Determination of cross relaxation rates by 3D NOE-NOE spectroscopy

Summary A method for quantitative determination of cross-relaxation rates of macromolecules in solution is developed. The method is based on the analysis of the intensities of cross peaks in 3D NOE-NOE spectra. The linear combination of the intensities of 3D peaks (spin-diffusion peaks, back-transfer peaks) results in an expression directly proportional to the cross-relaxation rate. The proposed approach allows to determine interproton distances in macromolecules more accurately.     

Structure determination of biological macromolecules in solution using nuclear magnetic resonance spectroscopy

A detailed understanding of the function of a biological macromolecule requires knowledge of its three-dimensional structure. Most atomic-resolution structures of biological macromolecules have been solved either by X-ray diffraction in single crystals or by nuclear magnetic resonance (NMR) in solution. This review surveys the method of NMR structure determination. First, a brief introduction to NMR and its basic concepts is presented. The main part of the article deals with the individual steps necessary for an NMR structure determination. At the end, the discussion turns to considerations on the influence of the molecular size of the macromolecules on the structure determination by NMR. New techniques are discussed that greatly enhance the possibilities of applying NMR to large molecular systems.     

108 Individual-particle electron tomography: a method for studying macromolecule dynamics

In solution, macromolecules are naturally flexible and dynamic. Dynamic personalities and structural heterogeneities of macromolecules are essential to understanding their proper function (Karplus & Kuriyan, 2005). However, structural determination of dynamic/heterogeneous macromolecules is limited by current technology such as: X-ray crystallography, nuclear magnetic resonance spectrum, small angle scattering, and electron microscopy single-particle reconstruction. A common weakness of all current techniques is requiring an averaged signal from thousands to millions of different macromolecules. Using averaged “signal” must involve in an assumption that macromolecules remain in identical structures or few identical conformations. This assumption is a good estimate for some macromolecule that have a rigid body, but not for most macromolecules that have “soft”, flexible, and dynamic body, such as lipoproteins and antibodies. An ideal approach for structure determination regardless of macromolecular dynamics is to use non-averaged signal, i.e. the signal from a single macromolecule itself. We developed a ‘‘focused electron tomography reconstruction’’ (FETR) algorithm to improve the resolution by decreasing the reconstructing image size so that it contains only a single-instance macromolecule (Zhang & Ren, 2012). FETR can tolerate certain levels of image distortion and measuring tilt-errors, and can also precisely determine the translational parameters via an iterative refinement process that contains a series of automatically generated dynamic filters and masks. Since this approach can obtain the structure of a single-instance macromolecule, we named it individual-particle electron tomography (IPET) as a new robust strategy/approach that does not require a pre-given initial model, class averaging of multiple molecules or an extended ordered lattice, but can tolerate small tilt-errors for high-resolution single ‘‘snapshot’’ of molecule structure determination (Zhang & Ren, 2012). FETR/IPET provides a completely new opportunity for a single-macromolecule structure determination, and could be used to study the dynamic character, equilibrium fluctuation, to reveal macromolecular mechanism, and even to track the intermediate state of the reaction of macromolecules (Zhang et al., 2010; Zhang & Ren, 2010).     

Improved simulation of NOESY spectra by RELAX-JT2 including effects of J-coupling, transverse relaxation and chemical shift anisotropy

RELAX-JT2 is an extension of RELAX, a program for the simulation of 1H 2D NOESY spectra and (15)N or (13)C edited 3D NOESY-HSQC spectra of biological macromolecules. In addition to the already existing NOE-simulation it allows the proper simulation of line shapes by the integrated calculation of T(2) times and multiplet structures caused by J-couplings. Additionally the effects of relaxation mediated by chemical shift anisotropy are taken into account. The new routines have been implemented in the program AUREMOL, which aims at the automated NMR structure determination of proteins in solution. For a manual or automatic assignment of experimental spectra that is based on the comparison with the corresponding simulated spectra, the additional line shape information now available is a valuable aid. The new features have been successfully tested with the histidine-containing phosphocarrier protein HPr from Staphylococcus carnosus.     

