Single particle cryo-EM of mitochondrial supercomplexes

Thorsten Althoff, Max-Planck-Institut für Biophysik, Frankfurt, Germany

Co-Authors: Deryck Mills, Jean-Luc Popot, Werner Kühlbrandt

Abstract: The mitochondrial respiratory chain is the key player in cellular energy production. The major steps in this process are the establishment of a proton gradient across the mitochondrial membrane and its utilization for the synthesis of ATP. For a long time there was a controversy whether the different proteins involved in this process exist as individual complexes diffusing freely in the inner membrane of the mitochondria or whether they form larger arrays, so called supercomplexes. In the latter case the close association of the different complexes would facilitate electron transfer along the respiratory chain and therefore enhance and speed up energy production. In the last decade there was growing evidence that there indeed exist various populations of supercomplexes. After the first identification and isolation of supercomplexes by BN-PAGE and biochemical characterization, research in this field culminated so far in the 3D structure determination of supercomplex I₁III₂IV₁ by negative stain single particle analysis. But as the resolution of this structure was not yet sufficient for automated fitting of high resolution X-ray structures into the EM volume, the exact interaction sites between the complexes could not be mapped. To achieve this, a higher resolution structure needs to be obtained by cryo-EM.

A Maximum-Likelihood Approach to Random Spherically Constrained cryo-EM Reconstruction

Andrew Barthel, Biomedical Engineering, Yale University

Co-Authors: Liguo Wang, Hemant Tagare, Fred Sigworth

Abstract: Random spherically constrained cryo-electron microscopy (EM) is a new form of single particle reconstruction (SPR) using EM images of membrane proteins embedded in lipid vesicles. The method has many advantages over the more conventional SPR:

-proteins are in a more native environment and formation; conditions across the vesicle membrane can be manipulated for more precise targeting of open, closed, and intermediate states.

-extra orientational information is gathered since we can precisely determine the center and radius of the vesicles and therefore two of the rotational degrees of freedom. These advances allow us to determine structures of smaller proteins or acquire structure estimates with less data. We can also accelerate our algorithm as we have a smaller search space due to fewer unknown degrees of freedom. We propose a maximum-likelihood framework to estimate the structure of the embedded proteins that relies on the algebraic reconstruction technique developed by Kaczmarz.

Structural Analysis of tubular crystals of a retroviral Gag protein

Tanmay Bharat, EMBL Heidelberg

Co-Authors: James Riches, Pavel Ulbrich, Thomas Ruml and John A. G. Briggs

Abstract: Mason-Pfizer Monkey virus (MPMV) assembles its capsid protein inside the host cytoplasm. Minimum assembly competent domains of the Gag protein may be induced to form spheres or tubes (Ulbrich et al, 2006). We have studied the three-dimensional structure of the MPMV Gag tubes using two distinct methodologies - cryo-electron tomography (cryoET) with subtomogram averaging and conventional cryo-electron microscopy (cryoEM) with real space helical reconstruction. CryoET allowed the determination of the helical handedness and the range of symmetries adopted by the tubes. It also provided a first estimate as to the level and characteristics of the heterogeneity present in the sample. These things allowed us to assess the feasibility of a conventional cryoEM approach, and allowed us to apply it in an attempt to improve the resolution.

Studying transient intermediate states of transcription initiation using single particle cryo-EM

Daniel Bose, Zhang Lab, Imperial College London

Co-Authors: Tim Grant, Tillmann Pape, Martin Buck, Xiaodong Zhang

Abstract: Transcription initiation is a multi-stage process encompassing binding and melting of promoter DNA by the RNA polymerase (RNAP) holoenzyme, and loading of the template strand into the active site. Ususally this occurs spontaneously and is driven by complex re-organisations of the holoenzyme, proceeding through at least two transient intermediate states. The short-lived nature of the intermediates on the initiation pathway makes their study by structural techniques challenging. In contrast, holoenzymes formed with an alternative class of sigma factor, sigma 54 (s54), do not spontaneously isomerise and must be re-modelled by s54-activator proteins, using the energy released from ATP hydrolysis. The unique properties of s54 holoenzymes enable us to capture intermediates on the initiation pathway using cryo-EM, revealing the conformational changes within RNAP that drive transcription initiation.

