Assessing the benefits of focal pair cryo-electron tomography

Cryo electron tomography provides nanometer-scale information on biological matter preserved in a close-to native state. The resolution of tomograms and structures resolved by sub-tomogram averaging is typically limited by the contrast transfer function of the electron microscope, which is especially critical for thick samples. Here, we report a method to increase the attainable resolution by recording tomographic 'focal pairs', which are pairs of tilt series of the same object acquired in complementary defocus conditions. Low defocus imaging provides high resolution at low contrast, while high defocus imaging yields high contrast at the price of limited resolution. Quantitative assessment of the quality of lipid bilayer reconstructions in the resulting tomograms demonstrates stable resolution preservation beyond 3 nm for cells thicker than 500 nm. Further, in computational simulations on synthetic datasets we show the applicability of the method to sub-tomogram averaging, demonstrating its potential for achieving higher resolution.     

Bacterial Na(+)-ATP synthase has an undecameric rotor

Synthesis of adenosine triphosphate (ATP) by the F₁F₀ ATP synthase involves a membrane-embedded rotary engine, the F₀ domain, which drives the extra-membranous catalytic F₁ domain. The F₀ domain consists of subunits a₁b₂ and a cylindrical rotor assembled from 9–14 α-helical hairpin-shaped c-subunits. According to structural analyses, rotors contain 10 c-subunits in yeast and 14 in chloroplast ATP synthases. We determined the rotor stoichiometry of Ilyobacter tartaricus ATP synthase by atomic force microscopy and cryo-electron microscopy, and show the cylindrical sodium-driven rotor to comprise 11 c-subunits.     

High resolution AFM topographs of the Escherichia coli water channel aquaporin Z.

Aquaporins form a large family of membrane channels involved in osmoregulation. Electron crystallography has shown monomers to consist of six membrane spanning alpha-helices confirming sequence based predictions. Surface exposed loops are the least conserved regions, allowing differentiation of aquaporins. Atomic force microscopy was used to image the surface of aquaporin Z, the water channel of Escherichia coli. Recombinant protein with an N-terminal fragment including 10 histidines was isolated as a tetramer by Ni-affinity chromatography, and reconstituted into two-dimensional crystals with p42(1)2 symmetry. Small crystalline areas with p4 symmetry were found as well. Imaging both crystal types before and after cleavage of the N-termini allowed the cytoplasmic surface to be identified; a drastic change of the cytoplasmic surface accompanied proteolytic cleavage, while the extracellular surface morphology did not change. Flexibility mapping and volume calculations identified the longest loop at the extracellular surface. This loop exhibited a reversible force-induced conformational change.     

Surface tongue-and-groove contours on lens MIP facilitate cell-to-cell adherence

The lens major intrinsic protein (MIP, AQP0) is known to function as a water and solute channel. However, MIP has also been reported to occur in close membrane contacts between lens fiber cells, indicating that it has adhesive properties in addition to its channel function. Using atomic force and cryo-electron microscopy we document that crystalline sheets reconstituted from purified ovine lens MIP mostly consisted of two layers. MIP lattices in the apposing membranes were in precise register, and determination of the membrane sidedness demonstrated that MIP molecules bound to each other via their extracellular surfaces. The surface structure of the latter was resolved to 0.61 nm and revealed two protruding domains providing a tight "tongue-and-groove" fit between apposing MIP molecules. Cryo-electron crystallography produced a projection map at 0.69 nm resolution with a mirror symmetry axis at 45 degrees to the lattice which was consistent with the double-layered nature of the reconstituted sheets. These data strongly suggest an adhesive function of MIP, and strengthen the view that MIP serves dual roles in the lens.     

Yersinia enterocolitica type III secretion injectisomes form regularly spaced clusters,which incorporate new machines upon activation

Bacterial type III secretion systems or injectisomes are multiprotein complexes directly transporting bacterial effector proteins into eukaryotic host cells. To investigate the distribution of injectisomes in the bacterium and the influence of activation of the system on that distribution, we combined in vivo fluorescent imaging and high‐resolution in situ visualization of Yersinia enterocolitica injectisomes by cryo‐electron tomography. Fluorescence microscopy showed the injectisomes as regularly distributed spots around the bacterial cell. Under secreting conditions (absence of Ca²⁺), the intensity of single spots significantly increased compared with non‐secreting conditions (presence of Ca²⁺), in line with an overall up‐regulation of expression levels of all components. Single injectisomes observed by cryo‐electron tomography tended to cluster at distances less than 100 nm, suggesting that the observed fluorescent spots correspond to evenly distributed clusters of injectisomes, rather than single injectisomes. The up‐regulation of injectisome components led to an increase in the number of injectisomes per cluster rather than the formation of new clusters. We suggest that injectisome clustering may allow more effective secretion into the host cells.     

