Intense femtosecond x-ray pulses from free-electron laser sources allow the imaging of individual particles in a single shot. Early experiments at the Linac Coherent Light Source (LCLS) have led to ...rapid progress in the field and, so far, coherent diffractive images have been recorded from biological specimens, aerosols, and quantum systems with a few-tens-of-nanometers resolution. In March 2014, LCLS held a workshop to discuss the scientific and technical challenges for reaching the ultimate goal of atomic resolution with single-shot coherent diffractive imaging. This paper summarizes the workshop findings and presents the roadmap toward reaching atomic resolution, 3D imaging at free-electron laser sources.
Knowledge about the synthesis, growth mechanisms, and physical properties of colloidal nanoparticles has been limited by technical impediments. We introduce a method for determining three-dimensional ...(3D) structures of individual nanoparticles in solution. We combine a graphene liquid cell, high-resolution transmission electron microscopy, a direct electron detector, and an algorithm for single-particle 3D reconstruction originally developed for analysis of biological molecules. This method yielded two 3D structures of individual platinum nanocrystals at near-atomic resolution. Because our method derives the 3D structure from images of individual nanoparticles rotating freely in solution, it enables the analysis of heterogeneous populations of potentially unordered nanoparticles that are synthesized in solution, thereby providing a means to understand the structure and stability of defects at the nanoscale.
We have developed methods for ab initio three-dimensional (3D) structure determination from projection images of randomly oriented single molecules coexisting in multiple functional states, to aid ...the study of complex samples of macromolecules and nanoparticles by electron microscopy (EM). New algorithms for the determination of relative 3D orientations and conformational state assignment of single-molecule projection images are combined with well-established techniques for alignment and statistical image analysis. We describe how the methodology arrives at homogeneous groups of images aligned in 3D and discuss application to experimental EM data sets of the
Escherichia coli ribosome and yeast RNA polymerase II.
► New technique for structure determination of large macromolecules ► No a priori information needed to reconstruct coexisting conformational states
Mg-chelatase catalyzes the first committed step of the chlorophyll biosynthetic pathway, the ATP-dependent insertion of Mg
2+ into protoporphyrin IX (PPIX). Here we report the reconstruction using ...single-particle cryo-electron microscopy of the complex between subunits BchD and BchI of
Rhodobacter capsulatus Mg-chelatase in the presence of ADP, the nonhydrolyzable ATP analog AMPPNP, and ATP at 7.5 Å, 14 Å, and 13 Å resolution, respectively. We show that the two AAA+ modules of the subunits form a unique complex of 3 dimers related by a three-fold axis. The reconstructions demonstrate substantial differences between the conformations of the complex in the presence of ATP and ADP, and suggest that the C-terminal integrin-I domains of the BchD subunits play a central role in transmitting conformational changes of BchI to BchD. Based on these data a model for the function of magnesium chelatase is proposed.
► The first quasi-atomic model of the Mg-chelatase AAA+ unit in the presence of ADP ► Cryo-EM three-dimensional reconstructions of the AAA+ complex in the presence of AMPPNP and ATP ► Conformational dynamics involving IntegrinI domains
CDK8 (cyclin-dependent kinase 8), along with CycC, Med12, and Med13, form a repressive module (the Cdk8 module) that prevents RNA polymerase II (pol II) interactions with Mediator. Here, we report ...that the ability of the Cdk8 module to prevent pol II interactions is independent of the Cdk8-dependent kinase activity. We use electron microscopy and single-particle reconstruction to demonstrate that the Cdk8 module forms a distinct structural entity that binds to the head and middle region of Mediator, thereby sterically blocking interactions with pol II.
Three-dimensional (3D) structure determination from electron microscopic images of single molecules can be difficult for particles with low or no internal symmetry, and for images with low ...signal-to-noise ratio (SNR), due to the existence of false maxima in the scoring function used for orientation search. In attempt to improve robustness of orientation parameter refinement towards noise and poor starting reconstruction quality, we have developed a method for common lines-based orientation search in Fourier space. The Fourier-space formulation enables inclusion of resolution (spatial frequency of the low-pass limit) as a variable that is adjusted in a particle-dependent, self-adaptive manner. The method allows for the underlying 3D structure to be estimated to high resolution, and requires only a crude, low-resolution reconstruction as starting-point for refinement. Benchmarking of the method is performed on experimental and synthetic data.