Single Particle Electron Microscopy Analysis of Mediator Structure and Functional Interactions

Gang Cai, Department of Cell Biology, The Scripps Research Institute

Co-Authors: Tsuyoshi Imasaki, Kentaro Yamada, Francesco Cardelli, Yuichiro Takagi, and Francisco J. Asturias

Abstract: Single particle electron microscopy (EM) analysis demonstrated that both Mediator and Mediator Head module have remarkable conformational malleability. Detailed analysis of the cryo-EM Mediator structure revealed the specific conformational changes required for interaction with RNA polymerase II (Pol II), which mirror conformational changes observed in human Mediator upon interaction with nuclear receptors, suggesting that Mediator conformational flexibility is important for its functional interactions and highly conserved from yeast to human system. The specific functional interactions of Head module with the Pol II Rpb4/Rpb7 subunit complex and TATA-binding protein (TBP) were characterized by EM, which argued that Mediator conformational variability especially Head module flexibility probably has important implications for initiation regulation of transcription.

Imaging large macromolecular assemblies involved in bacterial transcription activation

Sacha De Carlo, City University of New York

Co-Authors: Prof B. Tracy Nixon

Abstract: The overall goal of this project is to gain structural insight into the mechanism of conversion of ATP hydrolysis into mechanical work, with emphasis on the study of the regulated assembly and function of macromolecular motors involved in bacterial transcription activation. To this end, we plan to employ novel cryo-electron microscopy (cryo-EM) specimen preparation and image processing approaches, which in combination with molecular docking techniques will allow us to visualize and interpret the structural changes underlying the function of these large macromolecular motors. We aim at elucidating the molecular mechanism of promoter DNA melting that occurs only in the presence of the active ATPase, its target protein sigma-54, the bacterial RNA polymerase, and promoter DNA.

Structural analysis of TFIID bound to TFIIA and promoter DNA provides regulatory insight into transcription initiation

Michael A. Cianfrocco, Biophysics Graduate Group, University of California Berkeley, Berkeley, CA

Co-authors: Patricia Grob, Eva Nogales

Abstract: The multisubunit transcription factor complex TFIID is essential for the regulation of eukaryotic transcription. Despite its central role in transcription initiation, structural evidence for TFIID’s promoter recognition and regulation by co-activators remains severely lacking. To provide the insight into this critical process, single-particle image analysis has been performed on negatively stained samples of TFIID in complex with the co-activator TFIIA and super core promoter DNA. Advanced statistical analysis revealed two conformational states representative of TFIID-IIA-DNA and holo-TFIID complexes. These data suggest a model of transcription activation where TFIIA shifts TFIID’s conformational landscape towards a “DNA-bound” state by directing long-range rearrangements of TFIID’s arms.

Purification and structural characterization of the human chromatin remodeler PBAF

Claudio Ciferri & Gabriel C Lander, UC Berkeley

Co-authors: Eva Nogales

Abstract: Nucleosome remodeling, a necessary step in gene expression and repression, is performed by an array of chromatin remodelers.   In humans, the PBAF complex represents one of the subfamilies of these complexes, although the mechanism by which it binds the nucleosome and achieves chromatin remodeling is not yet well understood.  Here, we present a one-step protocol for the purification of the PBAF complex from endogenous sources, producing suitable amounts and quality of sample for cryo-EM investigations, as well as preliminary structural analyses of the PBAF complex in its frozen-hydrated state.

