Single-particle Reconstruction Techniques Research Articles

Fine details in complex environments: the power of cryo-electron tomography.

Cryo-electron tomography (CET) is uniquely suited to obtain structural information from a wide range of biological scales, integrating and bridging knowledge from molecules to cells. In particular, CET can be used to visualise molecular structures in their native environment. Depending on the experiment, a varying degree of resolutions can be achieved, with the first near-atomic molecular structures becoming recently available. The power of CET has increased significantly in the last 5 years, in parallel with improvements in cryo-EM hardware and software that have also benefited single-particle reconstruction techniques. In this review, we cover the typical CET pipeline, starting from sample preparation, to data collection and processing, and highlight in particular the recent developments that support structural biology in situ. We provide some examples that highlight the importance of structure determination of molecules embedded within their native environment, and propose future directions to improve CET performance and accessibility.

3D Motion Maps of TRPV1 Cation Channel Depicted by Diffracted X-ray Tracking Method

The TRPV1 is a nonselective cation channel that responds to various signals, including high temperature with threshold at 43 °C, acidic conditions, as well as chemical compounds such as capsaicin and allylisothiocyanate. Because TRPV1 is a membrane protein with large molecular mass, crystallography technique has not been succeeded for a long time. However recent progress in the cryoelectron microscopy enabled depiction of the TRPV1 structure in atomic resolution. Cryo-EM and the single particle reconstruction technique showed structures of apo- and ligand binding forms of TRPV1 with various agonists, antagonists and toxins. In spite of accumulation of structural data, gating mechanisms of TRPV1 is not clearly understood yet. Information about state-to-state transition is missing. To understand the dynamics of TRPV1 in channel function, we adopted the Diffracted X-ray Tracking (DXT) technique to this protein. In DXT, individual protein was labeled with gold nanocrystals, and the motion of X-ray diffraction spots from the crystal were investigated as intramolecular movement of TRPV1 in real time. We introduced “Met tag” for labeling nanocrystal and “His tag” for substrate absorption to TRPV1. It was expressed in HEK293 cells, purified, and immobilized on the Ni-NTA coated polyimide substrates. Intramolecular motion was recorded by tracking the movement of diffraction spots from the gold nanocrystals. Molecular dynamics of TRPV1 against capsaicin, pH change, and temperature activation were investigated.

Spherical deconvolution improves quality of single particle reconstruction.

One single-particle reconstruction technique is the reconstruction of macromolecules from projection images of randomly oriented particles (SPRR). In SPRR the reliability and consequent interpretation of the final reconstruction is affected by errors arising from incorrect assignment of projection angles to individual particles. In order to improve the resolution of SPRR we studied the influence of imperfect assignment on 3D blurring. We find that this blurring can be described as a Point Spread Function (PSF) that depends on the distance from geometrical center of the reconstructed volume and that blurring is higher at the periphery. This particular PSF can be described by an almost pure tangential angular function with a negligible radial component. We have developed a reliable algorithm for spherical deconvolution of the 3D reconstruction. This spherical deconvolution operation was tested on reconstructions of GroEL and mitochondrial ribosomes. We show that spherical deconvolution improves the quality of SPRR by reducing blurring and enhancing high frequency components, particularly near the periphery of the reconstruction.

A CRYO-EM Structure of a Minimal Translation Initiation System: Deletion in Self-Sufficient IRES Prevents Translocation

