Angstrom Resolution Research Articles

Characteristics of channels and arrangement of cofactors in the 1.9 angstrom resolution structure of photosystem II

Characteristics of channels and arrangement of cofactors in the 1.9 angstrom resolution structure of photosystem II

Crystal Structure of the Eukaryotic Ribosome

Crystal structures of prokaryotic ribosomes have described in detail the universally conserved core of the translation mechanism. However, many facets of the translation process in eukaryotes are not shared with prokaryotes. The crystal structure of the yeast 80S ribosome determined at 4.15 angstrom resolution reveals the higher complexity of eukaryotic ribosomes, which are 40% larger than their bacterial counterparts. Our model shows how eukaryote-specific elements considerably expand the network of interactions within the ribosome and provides insights into eukaryote-specific features of protein synthesis. Our crystals capture the ribosome in the ratcheted state, which is essential for translocation of mRNA and transfer RNA (tRNA), and in which the small ribosomal subunit has rotated with respect to the large subunit. We describe the conformational changes in both ribosomal subunits that are involved in ratcheting and their implications in coordination between the two associated subunits and in mRNA and tRNA translocation.

Structural mechanism of the antigen recognition by the L1 cell adhesion molecule antibody A10-A3

The L1CAM antibody A10-A3 efficiently reduces tumor growth in a nude mouse model. Here, we describe the crystal structure of the Fab fragment of A10-A3 determined at 2.0 angstrom resolution. The A10-A3 antibody H3 loop reveals a characteristic arrangement of exposed aromatic residues that may play an important role in antigen binding. A structure model of the complex between L1CAM Ig1-4 and A10-A3 Fab indicates that the Fab binds to three small loops outside Ig1 and a residue between Ig1 and Ig2, consistent with an epitope mapping result. The data presented here should contribute to the design of high-affinity antibody for therapeutic purposes as well as to the understanding of neural cell remodeling and cancer progression mechanism mediated by L1CAM.

Realization of cantilever arrays for parallel proximity imaging

This paper reports on the fabrication and characterisation of self-actuating, and self-sensing cantilever arrays for large-scale parallel surface scanning. Each cantilever is integrated with a sharp silicon tip, a thermal-driven bimorph actuator, and a piezoresistive deflection sensor. Thus, the tip to the sample distance can be controlled individually for each cantilever. A radius of the tips below 10 nm is obtained, which enables nanometre in-plane surface imaging by Angstrom resolution in vertical direction. The fabricated cantilever probe arrays are also applicable for large-area manipulation, sub-10 nm metrology, bottom-up synthesis, high-speed gas analysis, for different bio-applications like recognition of DNA, RNA, or various biomarkers of a single disease, etc.

Atomic Structure of Human Adenovirus by Cryo-EM Reveals Interactions Among Protein Networks

Construction of a complex virus may involve a hierarchy of assembly elements. Here, we report the structure of the whole human adenovirus virion at 3.6 angstroms resolution by cryo-electron microscopy (cryo-EM), revealing in situ atomic models of three minor capsid proteins (IIIa, VIII, and IX), extensions of the (penton base and hexon) major capsid proteins, and interactions within three protein-protein networks. One network is mediated by protein IIIa at the vertices, within group-of-six (GOS) tiles--a penton base and its five surrounding hexons. Another is mediated by ropes (protein IX) that lash hexons together to form group-of-nine (GON) tiles and bind GONs to GONs. The third, mediated by IIIa and VIII, binds each GOS to five surrounding GONs. Optimization of adenovirus for cancer and gene therapy could target these networks.

Crystal Structure of Human Adenovirus at 3.5 Å Resolution

Rational development of adenovirus vectors for therapeutic gene transfer is hampered by the lack of accurate structural information. Here, we report the x-ray structure at 3.5 angstrom resolution of the 150-megadalton adenovirus capsid containing nearly 1 million amino acids. We describe interactions between the major capsid protein (hexon) and several accessory molecules that stabilize the capsid. The virus structure also reveals an altered association between the penton base and the trimeric fiber protein, perhaps reflecting an early event in cell entry. The high-resolution structure provides a substantial advance toward understanding the assembly and cell entry mechanisms of a large double-stranded DNA virus and provides new opportunities for improving adenovirus-mediated gene transfer.