Single Particle Electron Microscopy Structure of Human Cop9 Signalosome (CSN)

Radoslav Enchev, Institute of Cancer Research, London, UK

Co-Authors: Anne Schreiber, Fabienne Beuron, Ed Morris

Abstract: The COP9 Signalosome (CSN) is an eight-subunit protein complex which deneddylates cullin-ring E3 ligases (e.g. SCF). It is known to interact with kinases and a deubiquitinating enzyme, thus functioning at the interface between cell signalling and protein degradation. Its subunits exhibit a one-to-one sequence homology to the subunits of the 19S proteasome lid and the isopeptidase activity is conserved among the two. In principle electron microscopy (EM) and single particle analysis can provide structures at near atomic resolution. A major difficulty, however, is the analysis of asymmetrical biological macromolecules in the absence of prior structural information. The work presented here is two-fold. Theoretical advances in angular reconstitution methods for single particle EM are illustrated with a cryo-EM dataset of a 70S ribosome. The newly developed methods have been applied to both native and recombinant human CSN yielding its first 3D structure at 25 Å resolution. It exhibits the long anticipated similarity to the 26S proteasome lid (26S lid). Furthermore, the recombinant complex is shown to be biochemically and structurally identical to the native CSN, thus opening possibilities for subunit deletions and mapping, study of higher order complexes and quantitative biochemical analysis.

Electron Crystallographic Studies of the Bacterial Transporter YdhE

MaryBeth Evans, The Scripps Research Institute La Jolla, California

Co-Authors: Andrew Ward, Christopher Arthur, & Ron Milligan

Abstract: Multidrug transporters play an essential role in drug resistance in prokaryotes and humans. One family of bacterial multidrug transporters, classified as multidrug and toxic compound extrusion (MATE), include the proteins NorM and YdhE. Studies have shown NorM and YdhE to cause resistance to drugs such as Ciprofloxacin which is used to treat gonorrhea. Little is known about these transporters and to date no structural data exists. The objective of my work is to elucidate the structure of YdhE in context of the membrane. We have successfully grown 2D crystals of lipid reconstituted YdhE. Based on data collected we have generated 2D projection maps of YdhE. The crystals were processed by traditional electron crystallographic techniques and electron tomography including 3D volume averaging. Future studies will examine the relationship between substrates known to bind to YdhE and protein structure/conformation.

EM structure of the full length LRRK2: a key factor in Parkinson Disease

Renzi Fabiana, Dept Chemical and Structural Biology, Mount Sinai School of Medicine, MSSM and New York Structural Biology Center, NYSBC, New York, US

Co-Authors: Xianting L, Rice W, Zhenyu Y and Ubarretxena I

Abstract: Leucine-Reach-Repeat Kinase 2 (LRRK2) is the most recurrent genetic factor in Parkinson disease. Interestingly LRRK2 hosts in the same polypeptide an effector kinase and a regulative GTPase. Initial biochemical analyses suggest that LRRK2 can form dimers, probably mediated by the GTPase including a less characterized COR domain, and that dimerization affects the kinase activity. Atomic structures of human GTPase and of a prokaryote GTPase~COR domains form dimers, but the different interfaces suggest crystal packing artifacts. We present the first EM reconstruction of the full-length mouse LRRK2 purified from mice brain. We show that the full-length forms mostly dimers and by docking the GTPase structures our reconstruction supports only one dimerization model.

Analysis of Archaeal Protein Sorting and Translocation by Cryo-EM

Sibylle Franckenberg, Gene Center, LMU Munich

Co-author: Roland Beckmann

Abstract: Structural analysis of the processes that occur during translation of secretory and membrane proteins in thermophilic archaea: Protein sorting and translocation intermediates are reconstituted in vitro. Cryo-electron microscopy and single particle analysis methods are then used for studying the organization of translating ribosomes and their interaction with proteins that are involved in further steps of sorting and translocation

TDNA ejection revealed by cryo-electron Tomography of Bacillus anthracis Spore-Binding Phage 8a

Xiaofeng Fu, University of Texas, Houston

Co-Authors: Michael H Walter, Angel Paredes, Marc C. Morais and Jun Liu

Abstract: The three-dimensional structure of the recently discovered Bacillus anthracis spore-binding phage 8a (SBP8a) has been determined by cryo-electron tomography (cryo-ET). The resulting reconstruction shows the following mechanism for DNA ejection: 1) recognition of the host cell receptor causes a reorganization of the protein subunits in the baseplate from a closed, “fist”-like structure to an open, “palm”-like structure; 2) this rearrangement triggers tail sheath contraction, which drives the tail tube through the cell envelope and initiates injection of the phage genome into the host cell; 3) contraction of tail sheath also triggers a “gate” in the head-tail connector protein to “open”, allowing DNA to exit the head and travel through the tail tube en route to the host cell cytoplasm.

