KATP channel (Kir6.2/SUR1) at near-atomic resolution. Here, the author discusses the detailed picture of excitatory and inhibitory ligand binding to KATP that these structures present and suggests a possible mechanism by which channel activation may proceed from the ligand-binding domains of SUR to the channel pore.

New challenges to image processing posed by cryo-Electron microscopy of single particles

1991 ◽  
Vol 49 ◽  
pp. 422-423
Author(s):  
Joachim Frank
Keyword(s):  
Electron Microscopy ◽  
Phase Contrast ◽  
Particle Orientation ◽  
Single Particles ◽  
Contrast Transfer Function ◽  
Virtual Absence ◽  
Cryo Electron Microscopy ◽  
Spatial Frequencies ◽  
New Challenges ◽  
Particle Images

Compared with images of negatively stained single particle specimens, those obtained by cryo-electron microscopy have the following new features: (a) higher “signal” variability due to a higher variability of particle orientation; (b) reduced signal/noise ratio (S/N); (c) virtual absence of low-spatial-frequency information related to elastic scattering, due to the properties of the phase contrast transfer function (PCTF); and (d) reduced resolution due to the efforts of the microscopist to boost the PCTF at low spatial frequencies, in his attempt to obtain recognizable particle images.

Enhanced information from biological materials through technological improvements in cryo-electron microscopy

1992 ◽  
Vol 50 (1) ◽  
pp. 788-789
Author(s):  
Marc J.C. de Jong ◽  
Wim M. Busing ◽  
Max T. Otten
Keyword(s):  
Electron Microscopy ◽  
Electron Beam ◽  
Biological Materials ◽  
Electron Crystallography ◽  
Cryo Electron Microscopy ◽  
Transmission Electron ◽  
The Many ◽  
Technological Improvements ◽  
Beam Damage

Biological materials damage rapidly in the electron beam, limiting the amount of information that can be obtained in the transmission electron microscope. The discovery that observation at cryo temperatures strongly reduces beam damage (in addition to making it unnecessaiy to use chemical fixatives, dehydration agents and stains, which introduce artefacts) has given an important step forward to preserving the ‘live’ situation and makes it possible to study the relation between function, chemical composition and morphology.Among the many cryo-applications, the most challenging is perhaps the determination of the atomic structure. Henderson and co-workers were able to determine the structure of the purple membrane by electron crystallography, providing an understanding of the membrane's working as a proton pump. As far as understood at present, the main stumbling block in achieving high resolution appears to be a random movement of atoms or molecules in the specimen within a fraction of a second after exposure to the electron beam, which destroys the highest-resolution detail sought.

Study of eukaryotic flagellar components by cryo-electron microscopy

1986 ◽  
Vol 44 ◽  
pp. 98-101
Author(s):  
John M. Murray ◽  
Rob Ward
Keyword(s):  
Electron Microscopy ◽  
Primary Motion ◽  
Cryo Electron Microscopy ◽  
Cross Links ◽  
Cilia And Flagella

The eukaryotic flagellum is constructed from 11 parallel tubular elements arranged as 9 peripheral fibers (doublet microtubules) and 2 central fibers (singlet microtubules). The primary motion generating component has been found to be arranged as axially periodic “arms” bridging the adjacent doublets. The dynein, comprising the arms, has been isolated and characterized from several different cilia and flagella. Various radial and azimuthal cross-links stabilize the axially aligned microtubules, and probably play some role in controlling the form of the flagella beat cycle.

CRYO-Electron Microscopy of the Acto-Myosin-ATP Complex

1990 ◽  
Vol 48 (1) ◽  
pp. 248-249
Author(s):  
John Trinickt ◽  
Howard White
Keyword(s):  
Electron Microscopy ◽  
Atp Hydrolysis ◽  
Myosin Head ◽  
Bound State ◽  
Cross Linking ◽  
Weakly Bound ◽  
Cryo Electron Microscopy ◽  
Myosin Subfragment 1 ◽  
Attached State ◽  
High Fraction

The primary force of muscle contraction is thought to involve a change in the myosin head whilst attached to actin, the energy coming from ATP hydrolysis. This change in attached state could either be a conformational change in the head or an alteration in the binding angle made with actin. A considerable amount is known about one bound state, the so-called strongly attached state, which occurs in the presence of ADP or in the absence of nucleotide. In this state, which probably corresponds to the last attached state of the force-producing cycle, the angle between the long axis myosin head and the actin filament is roughly 45°. Details of other attached states before and during power production have been difficult to obtain because, even at very high protein concentration, the complex is almost completely dissociated by ATP. Electron micrographs of the complex in the presence of ATP have therefore been obtained only after chemically cross-linking myosin subfragment-1 (S1) to actin filaments to prevent dissociation. But it is unclear then whether the variability in attachment angle observed is due merely to the cross-link acting as a hinge.We have recently found low ionic-strength conditions under which, without resorting to cross-linking, a high fraction of S1 is bound to actin during steady state ATP hydrolysis. The structure of this complex is being studied by cryo-electron microscopy of hydrated specimens. Most advantages of frozen specimens over ambient temperature methods such as negative staining have already been documented. These include improved preservation and fixation rates and the ability to observe protein directly rather than a surrounding stain envelope. In the present experiments, hydrated specimens have the additional benefit that it is feasible to use protein concentrations roughly two orders of magnitude higher than in conventional specimens, thereby reducing dissociation of weakly bound complexes.