Automatic lattice determination for two-dimensional crystal images

Electron crystallography determines the structure of membrane proteins and other periodic samples by recording either images or diffraction patterns. Computer processing of recorded images requires the determination of the reciprocal lattice parameters in the Fourier transform of the image. We have developed a set of three programs 2dx_peaksearch, 2dx_findlat and 2dx_getlat, which can determine the reciprocal lattice from a Fourier transformation of a 2D crystal image automatically. 2dx_peaksearch determines a list of Fourier peak coordinates from a processed calculated diffraction pattern. These coordinates are evaluated by 2dx_findlat to determine one or more lattices, using a-priori knowledge of the real-space crystal unit cell dimensions, and the sample tilt geometry. If these are unknown, then the program 2dx_getlat can be used to obtain a guess for the unit cell dimensions. These programs are available as part of the 2dx software package for the image processing of 2D crystal images at http://2dx.org.     

The reaction centre of the photounit of Rhodospirillum rubrum is anchored to the light-harvesting complex with four-fold rotational disorder

The minimal photounit of the photosynthetic membranes of the purple non-sulphur bacterium Rhodospirillum rubrum, comprising the reaction centre and the light-harvesting complex has been purified and crystallised in two dimensions in the presence of added phospholipids, and subsequently visualised by electron microscopy after negatively-staining. The position of the reaction centres within the light-harvesting ring has been determined at low resolution by the application of a new analysis for rotationally disordered identical units (here the reaction centres) within a two-dimensional crystalline lattice comprised of perfectly aligned unit cells (here the light-harvesting complexes). The reaction centre was found to preferentially occupy one of four orientations within the light-harvesting complex. The light-harvesting complex appears to be distorted to C₄ symmetry, thus assuming a squarish shape when visualised by negative staining. A tentative structural model of the reaction centre-light-harvesting complex photounit which fits the experimental data is proposed.     

Projection structure of the secondary citrate/sodium symporter CitS at 6 Å resolution by electron crystallography

CitS from Klebsiella pneumoniae acts as a secondary symporter of citrate and sodium ions across the inner membrane of the host. The protein is the best characterized member of the 2-hydroxycarboxylate transporter family, while no experimental structural information at sub-nanometer resolution is available on this class of membrane proteins. Here, we applied electron crystallography to two-dimensional crystals of CitS. Carbon-film-adsorbed tubular two-dimensional crystals were studied by cryo-electron microscopy, producing the 6-?-resolution projection structure of the membrane-embedded protein. In the p22(1)2(1)-symmetrized projection map, the predicted dimeric structure is clearly visible. Each monomeric unit can tentatively be interpreted as being composed of 11 transmembrane α-helices. In projection, CitS shows a high degree of structural similarity to NhaP1, the Na(+)/H(+) antiporter of Methanococcus jannaschii. We discuss possible locations for the dimer interface and models for the helical arrangements and domain organizations of the symporter based on existing models.     

Graphene: Substrate preparation and introduction

This technical note describes the transfer of continuous, single-layer, pristine graphene to standard Quantifoil TEM grids. We compare the transmission properties of pristine graphene substrates to those of graphene oxide and thin amorphous carbon substrates. Positively stained DNA imaged across amorphous carbon is typically indiscernible and requires metal shadowing for sufficient contrast. However, in a practical illustration of the new substrates properties, positively stained DNA is imaged across pristine graphene in striking contrast without the need of metal shadowing. We go onto discuss technical considerations and the potential applications of pristine graphene substrates as well as their ongoing development.     

Thermal Unfolding of a Mammalian Pentameric Ligand-gated Ion Channel Proceeds at Consecutive,Distinct Steps

Pentameric ligand-gated ion channels (LGICs) play an important role in fast synaptic signal transduction. Binding of agonists to the β-sheet-structured extracellular domain opens an ion channel in the transmembrane α-helical region of the LGIC. How the structurally distinct and distant domains are functionally coupled for such central transmembrane signaling processes remains an open question. To obtain detailed information about the stability of and the coupling between these different functional domains, we analyzed the thermal unfolding of a homopentameric LGIC, the 5-hydroxytryptamine receptor (ligand binding, secondary structure, accessibility of Trp and Cys residues, and aggregation), in plasma membranes as well as during detergent extraction, purification, and reconstitution into artificial lipid bilayers. We found a large loss in thermostability correlating with the loss of the lipid bilayer during membrane solubilization and purification. Thermal unfolding of the 5-hydroxytryptamine receptor occurred in consecutive steps at distinct protein locations. A loss of ligand binding was detected first, followed by formation of different transient low oligomeric states of receptor pentamers, followed by partial unfolding of helical parts of the protein, which finally lead to the formation receptor aggregates. Structural destabilization of the receptor in detergents could be partially reversed by reconstituting the receptor into lipid bilayers. Our results are important because they quantify the stability of LGICs during detergent extraction and purification and can be used to create stabilized receptor proteins for structural and functional studies.