Falcon, a New 4k x 4k Direct Electron Detector for Transmission Electron Microscopy

Richard Gursky, FEI Company

Co-Authors: G. van Duinen, M. Storms, G. van Hoften, R. Gursky

Abstract : For many years, it has been a desire of Cryo TEM investigators to improve the quality of data acquisition from low dose imaging. CCD technology has been improved to it's practical limits and so another technology needed to be developed which was capable of better signal to noise ratios and better electron conversion without sacrificing resolution. The CMOS device had the right potential blend of characteristics for this challenge but it was not without it's own issues. This poster explores some current developments and preliminary data derived from the first Alpha unite of FEI's current CMOS Direct Electron Detector development.

A combined biochemical and computational approach for model-free determination of EM structures from heterogenous datasets

Florian Hauer, Molecular 3D Electron Cryomicroscopy, Max-Planck-Institut für biophysikalische Chemie, Goettingen, Germany

Co-Authors: Björn Sander, Christoph Gerle, Holger Stark

Abstract: Here, we present a model-free approach for the structural determination of biochemically sensitive and structurally heterogenous macromolecules. In this approach, biochemical and computational methods are combined to routinely solve EM structures of challenging complexes. First, samples are stabilized and purified using the GraFix protocol. A large number of tilt pairs for Random Conical Tilt analysis (RCT) are automatically correlated to receive a large number (>350) of RCT 3D reconstructions. These 3D RCTs are subjected to an iterative 3D alignment and weighted averaging procedure. After alignment, three-dimensional class averages can be calculated making use of multivariate statistical analysis and classification. The average 3D structures exhibit an improved signal-to-noise ratio, do not suffer from the missing cone and represent the 3D structural variations caused by compositional and conformational heterogeneity in the dataset. Subsequently, these 3D averages can be used for refinement of the complete dataset. Thus, this approach can be universally applied to any biomacromolecule suitable for EM analysis to resolve the structural heterogeneity without prior structural information or initial reference.

Cryo-electron tomography and volume averaging reveal new insights into the 3D structure of axonemes

Thomas Heuser, Biology Department, Brandeis University

Co-Authors: Milen Raytchev, Jeremy Krell, Mary E. Porter & Daniela Nicastro

Abstract: Cilia and flagella are highly conserved microtubule-based organelles with motile or sensory function. The core structure of motile cilia and flagella, the axoneme, has a highly conserved [9+2] organization. The dynein regulatory complex (DRC) has been identified as a key regulator for the coordination of thousands of dynein motors that drive axonemal movement, but structural details are lacking. We used cryo-electron tomography of intact and rapidly frozen Chlamydomonas axonemes to gain new insights into the 3D structure and molecular organization of the DRC in situ. Axonemes are highly repetitive so that we can use the PEET software (Particle Estimation for Electron Tomography) for 3D correlation averaging to increase the image resolution. By comparing tomograms of wild type and drc mutant flagella we localized key components of the DRC in unprecedented detail.

