Strategies for crystallization and structure determination of very large macromolecular assemblies

High-resolution structures of macromolecular assemblies are pivotal for our understanding of their biological functions in fundamental cellular processes. In the field of X-ray crystallography, recent methodological and instrumental advances have led to the structure determinations of macromolecular assemblies of increased size and complexity, such as those of ribosomal complexes, RNA polymerases, and large multifunctional enzymes. These advances include the use of robotic screening techniques that maximize the chances of obtaining well-diffracting crystals of large complexes through the fine sampling of crystallization space. Sophisticated crystal optimization and cryoprotection techniques and the use of highly brilliant X-ray beams from third-generation synchrotron light sources now allow data collection from weakly diffracting crystals with large asymmetric units. Combined approaches are used to derive phase information, including phases calculated from electron microscopy (EM) models, heavy atom clusters, and density modification protocols. New crystallographic software tools prove valuable for structure determination and model refinement of large macromolecular complexes.     

Defining and solving the essential protein-protein interactions in HIV infection

The structure determination of macromolecular complexes is entering a new era. The methods of optical microscopy, electron microscopy, X-ray crystallography, and nuclear magnetic resonance increasingly are being combined in hybrid method approaches to achieve an integrated view of macromolecular complexes that span from cellular context to atomic detail. A particularly important application of these hybrid method approaches is the structural analysis of the Human Immunodeficiency Virus (HIV) proteins with their cellular binding partners. High resolution structure determination of essential HIV - host cell protein complexes and correlative analysis of these complexes in the live cell can serve as critical guides in the design of a broad, new class of therapeutics that function by disrupting such complexes. Here, with the hope of stimulating some discussion, we will briefly review some of the literature in the context of what could be done to further apply structural methods to HIV research. We have chosen to focus our attention on certain aspects of the HIV replication cycle where we think that structural information would contribute substantially to the development of new therapeutic and vaccine targets for HIV.     

High-resolution electron cryomicroscopy of macromolecular assemblies

Electron cryomicroscopy is a high-resolution imaging technique that is particularly appropriate for the structural determination of large macromolecular assemblies, which are difficult to study by X-ray crystallography or NMR spectroscopy. For some biological molecules that form two-dimensional crystals, the application of electron cryomicroscopy and image reconstruction can help elucidate structures at atomic resolution. In instances where crystals cannot be formed, atomic-resolution information can be obtained by combining high-resolution structures of individual components determined by X-ray crystallography or NMR with image-derived reconstructions at moderate resolution. This can provide unique and crucial information on the mechanisms of these complexes. Finally, image reconstructions can be used to augment X-ray studies by providing initial models that facilitate phasing of crystals of large macromolecular machines such as ribosomes and viruses.     

Toward visualization of nanomachines in their native cellular environment

The cellular nanocosm is made up of numerous types of macromolecular complexes or biological nanomachines. These form functional modules that are organized into complex subcellular networks. Information on the ultra-structure of these nanomachines has mainly been obtained by analyzing isolated structures, using imaging techniques such as X-ray crystallography, NMR, or single particle electron microscopy (EM). Yet there is a strong need to image biological complexes in a native state and within a cellular environment, in order to gain a better understanding of their functions. Emerging methods in EM are now making this goal reachable. Cryo-electron tomography bypasses the need for conventional fixatives, dehydration and stains, so that a close-to-native environment is retained. As this technique is approaching macromolecular resolution, it is possible to create maps of individual macromolecular complexes. X-ray and NMR data can be ‘docked’ or fitted into the lower resolution particle density maps to create a macromolecular atlas of the cell under normal and pathological conditions. The majority of cells, however, are too thick to be imaged in an intact state and therefore methods such as ‘high pressure freezing’ with ‘freeze-substitution followed by room temperature plastic sectioning’ or ‘cryo-sectioning of unperturbed vitreous fully hydrated samples’ have been introduced for electron tomography. Here, we review methodological considerations for visualizing nanomachines in a close-to-physiological, cellular context. EM is in a renaissance, and further innovations and training in this field should be fully supported. Robert Feulgen Lecture 2009 presented at the 51st symposium of the Society for Histochemistry in Stubai, Austria, October 7–10, 2009.     