Translation occurs on the ribosome, a ribonucleoprotein machine that exists in many copies per cell and is conserved among all forms of life. In canonical eukaryotic translation initiation, a suite of protein factors is required for many essential processes: for messenger RNA (mRNA) to bind the ribosome and to align itself with the translation initiation site, for the first aminoacylated tRNA to bind the start codon, for the ribosome to shift the mRNA downstream by one codon, and more. These processes occur on and near the mRNA's start codon immediately downstream of a 5' modified-nucleotide cap.In a departure from canonical catalytic and location requirements, some eukaryotes and many viruses encode regions of highly structured RNA that can initiate translation without a 5' cap and at least some initiation factors. These RNAs are called internal ribosomal entry sites, or IRESes. We study a type of IRES found in the Dicistroviridae virus class. This type of IRES initiates translation of the second of two gene clusters in the continuous viral RNA genome, and, uniquely, it requires no initiation factors to hijack eukaryotic host ribosomes.Here, we present a three-dimensional reconstruction of one such IRES--the Cricket Paralysis Virus intergenic IRES--bound to the eukaryotic yeast ribosome. The structure was obtained by cryo-electron microscopy and single-particle reconstruction techniques. This particular IRES sample is a mutant lacking three consecutive nucleotides in a loop region; it cannot undergo the initial translocation event, when mRNA shifts to allow a new aminoacylated tRNA into the A-site. We examine how the deletion prevents translocation, and thus how the intact IRES, a unique minimal translation system, might enable translocation, an essential part of initiating the universal translation process that is not wholly understood.

P117 3D cryo-electron microscopic analysis of the disease mechanism of α1-antitrypsin deficiency

α1-antitrypsin deficiency is characterised by predispositions to liver disease and severe, early-onset emphysema. It is caused by point mutations that destabilise the molecular structure of α1-antitrypsin, leading it to self-associate...

Molecular Architecture of the Human Mediator–RNA Polymerase II–TFIIF Assembly

Author SummaryTranscription initiation in humans is regulated by a macromolecular complex formed by the RNA polymerase II enzyme (pol II), Mediator, and the general transcription factors TFIIA, TFIIB, TFIID, TFIIE, TFIIF, and TFIIH. Collectively, these factors are known as the Pre-Initiation Complex (PIC). Although the 1.2 MDa Mediator seems to have a major role in regulating assembly and function of the PIC, a structural understanding of the complex has yet to be established. This study outlines a cryo-EM analysis of the Mediator–pol II assembly in the presence or absence of the dimeric TFIIF complex. We observe that TFIIF is required to stably orient the pol II enzyme within the Mediator–pol II assembly, indicating a novel structural role for TFIIF in transcription initiation. Additionally, we accurately dock the pol II crystal structure within the human Mediator–pol II–TFIIF cryo-EM map. The locations of TFIIH, TBP (a subunit within TFIID), TFIIA, TFIIB, TFIIE, and TFIIF relative to the pol II enzyme itself have been determined by previous studies. These data in combination with the Mediator–pol II–TFIIF structure described here allow us to propose the structural organization of the entire 3.5 MDa human PIC.

Mitsugumin23, a Protein Associated with Intracellular Calcium Stores, Behaves as an Ion-Channel that can Conduct Calcium

Mitsugumin23 (MG23) is localized to ER/SR and nuclear membranes in a wide range of cell types. It has recently been linked with apoptosis (Yamazaki et al. (2010) 392, 196-200) but its underlying physiological function is unknown. Proteolytic assay of purified MG23 predicts three transmembrane segments and chemical crosslinking experiments provide evidence of a multimeric complex. Reconstitution of MG23 into artificial membranes under voltage-clamp conditions revealed that MG23 behaves as a cation channel that is permeable to both monovalent and divalent cations. A characteristic of MG23 channels was that multiple channels always appeared to be gating together in the bilayer. We frequently observed long episodes of the apparent co-ordinated gating of multiple channels but we have not identified any tendency for a preferred number of MG23 channels to gate together in synchrony; this seems to be a random process. MG23 showed distinct voltage-dependence, being more open at negative holding potentials. Electron microscopy and single particle reconstruction techniques indicate that purified MG23 forms two types of structure; large shapes of 6-fold symmetry and smaller, asymmetrically-shaped particles. We suggest that the apparent coordinated opening of various numbers of MG23 channels may reflect reversible structural changes in subunit assembly. We propose that MG23 is a homo-multimeric complex which transitorily forms cation-channels in intracellular membranes, enabling rapid changes in the ionic fluxes across intracellular Ca2+-stores.Supported by the British Heart Foundation, Japan Society for the Promotion of Science, Japan New Energy and Industrial Technology Development Organization, Astellas Foundation, Mochida Memorial Foundation, Kanae Foundation, Sankyo Foundation of Life Science, Salt Science Research Foundation and Takeda Science Foundation.

