Sub-angstrom Resolution Research Articles

Zeolites are no longer a challenge: Atomic resolution data by Aberration-corrected STEM

Transmission electron microscopy is undoubtedly an indispensable tool for materials characterization, which can currently reach sub-angstrom resolution down to the elemental building blocks of matter, isolated single atoms of most elements. In addition to the phenomenal image resolution, if the material is strong enough, it can be accompanied with chemical information, converting electron microscopy into a unique method for the analysis of a great variety of materials. Unfortunately, extracting all this valuable information is not simple as most materials in one way or another are affected by the strong and localized electron beam. Radiolysis is one kind of reaction between electrons and matter than can cause irreversible structural transformations in our materials. This effect is the predominant factor in zeolites, zeotypes and the majority of molecular sieves. In the present work some results, taken at high voltage (300kV) and minimizing the exposure to the beam, are presented proving the feasibility of the technique to obtain unprecedented atomic resolution information of different zeolites and microporous solids.

Determination of damage-free crystal structure of an X-ray-sensitive protein using an XFEL.

We report a method of femtosecond crystallography for solving radiation damage-free crystal structures of large proteins at sub-angstrom spatial resolution, using a large single crystal and the femtosecond pulses of an X-ray free-electron laser (XFEL). We demonstrated the performance of the method by determining a 1.9-Å radiation damage-free structure of bovine cytochrome c oxidase, a large (420-kDa), highly radiation-sensitive membrane protein.

Conformational flexibility in the catalytic triad revealed by the high-resolution crystal structure of Streptomyces erythraeus trypsin in an unliganded state.

With more than 500 crystal structures determined, serine proteases make up greater than one-third of all proteases structurally examined to date, making them among the best biochemically and structurally characterized enzymes. Despite the numerous crystallographic and biochemical studies of trypsin and related serine proteases, there are still considerable shortcomings in the understanding of their catalytic mechanism. Streptomyces erythraeus trypsin (SET) does not exhibit autolysis and crystallizes readily at physiological pH; hence, it is well suited for structural studies aimed at extending the understanding of the catalytic mechanism of serine proteases. While X-ray crystallographic structures of this enzyme have been reported, no coordinates have ever been made available in the Protein Data Bank. Based on this, and observations on the extreme stability and unique properties of this particular trypsin, it was decided to crystallize it and determine its structure. Here, the first sub-angstrom resolution structure of an unmodified, unliganded trypsin crystallized at physiological pH is reported. Detailed structural analysis reveals the geometry and structural rigidity of the catalytic triad in the unoccupied active site and comparison to related serine proteases provides a context for interpretation of biochemical studies of catalytic mechanism and activity.

Breaking the Crowther limit: Combining depth-sectioning and tilt tomography for high-resolution, wide-field 3D reconstructions

To date, high-resolution (<1 nm) imaging of extended objects in three-dimensions (3D) has not been possible. A restriction known as the Crowther criterion forces a tradeoff between object size and resolution for 3D reconstructions by tomography. Further, the sub-Angstrom resolution of aberration-corrected electron microscopes is accompanied by a greatly diminished depth of field, causing regions of larger specimens (>6 nm) to appear blurred or missing. Here we demonstrate a three-dimensional imaging method that overcomes both these limits by combining through-focal depth sectioning and traditional tilt-series tomography to reconstruct extended objects, with high-resolution, in all three dimensions. The large convergence angle in aberration corrected instruments now becomes a benefit and not a hindrance to higher quality reconstructions. A through-focal reconstruction over a 390 nm 3D carbon support containing over 100 dealloyed and nanoporous PtCu catalyst particles revealed with sub-nanometer detail the extensive and connected interior pore structure that is created by the dealloying instability.