Lipid Polymorphism and Virus-Membrane Fusion as Observed in Vitrified Thin Films by Cryo-Electron Microscopy

1990 ◽  
Vol 48 (1) ◽  
pp. 492-493
Author(s):  
P.M. Frederik ◽  
K.N.J. Burger ◽  
M.C.A. Stuart ◽  
A.J. Verkleij
Keyword(s):  
Electron Microscopy ◽  
Thin Films ◽  
Membrane Fusion ◽  
Film Formation ◽  
Molar Ratio ◽  
Influenza B ◽  
Complex Structures ◽  
Type Ii ◽  
Suspended Material ◽  
Cryo Electron Microscopy

Cellular membranes are often composed of phospholipid mixtures in which one or more components have a tendency to adopt a type II non-bilayer lipid structure such as the inverted hexagonal (H||) phase. The formation of a type II non-bilayer intermediate, the inverted lipid micel is proposed as the initial step in membrane fusion (Verkleij 1984, Siegel, 1986). In the various forms of cellular transport mediated by carrier vesicles (e.g. exocytosis, endocytosis) the regulation of membrane fusion, and hence of inverted lipid micel formation, is of vital importance.We studied the phase behaviour of simple and complex lipid mixtures by cryo-electron microscopy to gain more insight in the ultrastructure of different lipid phases (e.g. Pβ’, Lα, H||) and in the complex membrane structures arising after Lα < - > H|| phase changes (e.g. isotropic, cubic). To prepare hydrated thin films a 700 mesh hexagonal grid (without supporting film) was dipped into and withdrawn from a liposome suspension. The excess fluid was blotted against filter paper and the thin films that form between the bars of the specimen grid were immediately (within 1 second) vitrified by plunging of the carrier grids into ethane cooled to its melting point by liquid nitrogen (Dubochet et al., 1982). Surface active molecules such as phospholipids play an important role in the formation and thinning of these aqueous thin films (Frederik et al., 1989). The formation of two interfacial layers at the air-water interfaces requires transport of surface molecules from the suspension as well as the orientation of these molecules at the interfaces. During the spontaneous thinning of the film the interfaces approach each other, initially driven by capillary forces later by Van der Waals attraction. The process of thinning results in the sorting by size of the suspended material and is also accompanied by a loss of water from the thinner parts of the film. This loss of water may result in the concentration and eventually in partial dehydration of suspended material even if thin films are vitrified within 1 sec after their formation. Film formation and vitrification were initiated at temperatures between 20-60°C by placing die equipment in an incubator provided widi port holes for the necessary manipulations. Unilamellar vesicles were made from dipalmitoyl phosphatidyl choline (DPPC) by an extrusion method and showed a smooth (Lα) or a rippled (PB’.) structure depending on the temperature of the suspensions and the temperature of film formation (50°C resp. 39°C) prior to vitrification. The thermotropic phases of hydrated phospholipids are thus faithfully preserved in vitrified thin films (fig. a,b). Complex structures arose when mixtures of dioleoylphosphatidylethanol-amine (DOPE), dioleoylphosphatidylcholine (DOPC) and cholesterol (molar ratio 3/1/2) are heated and used for thin film formation. The tendency of DOPE to adopt the H|| phase is responsible for the formation of complex structures in this lipid mixture. Isotropic and cubic areas (fig. c,d) having a bilayer structure are found in coexistence with H|| cylinders (fig. e). The formation of interlamellar attachments (ILA’s) as observed in isotropic and cubic structures is also thought to be of importance in biological fusion events. Therefore the study of the fusion activity of influenza B virus with liposomes (DOPE/DOPC/cholesterol/ganglioside in a molar ratio 1/1/2/0.2) was initiated. At neutral pH only adsorption of virus to liposomes was observed whereas 2 minutes after a drop in pH (7.4 - > 5.4) fusion between virus and liposome membranes was demonstrated (fig. f). The micrographs illustrate the exciting potential of cryo-electron microscopy to study lipid-lipid and lipid-protein interactions in hydrated specimens.

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