Do's and Don'ts of Cryo-electron Microscopy: A Primer on Sample Preparation and High Quality Data Collection for Macromolecular 3D Reconstruction

Cryo-electron microscopy (cryoEM) entails flash-freezing a thin layer of sample on a support, and then visualizing the sample in its frozen hydrated state by transmission electron microscopy (TEM). This can be achieved with very low quantity of protein and in the buffer of choice, without the use of any stain, which is very useful to determine structure-function correlations of macromolecules. When combined with single-particle image processing, the technique has found widespread usefulness for 3D structural determination of purified macromolecules. The protocol presented here explains how to perform cryoEM and examines the causes of most commonly encountered problems for rational troubleshooting; following all these steps should lead to acquisition of high quality cryoEM images. The technique requires access to the electron microscope instrument and to a vitrification device. Knowledge of the 3D reconstruction concepts and software is also needed for computerized image processing. Importantly, high quality results depend on finding the right purification conditions leading to a uniform population of structurally intact macromolecules. The ability of cryoEM to visualize macromolecules combined with the versatility of single particle image processing has proven very successful for structural determination of large proteins and macromolecular machines in their near-native state, identification of their multiple components by 3D difference mapping, and creation of pseudo-atomic structures by docking of x-ray structures. The relentless development of cryoEM instrumentation and image processing techniques for the last 30 years has resulted in the possibility to generate de novo 3D reconstructions at atomic resolution level.     

Version 1.2 of the Crystallography and NMR system

Version 1.2 of the software system, termed Crystallography and NMR system (CNS), for crystallographic and NMR structure determination has been released. Since its first release, the goals of CNS have been (i) to create a flexible computational framework for exploration of new approaches to structure determination, (ii) to provide tools for structure solution of difficult or large structures, (iii) to develop models for analyzing structural and dynamical properties of macromolecules and (iv) to integrate all sources of information into all stages of the structure determination process. Version 1.2 includes an improved model for the treatment of disordered solvent for crystallographic refinement that employs a combined grid search and least-squares optimization of the bulk solvent model parameters. The method is more robust than previous implementations, especially at lower resolution, generally resulting in lower R values. Other advances include the ability to apply thermal factor sharpening to electron density maps. Consistent with the modular design of CNS, these additions and changes were implemented in the high-level computing language of CNS.     

Thermodynamics and the structure of biological macromolecules. Rozhinkes mit mandeln

In this review, I will discuss the role of thermodynamics in both the determination and evaluation of the structure of biological macromolecules. The presentation relates to the historical context, state-of-the-art and projection into the future. Fundamental features relate to the effect of charge, exemplified in the study of synthetic and natural polyelectrolytes. Hydrogen bonding and water structure constitute basic aspects of the medium in which biological reactions occur. Viscosity is a classical tool to determine the shape and size of biological macromolecules. The thermodynamic analysis of multicomponent systems is essential fo the correct understanding of the behavior of biological macromolecules in solution and for the evaluation of results from powerful experimental techniques such as ultracentrifugation, light, X-ray and neutron scattering. The hydration, shape and flexibility of DNA have been studied, as well as structural transitions in nucleosomes and chromatin. A particularly rewarding field of activity is the study of unusual structural features of enzymes isolated from the extreme halophilic bacteria of the Dead Sea, which have adapted to saturated concentrations of salt. Future studies in various laboratories will concentrate on nucleic-acid--protein interactions and on the so-called 'crowding effect', distinguishing the behavior in bacteria, or other cells, from simple test-tube experiments.     

Restoring low resolution structure of biological macromolecules from solution scattering using simulated annealing.

A method is proposed to restore ab initio low resolution shape and internal structure of chaotically oriented particles (e.g., biological macromolecules in solution) from isotropic scattering. A multiphase model of a particle built from densely packed dummy atoms is characterized by a configuration vector assigning the atom to a specific phase or to the solvent. Simulated annealing is employed to find a configuration that fits the data while minimizing the interfacial area. Application of the method is illustrated by the restoration of a ribosome-like model structure and more realistically by the determination of the shape of several proteins from experimental x-ray scattering data.     

Low-resolution density maps from atomic models: how stepping "back" can be a step "forward"

Atomic-resolution structures have had a tremendous impact on modern biological science. Much useful information also has been gleaned by merging and correlating atomic-resolution structural details with lower-resolution (15-40 A), three-dimensional (3D) reconstructions computed from images recorded with cryo-transmission electron microscopy (cryoTEM) procedures. One way to merge these structures involves reducing the resolution of an atomic model to a level comparable to a cryoTEM reconstruction. A low-resolution density map can be derived from an atomic-resolution structure by retrieving a set of atomic coordinates editing the coordinate file, computing structure factors from the model coordinates, and computing the inverse Fourier transform of the structure factors. This method is a useful tool for structural studies primarily in combination with 3D cryoTEM reconstructions. It has been used to assess the quality of 3D reconstructions, to determine corrections for the phase-contrast transfer function of the transmission electron microscope, to calibrate the dimensions and handedness of 3D reconstructions, to produce difference maps, to model features in macromolecules or macromolecular complexes, and to generate models to initiate model-based determination of particle orientation and origin parameters for 3D reconstruction.     