Experimental measurements and theoretical calculations of the atomic structure of materials with subangstrom resolution and picometer precision

Lattice defects are unavoidable structural units in materials and play an important role in determining material properties. Compared with the periodic structure of crystals, the atomic configurations of the lattice defects are determined by the coordinates of a large number of atoms, making it difficult to experimentally investigate them. In computational materials science, multiparameter optimization is also a difficult problem and experimental verification is usually required to determine the possibility of obtaining the structure and properties predicted by calculations. Using our recent studies on oxide surfaces as examples, we introduce the method of integrated aberration-corrected electron microscopy and the first-principles calculations to analyze the atomic structure of lattice defects. The atomic configurations of defects were measured using quantitative high-resolution electron microscopy at subangstrom resolution and picometer precision, and then the electronic structure and dynamic behavior of materials can be studied at the atomic scale using the first-principles calculations. The two methods complement each other and can be combined to increase the understanding of the atomic structure of materials in both the time and space dimensions, which will benefit materials design at the atomic scale.

Subpicosecond and Sub-Angstrom Time and Space Studies by Means of Light, X-ray, and Electron Interaction with Matter.

This Perspective article considers an experimental system that consists of ultrafast optical, electron, and X-ray time-resolved components. These techniques are used simultaneously on the same sample to study, in real time, the events that occur immediately upon disturbance with an ultrafast optical pulse. Excited states and metastable species are generated on the surface, and the electrical and mechanical waves propagating through the sample are recorded with subpicosecond and sub-Angstrom resolution. The characteristic of each technique is briefly described as a means of introducing the experimental system that intergrates these techniques. The processes evolved after femtosecond excitation of a Au single crystal have been monitored by these techniques. The data presented show changes with a resolution of 0.3 ± 0.1 ps in optical thermoreflectance, 1.0 ± 0.2 ps in electron Bragg diffraction, and 0.6 ± 0.1 ps in X-ray diffraction intensity accompanying shift and broadening.

Atomic Scale Structure Changes Induced by Charged Domain Walls in Ferroelectric Materials

Charged domain walls (CDWs) are of significant scientific and technological importance as they have been shown to play a critical role in controlling the switching mechanism and electric, photoelectric, and piezoelectric properties of ferroelectric materials. The atomic scale structure and properties of CDWs, which are critical for understanding the emergent properties, have, however, been rarely explored. In this work, using a spherical-aberration-corrected transmission electron microscope with subangstrom resolution, we have found that the polarization bound charge of the CDW in rhombohedral-like BiFeO3 thin films not only induces the formation of a tetragonal-like crystal structure at the CDW but also stabilizes unexpected nanosized domains with new polarization states and unconventional domain walls. These findings provide new insights on the effects of bound charge on ferroelectric domain structures and are critical for understanding the electrical switching in ferroelectric thin films as well as in memory devices.

A two-axis in-plane motion measurement system based on optical beam deflection

Measurement of in-plane motion with high resolution and large bandwidth enables model-identification and real-time control of motion-stages. This paper presents an optical beam deflection based system for measurement of in-plane motion of both macro- and micro-scale motion stages. A curved reflector is integrated with the motion stage to achieve sensitivity to in-plane translational motion along two axes. Under optimal settings, the measurement system is shown to theoretically achieve sub-angstrom measurement resolution over a bandwidth in excess of 1 kHz and negligible cross-sensitivity to linear motion. Subsequently, the proposed technique is experimentally demonstrated by measuring the in-plane motion of a piezo flexure stage and a scanning probe microcantilever. For the former case, reflective spherical balls of different radii are employed to measure the in-plane motion and the measured sensitivities are shown to agree with theoretical values, on average, to within 8.3%. For the latter case, a prototype polydimethylsiloxane micro-reflector is integrated with the microcantilever. The measured in-plane motion of the microcantilever probe is used to identify nonlinearities and the transient dynamics of the piezo-stage upon which the probe is mounted. These are subsequently compensated by means of feedback control.