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.     

The emerging role of native mass spectrometry in characterizing the structure and dynamics of macromolecular complexes

Mass spectrometry (MS) is a powerful tool for determining the mass of biomolecules with high accuracy and sensitivity. MS performed under so-called “native conditions” (native MS) can be used to determine the mass of biomolecules that associate noncovalently. Here we review the application of native MS to the study of protein−ligand interactions and its emerging role in elucidating the structure of macromolecular assemblies, including soluble and membrane protein complexes. Moreover, we discuss strategies aimed at determining the stoichiometry and topology of subunits by inducing partial dissociation of the holo-complex. We also survey recent developments in "native top-down MS", an approach based on Fourier Transform MS, whereby covalent bonds are broken without disrupting non-covalent interactions. Given recent progress, native MS is anticipated to play an increasingly important role for researchers interested in the structure of macromolecular complexes.     

Direct observation of unstained wet biological samples by scanning-electron generation X-ray microscopy

Analytical tools of nanometre-scale resolution are indispensable in the fields of biology, physics and chemistry. One suitable tool, the soft X-ray microscope, provides high spatial resolution of visible light for wet specimens. For biological specimens, X-rays of water-window wavelength between carbon (284 eV; 4.3 nm) and oxygen (540 eV; 2.3 nm) absorption edges provide high-contrast imaging of biological samples in water. Among types of X-ray microscope, the transmission X-ray microscope using a synchrotron radiation source with diffractive zone plates offers the highest spatial resolution, approaching 15-10 nm. However, even higher resolution is required to measure proteins and protein complexes in biological specimens; therefore, a new type of X-ray microscope with higher resolution that uses a simple light source is desirable. Here we report a novel scanning-electron generation X-ray microscope (SGXM) that demonstrates direct imaging of unstained wet biological specimens. We deposited wet yeasts in the space between two silicon nitride (Si₃N₄) films. A scanning electron beam of accelerating voltage 5 keV and current 1.6 nA irradiates the titanium (Ti)-coated Si₃N₄ film, and the soft X-ray signal from it is detected by an X-ray photodiode (PD) placed below the sample. The SGXM can theoretically achieve better than 5 nm resolution. Our method can be utilized easily for various wet biological samples of bacteria, viruses, and protein complexes.     

Exploring overlapping functional units with various structure in protein interaction networks

Revealing functional units in protein-protein interaction (PPI) networks are important for understanding cellular functional organization. Current algorithms for identifying functional units mainly focus on cohesive protein complexes which have more internal interactions than external interactions. Most of these approaches do not handle overlaps among complexes since they usually allow a protein to belong to only one complex. Moreover, recent studies have shown that other non-cohesive structural functional units beyond complexes also exist in PPI networks. Thus previous algorithms that just focus on non-overlapping cohesive complexes are not able to present the biological reality fully. Here, we develop a new regularized sparse random graph model (RSRGM) to explore overlapping and various structural functional units in PPI networks. RSRGM is principally dominated by two model parameters. One is used to define the functional units as groups of proteins that have similar patterns of connections to others, which allows RSRGM to detect non-cohesive structural functional units. The other one is used to represent the degree of proteins belonging to the units, which supports a protein belonging to more than one revealed unit. We also propose a regularizer to control the smoothness between the estimators of these two parameters. Experimental results on four S. cerevisiae PPI networks show that the performance of RSRGM on detecting cohesive complexes and overlapping complexes is superior to that of previous competing algorithms. Moreover, RSRGM has the ability to discover biological significant functional units besides complexes.     

Solid-state magic-angle spinning NMR of membrane proteins and protein-ligand interactions

Structural biology is developing into a universal tool for visualizing biological processes in space and time at atomic resolution. The field has been built by established methodology like X-ray crystallography, electron microscopy and solution NMR and is now incorporating new techniques, such as small-angle X-ray scattering, electron tomography, magic-angle-spinning solid-state NMR and femtosecond X-ray protein nanocrystallography. These new techniques all seek to investigate non-crystalline, native-like biological material. Solid-state NMR is a relatively young technique that has just proven its capabilities for de novo structure determination of model proteins. Further developments promise great potential for investigations on functional biological systems such as membrane-integrated receptors and channels, and macromolecular complexes attached to cytoskeletal proteins. Here, we review the development and applications of solid-state NMR from the first proof-of-principle investigations to mature structure determination projects, including membrane proteins. We describe the development of the methodology by looking at examples in detail and provide an outlook towards future 'big' projects.     