Cryo-Electron Microscopy, Tomography, and Single-Particle Analysis of MSP Filaments Derived from the Amoeboid Sperm of Ascaris Suum

Locomotion of nematode sperm is remarkably similar to that of most other crawling cells but is powered by a system of filaments composed of major sperm protein (MSP) instead of the actin-myosin machinery typically associated with amoeboid motility. The MSP motility apparatus has been reconstituted in vitro, and individual MSP filaments can be generated by the addition of ATP to detergent-treated Ascaris sperm cytosol. Filaments formed in this way have been successfully vitrified and examined using cryo-electron microscopy, and this has allowed for the structural analysis of filaments formed in the presence of MSP accessory proteins. Single particle reconstruction techniques have been applied to individual filaments sampled along their lengths, and whole filament meshworks have been reconstructed using electron cryo-tomography. These resulting physiological models were compared to earlier models derived using purified MSP, including a helical reconstruction of filaments polymerized in ethanol (King et al. 1992. JCS 101:847) and an x-ray crystal model of MSP subfilaments (Bullock et al. 1998. NSB 5:184). Comparisons suggest important differences between filaments formed under physiological and nonphysiological conditions. There are currently six Ascaris sperm proteins known to modulate MSP filament dynamics in sperm; these same proteins are absent from filaments prepared using purified MSP. Comparing MSP filament models with and without these accessory proteins allows us to map the binding sites of these accessory proteins and provides a greater understanding of how they effect MSP filament dynamics and influence motility. Supported by NIH Grant R37 GM29994 and by the American Heart Assoc.

The structural landscape of native editosomes in African trypanosomes.

The majority of mitochondrial pre-messenger RNAs in African trypanosomes are substrates of a U-nucleotide-specific insertion/deletion-type RNA editing reaction. The process converts nonfunctional pre-mRNAs into translation-competent molecules and can generate protein diversity by alternative editing. High molecular mass protein complexes termed editosomes catalyze the processing reaction. They stably interact with pre-edited mRNAs and small noncoding RNAs, known as guide RNAs (gRNAs), which act as templates in the reaction. Editosomes provide a molecular surface for the individual steps of the catalytic reaction cycle and although the protein inventory of the complexes has been studied in detail, a structural analysis of the processing machinery has only recently been accomplished. Electron microscopy in combination with single particle reconstruction techniques has shown that steady state isolates of editosomes contain ensembles of two classes of stable complexes with calculated apparent hydrodynamic sizes of 20S and 35-40S. 20S editosomes are free of substrate RNAs, whereas 35-40S editosomes are associated with endogenous mRNA and gRNA molecules. Both complexes are characterized by a diverse structural landscape, which include complexes that lack or possess defined subdomains. Here, we summarize the consensus models and structural landmarks of both complexes. We correlate structural features with functional characteristics and provide an outlook into dynamic aspects of the editing reaction cycle.

Molecular basis of transcription initiation in Archaea

Compared with eukaryotes, the archaeal transcription initiation machinery—commonly known as the Pre-Initiation Complex—is relatively simple. The archaeal PIC consists of the TFIIB ortholog TFB, TBP, and an 11-subunit RNA polymerase (RNAP). The relatively small size of the entire archaeal PIC makes it amenable to structural analysis. Using purified RNAP, TFB, and TBP from the thermophile Pyrococcus furiosus, we assembled the biochemically active PIC at 65ºC. The intact archaeal PIC was isolated by implementing a cross-linking technique followed by size-exclusion chromatography, and the structure of this 440 kDa assembly was determined using electron microscopy and single-particle reconstruction techniques. Combining difference maps with crystal structure docking of various sub-domains, TBP and TFB were localized within the macromolecular PIC. TBP/TFB assemble near the large RpoB subunit and the RpoD/L “foot” domain behind the RNAP central cleft. This location mimics that of yeast TBP and TFIIB in complex with yeast RNAP II. Collectively, these results define the structural organization of the archaeal transcription machinery and suggest a conserved core PIC architecture.