In-situ heating studies of gold nanoparticles in an aberration corrected transmission electron microscope

Gold nanoparticles have a high catalytic activity for CO oxidation at low temperatures providing they remain less than 5nm in diameter. Their structure and stability and the growth processes that occur during heating have been investigated using Angstrom resolution in-situ double aberration corrected transmission electron microscopy with a JEOL JEM-2200FS. Colloidal Au nanoparticles suspended in water, with mean diameters of 2nm and 5.6nm, have been deposited onto 3.5nm thin carbon supported on holey carbon grids and onto Si3N4 membranes. Dynamic in-situ high resolution AC-TEM images show competitive sintering processes on the different supports. Whilst the 5.6nm particles were observed to be very stable on the carbon, the 2nm particles showed sintering predominantly through particle migration and coalescence, with particle migration occurring as early as ~200°C, peaking at ~500°C. In contrast Au nanoparticles on Si3N4 membranes were observed to coalesce at ~180°C, before Ostwald Ripening became the dominant growth process at higher temperatures. It is believed that atoms and small clusters migrate away from their original particle before becoming trapped on the Si3N4 substrate. These trapped clusters then build up to form the small particles observed, before having sufficient energy to continue to migrate and join another larger particle at ~500°C.

Molecular basis of alternating access membrane transport by the sodium-hydantoin transporter Mhp1.

The structure of the sodium-benzylhydantoin transport protein Mhp1 from Microbacterium liquefaciens comprises a five-helix inverted repeat, which is widespread among secondary transporters. Here, we report the crystal structure of an inward-facing conformation of Mhp1 at 3.8 angstroms resolution, complementing its previously described structures in outward-facing and occluded states. From analyses of the three structures and molecular dynamics simulations, we propose a mechanism for the transport cycle in Mhp1. Switching from the outward- to the inward-facing state, to effect the inward release of sodium and benzylhydantoin, is primarily achieved by a rigid body movement of transmembrane helices 3, 4, 8, and 9 relative to the rest of the protein. This forms the basis of an alternating access mechanism applicable to many transporters of this emerging superfamily.

Structural insights into the assembly and function of the SAGA deubiquitinating module.

SAGA is a transcriptional coactivator complex that is conserved across eukaryotes and performs multiple functions during transcriptional activation and elongation. One role is deubiquitination of histone H2B, and this activity resides in a distinct subcomplex called the deubiquitinating module (DUBm), which contains the ubiquitin-specific protease Ubp8, bound to Sgf11, Sus1, and Sgf73. The deubiquitinating activity depends on the presence of all four DUBm proteins. We report here the 1.90 angstrom resolution crystal structure of the DUBm bound to ubiquitin aldehyde, as well as the 2.45 angstrom resolution structure of the uncomplexed DUBm. The structure reveals an arrangement of protein domains that gives rise to a highly interconnected complex, which is stabilized by eight structural zinc atoms that are critical for enzymatic activity. The structure suggests a model for how interactions with the other DUBm proteins activate Ubp8 and allows us to speculate about how the DUBm binds to monoubiquitinated histone H2B in nucleosomes.

Attosecond imaging of molecular electronic wavepackets

A strong laser field may tunnel ionize a molecule from several orbitals simultaneously, forming an attosecond electron–hole wavepacket. Both temporal and spatial information on this wavepacket can be obtained through the coherent soft X-ray emission resulting from the laser-driven recollision of the liberated electron with the core. By characterizing the emission from aligned N2 molecules, we demonstrate the attosecond contributions of the two highest occupied molecular orbitals. We determine conditions where they are disentangled in the real and imaginary parts of the emission dipole moment. This allows us to carry out a tomographic reconstruction of both orbitals with angstrom spatial resolution. Their coherent superposition provides experimental images of the attosecond wavepacket created in the ionization process. Our results open the prospect of imaging ultrafast intramolecular dynamics combining attosecond and angstrom resolutions. Advances in X-ray imaging are allowing the investigation of molecular dynamics on an attosecond timescale and at angstrom-scale spatial resolution. It is now even possible to reconstruct images of the molecular orbitals, which provides us with a better understanding of how molecules respond to intense fields.