Synchrotron Protein Footprinting: A Technique to Investigate Protein-Protein Interactions

Abstract

Traditional approaches for macromolecular structure elucidation, including NMR, crystallography and cryo-EM have made significant progress in defining the structures of protein-protein complexes. A substantial number of macromolecular structures, however, have not been examined with atomic detail due to sample size and heterogeneity, or resolution limitations of the technique; therefore, the general applicability of each method is greatly reduced. Synchrotron footprinting attempts to bridge the gap in these methods by monitoring changes in accessible surface areas of discrete macromolecular moieties. As evidenced by our previous studies on RNA folding and DNA-protein interactions, the three-dimensional structure is probed by examining the reactions of these moieties with hydroxyl radicals generated by synchrotron X-rays. Here we report the application of synchrotron foot- printing to the investigation of protein-protein interactions, as the novel technique has been utilized to successfully map the contact sites of gelsolin segment-1 in the gelsolin segment 1/actin complex. Footprinting results demonstrate that phenylalanine 104, located on the actin binding helix of gelsolin segment 1, is protected from hydroxyl radical modification in the presence of actin. This change in reactivity results from the specific protection of gel- solin segment-1, consistent with the substantial decrease in solvent accessibility of F104 upon actin binding, as calculated from the crystal structural of the gelsolin segment 1/actin complex. The results presented here establish synchrotron footprinting as a broadly applicable method to probe structural features of macromolecular complexes that are not amenable to conventional approaches.     

(Questions) on phloem biology. 2. Mass flow, molecular hopping, distribution patterns and macromolecular signalling

This review speculates on correlations between mass flow in sieve tubes and the distribution of photoassimilates and macromolecular signals. Since micro- (low-molecular compounds) and macromolecules are withdrawn from, and released into, the sieve-tube sap at various rates, distribution patterns of these compounds do not strictly obey mass-flow predictions. Due to serial release and retrieval transport steps executed by sieve tube plasma membranes, micromolecules are proposed to “hop” between sieve element/companion cell complexes and phloem parenchyma cells under source-limiting conditions (apoplasmic hopping). Under sink-limiting conditions, micromolecules escape from sieve tubes via pore-plasmodesma units and are temporarily stored. It is speculated that macromolecules “hop” between sieve elements and companion cells using plasmodesmal trafficking mechanisms (symplasmic hopping). We explore how differential tagging may influence distribution patterns of macromolecules and how their bidirectional movement could arise. Effects of exudation techniques on the macromolecular composition of sieve-tube sap are discussed.     

Deriving correlated motions in proteins from X-ray structure refinement by using TLS parameters

Dynamic information in proteins may provide valuable information for understanding allosteric regulation of protein complexes or long-range effects of the mutations on enzyme activity. Experimental data such as X-ray B-factors or NMR order parameters provide a convenient estimate of atomic fluctuations (or atomic auto-correlated motions) in proteins. However, it is not as straightforward to obtain atomic cross-correlated motions in proteins — one usually resorts to more sophisticated computational methods such as Molecular Dynamics, normal mode analysis or atomic network models. In this report, we show that atomic cross-correlations can be reliably obtained directly from protein structure using X-ray refinement data. We have derived an analytic form of atomic correlated motions in terms of the original TLS parameters used to refine the B-factors of X-ray structures. The correlated maps computed using this equation are well correlated with those of the method based on a mechanical model (the correlation coefficient is 0.75) for a non-homologous dataset comprising 100 structures. We have developed an approach to compute atomic cross-correlations directly from X-ray protein structure. Being in analytic form, it is fast and provides a feasible way to compute correlated motions in proteins in a high throughput way. In addition, avoiding sophisticated computational operations; it provides a quick, reliable way, especially for non-computational biologists, to obtain dynamics information directly from protein structure relevant to its function.     