Chapter One - Fundamentals of Three-Dimensional Reconstruction from Projections

Three-dimensional (3D) reconstruction of an object mass density from the set of its 2D line projections lies at a core of both single-particle reconstruction technique and electron tomography. Both techniques utilize electron microscope to collect a set of projections of either multiple objects representing in principle the same macromolecular complex in an isolated form, or a subcellular structure isolated in situ. Therefore, the goal of macromolecular electron microscopy is to invert the projection transformation to recover the distribution of the mass density of the original object. The problem is interesting in that in its discrete form it is ill-posed and not invertible. Various algorithms have been proposed to cope with the practical difficulties of this inversion problem and their differ widely in terms of their robustness with respect to noise in the data, completeness of the collected projection dataset, errors in projections orientation parameters, abilities to efficiently handle large datasets, and other obstacles typically encountered in molecular electron microscopy. Here, we review the theoretical foundations of 3D reconstruction from line projections followed by an overview of reconstruction algorithms routinely used in practice of electron microscopy.

Visualizing tmRNA after its accommodation in the Ribosome

In eubacteria, translation of an mRNA that lacks a stop codon produces defective polypeptide that stalls on the ribosome. Transfer-messenger RNA (tmRNA), a molecule in eubacteria that possesses functions of both mRNA and tRNA, rescues the stalled ribosome by “trans-translation,” a process by which the tmRNA is recruited to the ribosome with the help of EF-Tu and small protein B (smpB). Tanslation is resumed on the open reading frame of the mRNA-like domain (MLD) of the tmRNA. Several structures of tmRNA in complex with ribosome, in the accommodating and the accommodated states, have been studied by cryo-EM single-particle reconstruction (Gillet et al., 2007; Kaur et al., 2006; Valle et al., 2003). However, the structures of the complex in the subsequent stages of trans-translation remain unknown. Here, using mutagenesis, we have been able to trap the complex in different stages of trans-translation by substituting one of the sense codons of the MLD open reading frame with a stop codon. Structures of these complexes were obtained by the cryo-EM single particle reconstruction technique. To address the sample heterogeneity, we used the maximum-liklihood classification method (Scheres et al., 2007). The resulting density maps were analyzed by rigid body fitting in combination with biochemical data. We discovered that part of the tmRNA molecule maintains a relative defined structure during tran-translation. Also, we identified several possible binding sites of the tRNA like domain (TLD) of tmRNA and smpB on the ribosome.Gillet et al. (2007) J. Biol. Chem. 282: 6356-6363.Kaur S. et al. (2006) Proc. Natl. Acad. Sci. USA 103:16484-16489.Scheres S. et al. (2007) Nature Methods 4: 27-29.Valle M. et al. (2003) Science 300: 127-130.

Three-dimensional Reconstruction of Human Cystic Fibrosis Transmembrane Conductance Regulator Chloride Channel Revealed an Ellipsoidal Structure with Orifices beneath the Putative Transmembrane Domain

The cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel is a membrane-integral protein that belongs to an ATP-binding cassette superfamily. Mutations in the CFTR gene cause cystic fibrosis in which salt, water, and protein transports are defective in various tissues. Here we expressed wild-type human CFTR as a FLAG-fused protein in HEK293 cells heterologously and purified it in three steps: anti-FLAG and wheat germ agglutinin affinity chromatographies and size exclusion chromatography. The stoichiometry of the protein was analyzed using various biochemical approaches, including chemical cross-linking, blue-native PAGE, size exclusion chromatography, and electron microscopy (EM) observation of antibody-decorated CFTR. All these data support a dimeric assembly of CFTR. Using 5,039 automatically selected particles from negatively stained EM images, the three-dimensional structure of CFTR was reconstructed at 2-nm resolution assuming a 2-fold symmetry. CFTR, presumably in a closed state, was shown to be an ellipsoidal particle with dimensions of 120 x 106 x 162 A. It comprises a small dome-shaped extracellular and membrane-spanning domain and a large cytoplasmic domain with orifices beneath the putative transmembrane domain. EM observation of CFTR.anti-regulatory domain antibody complex confirmed that two regulatory domains are located around the bottom end of the larger oval cytoplasmic domain.

Achievable resolution from images of biological specimens acquired from a 4k×4k CCD camera in a 300-kV electron cryomicroscope

Bacteriorhodopsin and epsilon 15 bacteriophage were used as biological test specimens to evaluate the potential structural resolution with images captured from a 4k x 4k charge-coupled device (CCD) camera in a 300-kV electron cryomicroscope. The phase residuals computed from the bacteriorhodopsin CCD images taken at 84,000x effective magnification averaged 15.7 degrees out to 5.8-A resolution relative to Henderson's published values. Using a single-particle reconstruction technique, we obtained an 8.2-A icosahedral structure of epsilon 15 bacteriophage with the CCD images collected at an effective magnification of 56,000x. These results demonstrate that it is feasible to retrieve biological structures to a resolution close to 2/3 of the Nyquist frequency from the CCD images recorded in a 300-kV electron cryomicroscope at a moderately high but practically acceptable microscope magnification.

Characterization of Vibrio cholerae O1 ElTor typing phage S5

S5 (ATCC No. 51352-B2), a Vibrio cholerae O1 ElTor typing phage was characterized. The growth characteristics and inactivation kinetics (thermal, UV and pH) of this lytic phage were investigated. Phage morphology was examined by electron microscopy and was classified as belonging to the family Podoviridae. The S5 phage genome is shown to be a linear double-stranded 39-kb-long DNA as determined by electron microscopy and restriction digestion. Partial denaturation maps were constructed and were used to show that the DNA is non-permuted and terminally redundant. The replication origin of this T7-like phage was visualized by electron microscopy. The polarity of packaging of S5 DNA in the phage head was determined. SDS-PAGE of phage S5 shows two major structural polypeptides of 50 and 42 kDa. A 3D structure of the phage head was reconstructed at a resolution of 37 A using Cryo-EM and a single-particle reconstruction technique.

Structural insights into excitation?contraction coupling by electron cryomicroscopy

In muscle, excitation-contraction coupling is defined as the process linking depolarization of the surface membrane with Ca2+ release from cytoplasmic stores, which activates contraction of striated muscle. This process is primarily controlled by interplay between two Ca2+ channels--the voltage-gated L-type Ca2+ channel (dihydropyridine receptor, DHPR) localized in the t-tubule membrane and the Ca2+-release channel (ryanodine receptor, RyR) of the sarcoplasmic reticulum membrane. The structures of both channels have been extensively studied by several groups using electron cryomicroscopy and single particle reconstruction techniques. The structures of RyR, determined at resolutions of 22-30 A, reveal a characteristic mushroom shape with a bulky cytoplasmic region and the membrane-spanning stem. While the cytoplasmic region exhibits a complex structure comprising a multitude of distinctive domains with numerous intervening cavities, at this resolution no definitive statement can be made about the location of the actual pore within the transmembrane region. Conformational changes associated with functional transitions of the Ca2+ release channel from closed to open states have been characterized. Further experiments determined localization of binding sites for various channel ligands. The structural studies of the DHPR are less developed. Although four 3D maps of the DHPR were reported recently at 24-30 A resolution from studies of frozen-hydrated and negatively stained receptors, there are some discrepancies between reported structures with respect to the overall appearance and dimensions of the channel structure. Future structural studies at higher resolution are needed to refine the structures of both channels and to substantiate a proposed molecular model for their interaction.