Crystal Structure and Substrate Specificity of Drosophila 3,4-Dihydroxyphenylalanine Decarboxylase

Background3,4-Dihydroxyphenylalanine decarboxylase (DDC), also known as aromatic L-amino acid decarboxylase, catalyzes the decarboxylation of a number of aromatic L-amino acids. Physiologically, DDC is responsible for the production of dopamine and serotonin through the decarboxylation of 3,4-dihydroxyphenylalanine and 5-hydroxytryptophan, respectively. In insects, both dopamine and serotonin serve as classical neurotransmitters, neuromodulators, or neurohormones, and dopamine is also involved in insect cuticle formation, eggshell hardening, and immune responses.Principal FindingsIn this study, we expressed a typical DDC enzyme from Drosophila melanogaster, critically analyzed its substrate specificity and biochemical properties, determined its crystal structure at 1.75 Angstrom resolution, and evaluated the roles residues T82 and H192 play in substrate binding and enzyme catalysis through site-directed mutagenesis of the enzyme. Our results establish that this DDC functions exclusively on the production of dopamine and serotonin, with no activity to tyrosine or tryptophan and catalyzes the formation of serotonin more efficiently than dopamine.ConclusionsThe crystal structure of Drosophila DDC and the site-directed mutagenesis study of the enzyme demonstrate that T82 is involved in substrate binding and that H192 is used not only for substrate interaction, but for cofactor binding of drDDC as well. Through comparative analysis, the results also provide insight into the structure-function relationship of other insect DDC-like proteins.

Direct Imaging of the Structure of Lipid Rafts by Atomic Force Microscopy

According to the fluid mosaic model, lipid bilayers have been thought of as two-dimensional homogenous mixtures of lipids, embedded with membrane proteins1. This model has recently been extended by the lipid raft model in which biologically functional structural lipid domains, rich in sphingolipids and cholesterol.2Previously, a low-noise AFM system3 was developed within the our group, capable of imaging the surface structure of lipid bilayers in aqueous buffer with Angstrom resolution.4,5 We now extend this work to the structure of the these lipid raft components at Angstrom resolution and discover a subtle organisation of the lipids headgroups of the molecules in these structures, stabilised by a combination of salt bridges and hydrogen bonds.

Pore Architecture and ion Sites of Acid Sensing ion Channels and P2X Receptors

Acid-sensing ion channels are proton-activated, sodium-selective channels composed of three subunits, and members of the degenerin/epithelial sodium channel (DEG/ENaC) superfamily. These eukaryotic channels have essential roles in sodium homeostasis, taste, and pain. Despite their roles in biology, there is little knowledge of the structural and chemical principles underlying their ion channel architecture and ion-binding sites. Here we present the crystal structure of a functional acid-sensing ion channel in a desensitized state at 3 angstrom resolution, the location of the desensitization gate, and the trigonal antiprism coordination of cesium ions bound in the extracellular vestibule. Comparison of the acid-sensing ion channel structure with the P2X receptor reveals unanticipated similarities and mechanical principles in different ligand-gated ion channels.

Crystal Structure of the Eukaryotic Strong Inward-Rectifier K + Channel Kir2.2 at 3.1 Å Resolution

Inward-rectifier potassium (K+) channels conduct K+ ions most efficiently in one direction, into the cell. Kir2 channels control the resting membrane voltage in many electrically excitable cells, and heritable mutations cause periodic paralysis and cardiac arrhythmia. We present the crystal structure of Kir2.2 from chicken, which, excluding the unstructured amino and carboxyl termini, is 90% identical to human Kir2.2. Crystals containing rubidium (Rb+), strontium (Sr2+), and europium (Eu3+) reveal binding sites along the ion conduction pathway that are both conductive and inhibitory. The sites correlate with extensive electrophysiological data and provide a structural basis for understanding rectification. The channel's extracellular surface, with large structured turrets and an unusual selectivity filter entryway, might explain the relative insensitivity of eukaryotic inward rectifiers to toxins. These same surface features also suggest a possible approach to the development of inhibitory agents specific to each member of the inward-rectifier K+ channel family.

Three-dimensional structure determination from a single view

The ability to determine the structure of matter in three dimensions has profoundly advanced our understanding of nature. Traditionally, the most widely used schemes for three-dimensional (3D) structure determination of an object are implemented by acquiring multiple measurements over various sample orientations, as in the case of crystallography and tomography, or by scanning a series of thin sections through the sample, as in confocal microscopy. Here we present a 3D imaging modality, termed ankylography (derived from the Greek words ankylos meaning 'curved' and graphein meaning 'writing'), which under certain circumstances enables complete 3D structure determination from a single exposure using a monochromatic incident beam. We demonstrate that when the diffraction pattern of a finite object is sampled at a sufficiently fine scale on the Ewald sphere, the 3D structure of the object is in principle determined by the 2D spherical pattern. We confirm the theoretical analysis by performing 3D numerical reconstructions of a sodium silicate glass structure at 2 A resolution, and a single poliovirus at 2-3 nm resolution, from 2D spherical diffraction patterns alone. Using diffraction data from a soft X-ray laser, we also provide a preliminary demonstration that ankylography is experimentally feasible by obtaining a 3D image of a test object from a single 2D diffraction pattern. With further development, this approach of obtaining complete 3D structure information from a single view could find broad applications in the physical and life sciences.