Antimony-Mediated Control of Misfit Dislocations and Strain at the Highly Lattice Mismatched GaSb/GaAs Interface

Determining the atomic structure of misfit dislocations at highly lattice mismatched interface is essential to optimize the quality of the epitaxial layer. Here, with aberration corrected scanning transmission electron microscopy at sub-Angstrom resolution and molecular dynamics simulation, we investigated the atomic structure of misfit dislocations at GaSb/GaAs interface. New types of Lomer misfit dislocation formed on an Sb wetting monolayer were observed, in contrast to a conventional misfit dislocation whose core is located at interface. These Sb-mediated dislocations have highly localized cores and offer more capability to confine the mismatch strain at the interface. The low strain atomic configuration of Sb-mediated dislocations is driven by minimization of the core energy. This unveiled mechanism may pave the way to the growth of high quality hetero-epitaxial layers.

Subangstrom Resolution X-Ray Structure Details Aquaporin-Water Interactions

Aquaporins are membrane channels that facilitate the flow of water across biological membranes. Two conserved regions are central for selective function: the dual asparagine-proline-alanine (NPA) aquaporin signature motif and the aromatic and arginine selectivity filter (SF). Here, we present the crystal structure of a yeast aquaporin at 0.88 angstrom resolution. We visualize the H-bond donor interactions of the NPA motif's asparagine residues to passing water molecules; observe a polarized water-water H-bond configuration within the channel; assign the tautomeric states of the SF histidine and arginine residues; and observe four SF water positions too closely spaced to be simultaneously occupied. Strongly correlated movements break the connectivity of SF waters to other water molecules within the channel and prevent proton transport via a Grotthuss mechanism.

Relaxation calorimeter for hydrogen thermoporometry

A relaxation calorimeter for measuring the heat capacity of hydrogen isotopes in nanoporous solids is described. Apparatus' features include (i) cooling by a pulse tube refrigerator, (ii) a modular design, allowing for rapid reconfiguration and sample turn around, (iii) a thermal stability of ≲1 mK, and (iv) a bottom temperature of ~5 K. The calorimeter is tested on effective heat capacity measurements of H2 in Vycor (silica) nanoporous glass, yielding a very detailed pore size distribution analysis with an effectively sub-Angstrom resolution.

Lithium Atom and A-Site Vacancy Distributions in Lanthanum Lithium Titanate

Lanthanum lithium titanate (LLTO) is one of the most promising electrolyte materials for all-solid-state lithium-ion batteries. Despite numerous studies, the detailed crystal structure is still open to conjecture because of the difficulty of identifying precisely the positions of Li atoms and the distribution of intrinsic cation vacancies. Here we use subangstrom resolution scanning transmission electron microscopy (STEM) imaging methods and spatially resolved electron energy loss spectroscopy (EELS) analysis to examine the local atomic structure of LLTO. Direct annular bright-field (ABF) observations show Li locations on O4 window positions in Li-poor phase La0.62Li0.16TiO3 and near to A-site positions in Li-rich phase La0.56Li0.33TiO3. Local clustering of A-site vacancies results in aggregation of Li atoms, enhanced octahedral tilting and distortion, formation of O vacancies, and partial Ti4+ reduction. The results suggest local LLTO structures depend on a balance between the distribution of A-site vaca...

Functional Complex Point-Defect Structure in a Huge-Size-Mismatch System

Cubic boron nitride is a promising system for photonics and optoelectronics. Determining the inclusion mechanisms for dopants with a large size mismatch, such as luminous rare-earth elements, is prerequisite to understanding their functional properties and to effective doping control. Combining evidence from subangstrom resolution scanning transmission electron microscopy, imaging simulations, and first-principles calculations, we show that cationic Ce(3+) single dopants are not located at cationic B sites but rather at anionic N sites surrounded by B-site vacancies.