Advanced Scoring and Stochastic Sampling Methods for Sub Tomogram Averaging

Thomas Hrabe, Max Planck Institute of Biochemistry / Department Molecular Structural Biology

Co-Authors: Friedrich Förster

Abstract: Key to high resolution in cryoelectron tomography is the accurate alignment of sub-tomograms to a common coordinate system. Sub-tomogram alignment represents an optimization task: the optimal alignment of sub-tomograms maximizes (or minimizes) a score for their respective similarity. In this project, we explore different scoring and sampling schemes for sub-tomogram alignment. For example, common methods utilize cross correlation as a similarity measure and the ‘expectation maximization’ algorithm for sampling. In addition to these methods, we apply locally normalized correlation and weighted correlation as scoring functions. For sampling, we develop different stochastic sampling protocols (e.g., simulated annealing) to determine sub-tomogram alignment. Alignment quality is assessed using simulated data as well as experimental tomograms by measuring the accuracy of the determined alignment parameters and the resolution of the resulting reconstructions. 

Synthesis and Characterization of Discrete, Monodispersed Thiolated-Protected Gold Clusters

Yael Levi-Kalisman, Dept. of Structural Biology, Stanford University School of Medicine

Co-Authors: Pablo D. Jadzinsky, David A. Bushnell and Roger D. Kornberg

Abstract: Recently the synthesis of stable, water soluble monodispersed thiolated-protected gold clusters has been developed in our lab to be used as markers for 3D cryo-TEM reconstruction of protein molecules. In this study we focus on the synthesis and characterization of p-Mercaptobenzoic acid (pMBA)-protected gold clusters having core diameters of 1.5-2.3 nm by various techniques commonly used to study gold clusters. My study focuses primarily on the clusters which upon crystallization form the well-defined Au₁₀₂pMBA₄₄ crystals. Mass Spectrometry, Uv-Vis spectroscopy, Thermogravimetric analysis (TGA) and electron microscopy are used to characterize the gold nanoparticles. Reactivity towards biomaterials has been tested implying that these clusters can be conjugated with thiolated-DNA oligomers as well as with proteins having surface exposed cysteine residues and therefore may facilitate cryo-TEM 3D structure determination.

Analysis of molecular complexes in neuronal synapses by cryo-electron tomography

Zdravko Kochovski, Max Planck Institute of Biochemistry, Martinsried, Germany

Co-Authors: Vladan Lucic, Wolfgang Baumeister

Abstract: Cryo-electron tomography (cryo-ET) allows the investigation of frozen-hydrated samples that are free of artifacts caused by dehydration, chemical fixation and uneven stain accumulation and therefore allows studying the three dimensional structural features of biological material preserved in a near-to-physiological state. My work is focused on cryo-ET of synapses from the synaptosomal cellular fraction obtained from rat neocortex. This fraction contains synapses derived from pinched-off nerve terminals held together by synaptic cell-adhesion molecules and has often been used for structural and biochemical investigations. The synapses in my tomograms show the characteristic ultrastructural features: synaptic vesicles on the presynaptic side, dense thickening termed the postsynaptic density (PSD) apposed to the cellular membrane on the postsynaptic side, and the well-defined synaptic cleft between the terminals. In addition, many molecular complexes are visible, such as presynaptic membrane bound filaments, synaptic adhesion complexes and the intricately connected components of the PSD. Due to the nature of cryo-ET, the tomograms are quite noisy and their usable resolution is limited. In order to increase the usable resolution and analyze the structures present in the tomograms, I will work on the improvement of existing image processing methods such as denoising, segmentation and the morphological analysis of molecular complexes that are present in the tomograms.

YidC and Oxa1 Form Dimeric Insertion Pores on the Translating Ribosome

Rebecca Kohler, Institute for Molecular Biology and Biophysics, ETH Zurich,

Co-Authors: Daniel Boehringer, Basil Greber, Rouven Bingel-Erlenmeyer, Ian Collinson, Christiane Schaffitzel, Nenad Ban

Abstract: The YidC/Oxa1/Alb3 family of membrane proteins facilitates the insertion and assembly of membrane proteins in bacteria, mitochondria and chloroplasts. We present the structures of both Escherichia coli YidC and Saccharomyces cerevisiae Oxa1 bound to E. coli ribosome nascent chain complexes determined by cryo-electron microscopy. Crosslinking experiments show that the ribosome specifically stabilizes the dimeric state of the insertases. Both Oxa1 and YidC dimers contact the ribosome similarly to what was observed for the non-homologous SecYEG translocon. We suggest that dimers of the YidC and Oxa1 proteins form insertion pores and share a common overall architecture with the SecY monomer.