Structural analysis of membrane protein complexes by single particle electron microscopy

Single particle electron microscopy (EM) is an increasingly important tool for the structural analysis of macromolecular complexes. The main advantage of the technique over other methods is that it is not necessary to precede the analysis with the growth of crystals of the sample. This advantage is particularly important for membrane proteins and large protein complexes where generating crystals is often the main barrier to structure determination. Therefore, single particle EM can be employed with great utility in the study of large membrane protein complexes. Although the construction of atomic resolution models by single particle EM is possible in theory, currently the highest resolution maps are still limited to approximately 7-10A resolution and 15-30 A resolution is more typical. However, by combining single particle EM maps with high-resolution models of subunits or subcomplexes from X-ray crystallography and NMR spectroscopy it is possible to build up an atomic model of a macromolecular assembly. Image analysis procedures are almost identical for micrographs of soluble protein complexes and detergent solubilized membrane protein complexes. However, electron microscopists attempting to prepare specimens of a membrane protein complex for imaging may find that these complexes require different handling than soluble protein complexes. This paper seeks to explain how high-quality specimen grids of membrane protein complexes may be prepared to allow for the determination of their structure by EM and image analysis.     

Steps towards flexible docking: modeling of three-dimensional structures of the nuclear receptors bound with peptide ligands mimicking co-activators' sequences

We developed a fully flexible docking method that uses a reduced lattice representation of protein molecules, adapted for modeling peptide–protein complexes. The CABS model (Carbon Alpha, Carbon Beta, Side Group) employed here, incorporates three pseudo-atoms per residue—C, Cβ and the center of the side group instead of full-atomic protein representation. Force field used by CABS was derived from statistical analysis of non-redundant database of protein structures. Application of our method included modeling of the complexes between various nuclear receptors (NRs) and peptide co-activators, for which three-dimensional structures are known. We tried to rebuild the native state of the complexes, starting from separated components. Accuracy of the best obtained models, calculated as coordinate root-mean-square deviation (cRMSD) between the target and the modeled structures, was under 1 Å, which is competitive with experimental methods, such as crystallography or NMR. Forthcoming modeling study should lead to better understanding of mechanisms of macromolecular assembly and will explain co-activators’ effects on receptors activity, especially on vitamin D receptor and other nuclear receptors.     

Native gel analysis of macromolecular protein complexes in cultured mammalian cells

Native gel electrophoresis enables separation of cellular proteins in their non‐denatured state. In experiments aimed at analysing proteins in higher order or multimeric assemblies (i.e. protein complexes) it offers some advantages over rival approaches, particularly as an interface technology with mass spectrometry. Here we separated fractions from HEK293 cells by native electrophoresis in order to survey protein complexes in the cytoplasmic, nuclear and chromatin environments, finding 689 proteins distributed among 217 previously described complexes. As expected, different fractions contained distinct combinations of macromolecular complexes, with subunits of the same complex tending to co‐migrate. Exceptions to this observation could often be explained by the presence of subunits shared among different complexes. We investigated one identified complex, the Polycomb Repressor Complex 2 (PRC2), in more detail following affinity purification of the EZH2 subunit. This approach resulted in the identification of all previously reported members of PRC2. Overall, this work demonstrates that the use of native gel electrophoresis as an upstream separating step is an effective approach for analysis of the components and cellular distribution of protein complexes.     

Size distribution of native cytosolic proteins of Thermoplasma acidophilum

We used molecular sieve chromatography in combination with LC‐MS/MS to identify protein complexes that can serve as templates in the template matching procedures of visual proteomics approaches. By this method the sample complexity was lowered sufficiently to identify 464 proteins and – on the basis of size distribution and bioinformatics analysis – 189 of them could be assigned as subunits of macromolecular complexes over the size of 300 kDa. From these we purified six stable complexes of Thermoplasma acidophilum whose size and subunit composition – analyzed by electron microscopy and MALDI‐TOF‐MS, respectively – verified the accuracy of our method.