Novel Structural and Dynamical Features of Water Permeation through an Aquaporin Crystallized at Sub-Angstrom Resolution

Aquaporins are ubiquitous trans-membrane channels that maintain water homeostasis of the cell by facilitating selective diffusion of water across the membrane while preventing proton diffusion. The selectivity has been suggested to be achieved by two conserved regions located along the pore: the dual asparagine, proline, alanine (NPA) aquaporin signature motif, and the aromatic/arginine (ar/R) selectivity filter. Recently, our collaborators have crystallized a yeast aquaporin at sub-angstrom resolution, the highest resolution achieved to date for a membrane protein. The structure reveals a great deal of novel information on the structure of hydrogen-bonded network of water and protein side chains. In order to complement the experimental results by determining the dynamics and energetics of water diffusion along the channel, we performed molecular dynamics simulations of this impressively high quality crystal structure. The results show disruption of the water chain in both NPA and ar/R regions in this aquaporin, due to characteristic hydrogen-bonding patterns that dictate specific orientations to water molecules. The motion of water molecules is highly correlated on either side of the NPA region. On the other hand, the correlation is reduced at the NPA region, attesting yet another possible mechanism for this region to contribute to a barrier against proton transport. Besides, the NPA region appears as a barrier region with low occupancy for water, a feature not seen in other aquaporins. The correlated motion of adjacent water molecules along with their binary co-occupancies in the ar/R selectivity filter show that water molecules move in pairs in this region. Specific hydrogen-bonding patterns in the ar/R region may also play a role in exclusion of hydronium and/or hydroxide ions. These simulations have helped elucidate the dynamical basis of many intricate features revealed by this new structure.

Single Molecule Enzymology

We use single molecule force-clamp techniques to study the activity and chemical mechanisms of oxidases and oxidoreductase enzymes. In this approach the length of an extending protein is measured while the pulling force is actively kept constant with a feedback loop. Using the force-clamp technique we have investigated the force-dependency of protein folding, unfolding and of chemical reactions. For example, using various types of force pulses we can drive a single substrate protein to well defined extended states and monitor the reduction of its disulfide bonds by thioredoxin enzymes, with sub-Angstrom resolution (1). By varying the pulling force on the substrate we can also identify different chemical mechanisms of reduction. Using maximum likelihood techniques we have resuscitated ancient thioredoxin enzymes, going back billions of years. We have studied the activity of our resuscitated enzymes using our single molecule techniques in an effort to observe the evolution of the chemical mechanisms of reduction over time(2). Finally, we have used these single molecule techniques to study the activity of oxidases such as PDI and DsbA, and of glutharedoxin(3). Our results demonstrate that single molecule force-spectroscopy techniques provide an entirely novel and powerful approach to study enzymes, at an unprecedented resolution. These experiments show that force-clamp AFM probes dynamic rearrangements within an enzyme's active site, which cannot be resolved by any other current structural biological technique. We anticipate that these studies will be extended to a wide range of other enzymes.1.Wiita et al, (2007) Probing the chemistry of thioredoxin catalysis with force. Nature, 450:124-7.2.Perez-Jimenez, et al, (2011). Single-molecule paleoenzymology probes the chemistry of resurrected enzymes. Nat Struct Mol Biol, 18(5): 592-596.3.Kosuri, P et al, (2012). Protein folding drives disulfide formation. Cell, in press

Electron Transfer Mechanism in Organometallic Molecules Studied by Subpicosecond Extended X-ray Absorption Fine Structure Spectroscopy

The mechanism responsible for the redox reaction of [Co(III)(en)3]Ac3 to Co(II) complex has been determined to be intramolecular electron transfer. It was measured in real time by means of subpicosecond extended X-ray absorption fine structure spectra, EXAFS, and optical experiments and supported by density functional theory calculations. The proposed mechanism is based on histograms of bond length changes of the transient structures measured as a function of time, with subpicosecond time and sub-Angstrom resolution and femtosecond transient spectra and kinetics after excitation with a 267 nm femtosecond pulse. Even though four Fe and Co complexes were excited in the charge transfer band and the photoinduced redox reaction proceeds with similar high redox quantum yield, the dominant electron operating mechanism differs: intramolecular for amine metal complexes and intermolecular for oxalate metal complexes. The ligand orientation degree of freedom and counterion effect are proposed to provide tentative explanation for the electron transfer mechanism.