Structural and functional characterization of the human vasopressin V2 receptor

Krueger, A., University of Basel

Co-Authors: Engel, A.

Abstract: The vasopressin V2 receptor (V2R) belongs to the super family of G protein coupled receptors and has a seven transmembrane topology. Structural studies of V2R have been prevented, among others, by the limited availability in conventional overproduction systems. Structure and molecular mechanisms of V2R are still obscure. For this reason the overproduction of V2R is carried out either in mammalian cells, using the Semliki Forest Virus expression system or with a cell-free expression system to obtain samples suitable for single particle analysis, 2D crystallization and functional studies. Quality of cell free or cell based produced proteins is evaluated by electron microscopy. Once proteins are available in suitable amounts the next step is their characterization using different biophysical tools like cryo electron microscopy for single particle analysis and atomic force microscopy for single molecule force spectroscopy. The function should be investigated e.g. by ligand binding. Furthermore 2D crystallization and solving the structure of V2R at high resolution is a goal of our work.

Full-Atom Model of an Aquareovirus by CryoEM Using a Titan Krios Microscope

Lei Jin, Department of Microbiology, Immunology & Molecular Genetics and the California NanoSystems Institute, University of California at Los Angeles

Co-Authors: Xing Zhang, Wong H. Hui, Qin Fang, Z. Hong Zhou

Abstract:  We report the full-atom model of an aquareovirus determined by cryo-electron microscopy (cryoEM) and single-particle analysis of high-resolution images obtained from a recently installed Titan Krios microscope from the FEI Company. In the density map, most side-chain densities are clearly resolved. Based on the observed structural features, the effective resolution is estimated to be 3.3-Å for the inner shell proteins and 3.5-Å for outer shell proteins. This resolution difference is likely due to the limitations of the Ewald sphere curvature at different radii of the large virus particle. To our best knowledge, this is the first time that a full-atom model is derived from single-particle cryoEM and represents a major step forward in fulfilling the great promise of cryoEM.

Structure of Human Dicer by Single Particle Electron Microscopy

Pick-Wei Lau, NRAMM, The Scripps Research Institute

Co-Authors: Clinton S. Potter, Bridget Carragher, Ian J. MacRae

Abstract: Dicer is a specialized ribonuclease that initiates RNA interference (RNAi) by cleaving double-stranded RNA into RNA fragments about 22 nucleotides in length. Dicer also serves as a scaffold for the initiation of RNAi by bringing together multiple factors required for the correct processing of small RNAs. Here, we present the 3-D structure of human Dicer bound to the protein TRBP at ~20 angstrom resolution determined by electron microscopy (EM). A robust and reliable 3-D model of the complex was generated using a combination of random conical tilt and projection matching refinement methods. Our analysis reveals that the Dicer-TRBP complex is an L-shaped molecule. These results offer insights into the structure of Dicer proteins found in multi-cellular organisms and provide a conceptual framework for understanding the initiation of RNAi.

3D EM Structure of an intact activator-dependent transcription initiation complex

Cathy Lawson, Rutgers University

Co-authors: Brian Hudson, Samuel Lara-González, Helen Berman, Eddy Arnold, Richard Ebright, Joel Quispe

Abstract: We present the experimentally determined three-dimensional structure of an intact activator dependent transcription initiation complex comprising the Escherichia coli catabolite activator protein (CAP), RNA polymerase holoenzyme (RNAP), and a DNA fragment containing positions -78 to +20 of a Class I CAP-dependent promoter with a CAP site at position -61.5 and a pre-melted transcription bubble. A 20 Å electron microscopy reconstruction was obtained by iterative projection-based matching of single particles visualized in carbon-sandwich negative stain, and was fitted using atomic coordinate sets for CAP, RNAP, and DNA. The structure defines the organization of a Class I CAP-RNAP-promoter complex and supports previously proposed interactions of CAP with RNAP  subunit C-terminal domain (CTD), interactions of CTD with ⁷⁰ region 4, interactions of CAP and RNAP with promoter DNA, and phased DNA bend dependent partial wrapping of DNA around the complex. The structure also reveals the positions and shapes of species-specific domains within the RNAP ', , and ⁷⁰ subunits. (EMDB entry EMD-5127; PDBid 3iyd).