Structure of an RNA polymerase II-TFIIB complex and the transcription initiation mechanism.

Previous x-ray crystal structures have given insight into the mechanism of transcription and the role of general transcription factors in the initiation of the process. A structure of an RNA polymerase II-general transcription factor TFIIB complex at 4.5 angstrom resolution revealed the amino-terminal region of TFIIB, including a loop termed the "B finger," reaching into the active center of the polymerase where it may interact with both DNA and RNA, but this structure showed little of the carboxyl-terminal region. A new crystal structure of the same complex at 3.8 angstrom resolution obtained under different solution conditions is complementary with the previous one, revealing the carboxyl-terminal region of TFIIB, located above the polymerase active center cleft, but showing none of the B finger. In the new structure, the linker between the amino- and carboxyl-terminal regions can also be seen, snaking down from above the cleft toward the active center. The two structures, taken together with others previously obtained, dispel long-standing mysteries of the transcription initiation process.

Crystallization and preliminary crystallographic analysis of gallate dioxygenase DesB from Sphingobium sp. SYK-6.

Gallate dioxygenase (DesB) from Sphingobium sp. SYK-6, which belongs to the type II extradiol dioxygenase family, was purified and crystallized using the hanging-drop vapour-diffusion method. Two crystal forms were obtained. The form I crystal belonged to space group C2, with unit-cell parameters a = 136.2, b = 53.6, c = 55.1 angstrom, beta = 112.8 degrees, and diffracted to 1.6 angstrom resolution. The form II crystal belonged to space group P2(1), with unit-cell parameters a = 56.2, b = 64.7, c = 116.1 angstrom, beta = 95.1 degrees, and diffracted to 1.9 angstrom resolution. A molecular-replacement calculation using LigAB as a search model yielded a satisfactory solution for both crystal forms.

Crystallization and preliminary crystallographic studies of dihydrofolate reductase-thymidylate synthase from Trypanosoma cruzi, the Chagas disease pathogen.

Trypanosoma cruzi dihydrofolate reductase-thymidylate synthase (TcDHFR-TS) was crystallized in complexes with the dihydrotriazine-based or quinazoline-based antifolates C-448, cycloguanil (CYC) and Q-8 in order to gain insight into the interactions of this DHFR enzyme with classical and novel inhibitors. The TcDHFR-TS-C-448-NDP-dUMP crystal belonged to space group C222(1) with two molecules per asymmetric unit and diffracted to 2.37 angstrom resolution. The TcDHFR-TS-CYC, TcDHFR-TS-CYC-NDP and TcDHFR-TS-Q-8-NDP crystals belonged to space group P2(1) with four molecules per asymmetric unit and diffracted to 2.1, 2.6 and 2.8 angstrom resolution, respectively. Crystals belonging to the two different space groups were suitable for structure determination.

Crystallization of the head and galectin-like domains of porcine adenovirus isolate NADC-1 fibre.

The porcine adenovirus NADC-1 isolate, a strain of porcine adenovirus type 4, has a fibre with an atypical architecture. In addition to a classical virus attachment region, shaft and head domains, it contains an additional galectin like domain C-terminal to the head domain and connected to the head domain by a long RGD-containing loop. The galectin-like domain contains two putative carbohydrate-recognition domains. The head and galectin-like domains have been independently crystallized. Diffraction data have been obtained to 3.2 angstrom resolution from crystals of the head domain and to 1.9 angstrom resolution from galectin-like domain crystals.

Production, purification, crystallization and preliminary X-ray diffraction studies of the nucleoside diphosphate kinase b fromLeishmania major

Nucleoside diphosphate kinases (NDKs; EC 2.7.4.6) play an essential role in the synthesis of nucleotides from intermediates in the salvage pathway in all parasitic trypanosomatids and their structural studies will be instrumental in shedding light on the biochemical machinery involved in the parasite life cycle and host-parasite interactions. In this work, NDKb from Leishmania major was overexpressed in Escherichia coli, purified to homogeneity and crystallized using the sitting-drop vapour-diffusion method. The NDK crystal diffracted to 2.2 angstrom resolution and belonged to the trigonal crystal system, with unit-cell parameters a = 114.2, c = 93.9 angstrom. Translation-function calculations yielded an unambiguous solution in the enantiomorphic space group P3(2)21.