Spatial heterodyne interferometry with polarization gratings

The implementation of a polarization-based spatial heterodyne interferometer (SHI) is described. While a conventional SHI uses a Michelson interferometer and diffraction gratings, our SHI exploits mechanically robust Wollaston prisms and polarization gratings. A theoretical model for the polarization SHI is provided and validated with data from our proof of concept experiments. This device is expected to provide a compact monolithic sensor for subangstrom resolution spectroscopy in remote sensing, biomedical imaging, and machine vision applications.

Data Processing for Atomic Resolution Electron Energy Loss Spectroscopy

The high beam current and subangstrom resolution of aberration-corrected scanning transmission electron microscopes has enabled electron energy loss spectroscopy (EELS) mapping with atomic resolution. These spectral maps are often dose limited and spatially oversampled, leading to low counts/channel and are thus highly sensitive to errors in background estimation. However, by taking advantage of redundancy in the dataset map, one can improve background estimation and increase chemical sensitivity. We consider two such approaches--linear combination of power laws and local background averaging--that reduce background error and improve signal extraction. Principal component analysis (PCA) can also be used to analyze spectrum images, but the poor peak-to-background ratio in EELS can lead to serious artifacts if raw EELS data are PCA filtered. We identify common artifacts and discuss alternative approaches. These algorithms are implemented within the Cornell Spectrum Imager, an open source software package for spectroscopic analysis.

Les protéines DING : propriétés biochimiques, structurales, et capacité à inhiber la réplication du virus VIH-1

DING proteins comprise an intriguing phosphate-binding protein family present in all animal phyla. Five different DING representatives have been described in humans. Eukaryotic DING proteins are mostly involved in cellular processes such as cell cycle regulation, and also in pathological process such as rheumatoid arthritis and kidney stone formation. Although these proteins are ubiquitous in eukaryotes, no relevant locus or ORF has yet been found in sequenced genomes. This lack of sequence information has considerably hampered functional and structural studies of these proteins, and has required the use of novel and original techniques such as ab initio protein sequencing based on a combination of X-ray crystallography and mass spectrometry. Sub-Angstrom structural resolution has elucidated the molecular binding mechanism of phosphate ions by these high-affinity proteins. Immunohistochemical studies show that these proteins are present in a wide variety of mouse tissues. Some DING proteins, particularly human phosphate binding protein (HPBP), can inhibit HIV replication. This inhibition takes place at the transcriptional step, which is not targeted by any current antiretroviral drug. Initial studies suggest that HPBP warrants animal testing. This recent discovery opens new possibilities for the treatment of HIV infection.

A Supported Bilayer Model to Study the Effect of Membrane Composition on Bcl-2 Family Proteins

Apoptosis is a self mediated form of cell death that plays a significant role in the growth, development, and well being of multicellular organisms. If this process fails, potentially harmful cells are able to multiply uncontrollably within the organism forming harmful structures such as tumours. Although there are many signals and processes involved that may trigger apoptosis, the Bcl-2 family of proteins find themselves at a bottle neck of many of these pathways, as well as at the point after which the cell is no longer able to abort committing suicide. Bax is such a protein, and upon insertion and oligomerization within the outer mitochondrial membrane of mitochondria, apoptotic factors (such as cytochrome c) are released into the cytoplasm of the cell sealing its fate. Lipid composition is known to affect the process of membrane permeabilization by Bcl-2 family proteins, where different lipid compositions can block the process at different steps. Here we have investigated whether these different effects could be related to structural membrane properties. Solid supported lipid bilayers and aligned bilayer stacks are a convenient model system to study membranes structural properties. Not only do they allow for a rigid macroscopic sample size, they also offer a well defined geometry exploitable through techniques such as atomic force microscopy as well as x-ray and neutron scattering. Through Fourier analysis, we are able to utilize x-ray reflectivity data to analyze the electron density profile perpendicular to the plane of the membranes with sub-Angstrom resolution. We have characterized the structure of a membrane with a lipid composition mimicking that of mitochondrial membranes, and that of a series of membranes slightly differing from this original composition, and known to block the Bcl-2 family proteins pore formation process.