Seeing GroEL at 6 A resolution by single particle electron cryomicroscopy.

We present a reconstruction of native GroEL by electron cryomicroscopy (cryo-EM) and single particle analysis at 6 A resolution. alpha helices are clearly visible and beta sheet density is also visible at this resolution. While the overall conformation of this structure is quite consistent with the published X-ray data, a measurable shift in the positions of three alpha helices in the intermediate domain is observed, not consistent with any of the 7 monomeric structures in the Protein Data Bank model (1OEL). In addition, there is evidence for slight rearrangement or flexibility in parts of the apical domain. The 6 A resolution cryo-EM GroEL structure clearly demonstrates the veracity and expanding scope of cryo-EM and the single particle reconstruction technique for macromolecular machines.

Distinct conformational states of nuclear receptor-bound CRSP-Med complexes.

The human CRSP-Med coactivator complex is targeted by a diverse array of sequence-specific regulatory proteins. Using EM and single-particle reconstruction techniques, we recently completed a structural analysis of CRSP-Med bound to VP16 and SREBP-1a. Notably, these activators induced distinct conformational states upon binding the coactivator. Ostensibly, these different conformational states result from VP16 and SREBP-1a targeting distinct subunits in the CRSP-Med complex. To test this, we conducted a structural analysis of CRSP-Med bound to either thyroid hormone receptor (TR) or vitamin D receptor (VDR), both of which interact with the same subunit (Med220) of CRSP-Med. Structural comparison of TR- and VDR-bound complexes (at a resolution of 29 A) indeed reveals a shared conformational feature that is distinct from other known CRSP- Med structures. Importantly, this nuclear receptor-induced structural shift seems largely dependent on the movement of Med220 within the complex.

Structure and Function of CRSP/Med2: A Promoter-Selective Transcriptional Coactivator Complex

The multi-subunit, human CRSP coactivator—also known as Mediator (Med)—regulates transcription by mediating signals between enhancer-bound factors (activators) and the core transcriptional machinery. Interestingly, different activators are known to bind distinct subunits within the CRSP/Med complex. We have isolated a stable, endogenous CRSP/Med complex (CRSP/Med2) that specifically lacks both the Med220 and the Med70 subunits. The three-dimensional structure of CRSP/Med2 was determined to 31 Å resolution using electron microscopy and single-particle reconstruction techniques. Despite lacking both Med220 and Med70, CRSP/Med2 displays potent, activator-dependent transcriptional coactivator function in response to VP16, Sp1, and Sp1/SREBP-1a in vitro using chromatin templates. However, CRSP/Med2 is unable to potentiate activated transcription from a vitamin D receptor-responsive promoter, which requires interaction with Med220 for coactivator recruitment, whereas VDR-directed activation by CRSP/Med occurs normally. Thus, it appears that CRSP/Med may be regulated by a combinatorial assembly mechanism that allows promoter-selective function upon exchange of specific coactivator targets.

Large T antigen on the simian virus 40 origin of replication: a 3D snapshot prior to DNA replication.

Large T antigen is the replicative helicase of simian virus 40. Its specific binding to the origin of replication and oligomerization into a double hexamer distorts and unwinds dsDNA. In viral replication, T antigen acts as a functional homolog of the eukaryotic minichromosome maintenance factor MCM. T antigen is also an oncoprotein involved in transformation through interaction with p53 and pRb. We obtained the three-dimensional structure of the full-length T antigen double hexamer assembled at its origin of replication by cryoelectron microscopy and single-particle reconstruction techniques. The double hexamer shows different degrees of bending along the DNA axis. The two hexamers are differentiated entities rotated relative to each other. Isolated strands of density, putatively assigned to ssDNA, protrude from the hexamer-hexamer junction mainly at two opposite sites. The structure of the T antigen at the origin of replication can be understood as a snapshot of the dynamic events leading to DNA unwinding. Based on these results a model for the initiation of simian virus 40 DNA replication is proposed.