The solution structure of a [3Fe-4S] ferredoxin: oxidised ferredoxin II from Desulfovibrio gigas

The use of standard 2D NMR experiments in combination with 1D NOE experiments allowed the assignment of 51 of the 58 spin systems of oxidised [3Fe-4S] ferredoxin isolated from Desulfovibrio gigas. The NMR solution structure was determined using data from 1D NOE and 2D NOESY spectra, as distance constraints, and information from the X-ray structure for the spin systems not detected by NMR in torsion angle dynamics calculations to produce a family of 15 low target function structures. The quality of the NMR family, as judged by the backbone r.m.s.d. values, was good (0.80?Å), with the majority of φ/ψ angles falling within the allowed region of the Ramachandran plot. A comparison with the X-ray structure indicated that the overall global fold is very similar in solution and in the solid state. The determination of the solution structure of ferredoxin II (FdII) in the oxidised state (FdII_ox) opens the way for the determination of the solution structure of the redox intermediate state of FdII (FdII_int), for which no X-ray structure is available.     

Developments in mid-infrared FT-IR spectroscopy of selected carbohydrates

FT-IR spectroscopy has become a powerful research tool for elucidating the structure, physical properties and interactions of carbohydrates. It provides a new interpretive and experimental framework for the study of complicated systems of natural polymers. This paper gives an overview over new infrared applications in the study of carbohydrates, both small compounds and macromolecules. These include a wide range of studies of carbohydrates in different physical states, from the crystalline solid state to aqueous solution, and special techniques, which expand the experimental framework to the in-muro studies of plant materials, and quantitative determination.     

Conformation of oligonucleotides in solution from data of the nuclear Overhauser effect

In this review the results of studies of oligonucleotides conformation in solution by 1D and 2D NOE over the last four years are presented. Theoretical basis of 2D NOE and other 2D techniques are briefly considered. The few attempts of determination of oligonucleotides structures in solution on the half-quantitative level and the perspectives of using 2D NOE for quantitative structure resolution are discussed.     

Single particle reconstruction of membrane proteins: A tool for understanding the 3D structure of disease-related macromolecules

Membrane proteins play important roles in cell functions such as neurotransmission, muscle contraction, and hormone secretion, but their structures are mostly undetermined. Several techniques have been developed to elucidate the structure of macromolecules; X-ray or electron crystallography, nuclear magnetic resonance spectroscopy, and high-resolution electron microscopy. Electron microscopy-based single particle reconstruction, a computer-aided structure determination method, reconstructs a three-dimensional (3D) structure from projections of monodispersed protein. A large number of particle images are picked up from EM films, aligned and classified to generate two-dimensional (2D) averages, and, using the Euler angle of each 2D average, reconstructed into a 3D structure. This method is challenging due to the necessity for close collaboration between classical biochemistry and innovative information technology, including parallel computing. However, recent progress in electron microscopy, mathematical algorithms, and computational ability has greatly increased the subjects that are considered to be primarily addressable using single particle reconstruction. Membrane proteins are one of these targets to which the single particle reconstruction is successfully applied for understanding of their structures. In this paper, we will introduce recently reconstructed channel-related proteins and discuss the applicability of this technique in understanding molecular structures and their roles in pathology.     

Low-Resolution Density Maps from Atomic Models: How Stepping “Back” Can Be a Step “Forward”

Atomic-resolution structures have had a tremendous impact on modern biological science. Much useful information also has been gleaned by merging and correlating atomic-resolution structural details with lower-resolution (15–40 Å), three-dimensional (3D) reconstructions computed from images recorded with cryo-transmission electron microscopy (cryoTEM) procedures. One way to merge these structures involves reducing the resolution of an atomic model to a level comparable to a cryoTEM reconstruction. A low-resolution density map can be derived from an atomic-resolution structure by retrieving a set of atomic coordinates editing the coordinate file, computing structure factors from the model coordinates, and computing the inverse Fourier transform of the structure factors. This method is a useful tool for structural studies primarily in combination with 3D cryoTEM reconstructions. It has been used to assess the quality of 3D reconstructions, to determine corrections for the phase-contrast transfer function of the transmission electron microscope, to calibrate the dimensions and handedness of 3D reconstructions, to produce difference maps, to model features in macromolecules or macromolecular complexes, and to generate models to initiate model-based determination of particle orientation and origin parameters for 3D reconstruction.     