Thioredoxin and thioredoxin reductase can regulate cell metabolism through redox regulation of disulfide bridges or through removal of H2O2. These two enzymatic functions are combined in ...NADPH-dependent thioredoxin reductase C (NTRC), which contains an N-terminal thioredoxin reductase domain fused with a C-terminal thioredoxin domain. Rice NTRC exists in different oligomeric states, depending on the absence or presence of its NADPH cofactor. It has been suggested that the different oligomeric states may have diverse activity. Thus, the redox status of the chloroplast could influence the oligomeric state of NTRC and thereby its activity. We have characterized the oligomeric states of NTRC from barley (Hordeum vulgare L.). This also includes a structural model of the tetrameric NTRC derived from cryo-electron microscopy and single-particle reconstruction. We conclude that the tetrameric NTRC is a dimeric arrangement of two NTRC homodimers. Unlike that of rice NTRC, the quaternary structure of barley NTRC complexes is unaffected by addition of NADPH. The activity of NTRC was tested with two different enzyme assays. The N-terminal part of NTRC was tested in a thioredoxin reductase assay. A peroxide sensitive Mg-protoporphyrin IX monomethyl ester (MPE) cyclase enzyme system of the chlorophyll biosynthetic pathway was used to test the catalytic ability of both the N- and C-terminal parts of NTRC. The different oligomeric assembly states do not exhibit significantly different activities. Thus, it appears that the activities are independent of the oligomeric state of barley NTRC.
Photosynthetic organisms require chlorophyll and bacteriochlorophyll to harness light energy and to transform water and carbon dioxide into carbohydrates and oxygen. The biosynthesis of these ...pigments is initiated by magnesium chelatase, an enzyme composed of BchI, BchD, and BchH proteins, which catalyzes the insertion of Mg2+ into protoporphyrin IX (Proto) to produce Mg-protoporphyrin IX. BchI and BchD form an ATP-dependent AAA+ complex that transiently interacts with the Proto-binding BchH subunit, at which point Mg2+ is chelated. In this study, controlled proteolysis, electron microscopy of negatively stained specimens, and single-particle three-dimensional reconstruction have been used to probe the structure and substrate-binding mechanism of the BchH subunit to a resolution of 25Å. The apo structure contains three major lobe-shaped domains connected at a single point with additional densities at the tip of two lobes termed the “thumb” and “finger.” With the independent reconstruction of a substrate-bound BchH complex (BchH·Proto), we observed a distinct conformational change in the thumb and finger subdomains. Prolonged proteolysis of native apo-BchH produced a stable C-terminal fragment of 45 kDa, and Proto was shown to protect the full-length polypeptide from degradation. Fitting of a truncated BchH polypeptide reconstruction identified the N- and C-terminal domains. Our results show that the N- and C-terminal domains play crucial roles in the substrate-binding mechanism.
The generation of
ab initio three-dimensional (3D) models is a bottleneck in the studies of large macromolecular assemblies by single-particle cryo-electron microscopy. We describe here a novel ...method, in which established methods for two-dimensional image processing are combined with newly developed programs for joint rotational 3D alignment of a large number of class averages (RAD) and calculation of 3D volumes from aligned projections (VolRec). We demonstrate the power of the method by reconstructing an ∼
660-kDa ATP-fueled AAA+ motor to 7.5 Å resolution, with secondary structure elements identified throughout the structure. We propose the method as a generally applicable automated strategy to obtain 3D reconstructions from unstained single particles imaged in vitreous ice.
The mitochondrial protein frataxin plays a central role in mitochondrial iron homeostasis, and frataxin deficiency is responsible for Friedreich ataxia, a neurodegenerative and cardiac disease that ...affects 1 in 40000 children. Here we present a single-particle reconstruction from cryoelectron microscopic images of iron-loaded 24-subunit oligomeric frataxin particles at 13 and 17 Å resolution. Computer-aided classification of particle images showed heterogeneity in particle size, which was hypothesized to result from gradual accumulation of iron within the core structure. Thus, two reconstructions were created from two classes of particles with iron cores of different sizes. The reconstructions show the iron core of frataxin for the first time. Compared to the previous reconstruction of iron-free particles from negatively stained images, the higher resolution of the present reconstruction allowed a more reliable analysis of the overall three-dimensional structure of the 24-meric assembly. This was done after docking the X-ray structure of the frataxin trimer into the EM reconstruction. The structure revealed a close proximity of the suggested ferroxidation sites of different monomers to the site proposed to serve in iron nucleation and mineralization. The model also assigns a new role to the N-terminal helix of frataxin in controlling the channel at the 4-fold axis of the 24-subunit oligomer. The reconstructions show that, together with some common features, frataxin has several unique features which distinguish it from ferritin. These include the overall organization of the oligomers, the way they are stabilized, and the mechanisms of iron core nucleation.
PDF