Multi-scale calculation and global-fit analysis of hydrodynamic properties of biological macromolecules: determination of the overall conformation of antibody IgG molecules

We present a scheme, based on existing and newly developed computational tools, for the determination of the overall conformation of biological macromolecules composed by domains or subunits, using from such structural determination easily available solution properties. In a multi-scale approach, atomic-level structures are used to provide simple shapes for the subunits, which are put together in a coarse grained model, with a few parameters that determine the overall shape of the macromolecule. Computer programs, like those in the HYDRO suite that evaluate the properties of either atomic or coarse-grained models. In this paper we present a new scheme for a global fit of multiple properties, implemented in a new computer program, HYDROFIT, which interfaces with the programs of the HYDRO suite to find an optimum, best-fitting structure in a robust but simple way. The determination of the overall structure of the native antibody IgG3, bearing a long hinge, and that of the hingeless mutant m15 is presented to test and confirm the validity of this simple, systematic and efficient scheme.     

Single particle macromolecular structure determination via electron microscopy

Three-dimensional structure determination of macromolecules and macromolecular complexes is an integral part of understanding biological functions. For large protein and macromolecular complexes structure determination is often performed using electron cryomicroscopy where projection images of individual macromolecular complexes are combined to produce a three-dimensional reconstruction. Single particle methods have been devised to perform this structure determination for macromolecular complexes with little or no underlying symmetry. These computational methods generally involve an iterative process of aligning unique views of the macromolecular images followed by determination of the angular components that define those views. In this review, this structure determination process is described with the aim of clarifying a seemingly complex structural method.     

Use of negative stain and single-particle image processing to explore dynamic properties of flexible macromolecules

Flexible macromolecules pose special difficulties for structure determination by crystallography or NMR. Progress can be made by electron microscopy, but electron cryo-microscopy of unstained, hydrated specimens is limited to larger macromolecules because of the inherently low signal-to-noise ratio. For three-dimensional structure determination, the single particles must be invariant in structure. Here, we describe how we have used negative staining and single-particle image processing techniques to explore the structure and flexibility of single molecules of two motor proteins: myosin and dynein. Critical for the success of negative staining is a hydrophilic, thin carbon film, because it produces a low noise background around each molecule, and stabilises the molecule against damage by the stain. The strategy adopted for single-particle image processing exploits the flexibility available within the SPIDER software suite. We illustrate the benefits of successive rounds of image alignment and classification, and the use of whole molecule averages and movies to analyse and display both structure and flexibility within the dynein motor.     

Excluded volume as a determinant of macromolecular structure and reactivity

The effect of excluded volume on the thermodynamic activity of globular macromolecules and macromolecular complexes in solution is studied in the hard-particle approximation. Activity coefficients are calculated as a function of the fraction of total volume occupied by macromolecules using relations obtained from scaled particle and lattice models. Significant and readily observable effects are predicted to occur as the fraction of volume occupied by globular macromolecules increases, including the following: (i) Compact quasi-spherical macromolecular conformations become increasingly energetically favored over extended anisometric conformations. (ii) Self- and heteroassociation processes are enhanced, particularly those leading to the formation of compact quasi-spherical aggregates. (iii) Depending upon the details of the reaction mechanism, the rate of an enzyme-catalyzed reaction may monotonically decrease, go through a maximum, or exhibit more complex behavior. A given degree of volume occupancy by larger macromolecules is predicted to have less effect on the structure and self-association of smaller macromolecules than the same degree of volume occupancy by smaller macromolecules has on the structure and self-association of larger macromolecules.     

Locating macromolecules and determining structures inside bacterial cells using electron cryotomography

Electron cryotomography (cryo-ET) is an imaging technique uniquely suited to the study of bacterial ultrastructure and cell biology. Recent years have seen a surge in structural and cell biology research on bacteria using cryo-ET. This research has driven major technical developments in the field, with applications emerging to address a wide range of biological questions. In this review, we explore the diversity of cryo-ET approaches used for structural and cellular microbiology, with a focus on in situ localization and structure determination of macromolecules. The first section describes strategies employed to locate target macromolecules within large cellular volumes. Next, we explore methods to study thick specimens by sample thinning. Finally, we review examples of macromolecular structure determination in a cellular context using cryo-ET. The examples outlined serve as powerful demonstrations of how the cellular location, structure, and function of any bacterial macromolecule of interest can be investigated using cryo-ET.     

Protein crystallization: from purified protein to diffraction-quality crystal

Determining the structure of biological macromolecules by X-ray crystallography involves a series of steps: selection of the target molecule; cloning, expression, purification and crystallization; collection of diffraction data and determination of atomic positions. However, even when pure soluble protein is available, producing high-quality crystals remains a major bottleneck in structure determination. Here we present a guide for the non-expert to screen for appropriate crystallization conditions and optimize diffraction-quality crystal growth.