EMDataBank.org: Unified Resource for CryoEM

Cathy Lawson, Rutgers University

Co-authors: Batsal Devkota, Raul Sala, Helen Berman, Christoph Best, Glen van Ginkel, Gerard Kleywegt, Matt Baker, Steve Ludtke, Wah Chiu, Powei Feng, Joe Warren

Abstract: EMDataBank.org is a joint effort of the Protein Databank in Europe (PDBe), the Research Collaboratory for Structural Bioinformatics Protein Data Bank (RCSB-PDB), and the National Center for Macromolecular Imaging (NCMI) to create a global deposition and retrieval network for cryoEM map, model, and associated metadata, and to provide a portal for software tools for standardized map format conversion, map, segmentation and model assessment, visualization, and data integration. The EMDataBank (EMDB) holds over 700 map entries, and PDB holds ~280 EM entries holding models fitted into EM maps. Recent improvements include joint map and coordinate model deposition, simple java map viewers, and remediation of EMDB and PDB metadata to improve uniformity. Please visit http://www.emdatabank.org to deposit or search for a map, browse EM software, look at our FAQ. Questions? Write to us at help@emdatabank.org.

Making use of a powerful global optimization method for extracting maximal information from experimental data such as NMR and EM

Jinwoo Lee, Department of Mathematics, Kwangwoon University, Seoul, Korea

Co-Authors: Jinhyuk Lee, Jooyoung Lee

Abstract : A powerful global optimization method called conformational space annealing (CSA) is recently implemented into CHARMM package. In its application to NMR structure determination, CSA could generate better structures than those deposited in the PDB in terms of various criteria such as crash score, NOE violation, and Ramachandran score. I will discuss potential applicability of CSA to EM structure determination in order to extract maximal information from EM data. Additionally, a new innovative de-noising method called Non-local de-noising and its preliminary testing result on EM images will be presented.

Binding of a neutralizing antibody to dengue virus resulted in an altered surface glycoproteins arrangement

Shee Mei Lok, Department of Biological Sciences, Purdue University

Co-Authors: Victor Kostyuchenko, Grant E. Nybakken, Heather A. Holdaway, Anthony J. Battisti, Soila Sukupolvi-Petty, Dagmar Sedlak, Daved H. Fremont, Paul R. Chipman, John T. Roehrig, Michael S. Diamond, Richard J. Kuhn, and Michael G. Rossmann

Abstract: The monoclonal antibody 1A1D 2 has been shown to strongly neutralize dengue virus serotypes 1, 2, and 3 primarily by inhibiting attachment to host cells. A crystal structure of its antigen-binding fragment (Fab) complexed with domain III of the viral envelope glycoprotein, E, showed that the epitope would be partially occluded in the known structure of the mature dengue virus. Nevertheless, antibody could bind to the virus at 37^oC, suggesting that the virus is in dynamic motion making hidden epitopes briefly available. A cryo-electron microscope image reconstruction of the virus-Fab complex showed large changes in the organization of the E protein that exposed the epitopes on two of the three E molecules in each of the sixty icosahedral asymmetric units of the virus. The changes in the structure of the viral surface are presumably responsible for inhibiting attachment to cells.

Automated Angular Reconstitution - A Method for Reproducible ab initio Reconstructions of 3-D Models Based on a Common Lines Approach

Dmitry Lyumkis, National Resource for Automated Molecular Microscopy and Department of Cell Biology, The Scripps Research Institute

Co-Authors: Ronald Milligan, Clinton S. Potter, and Bridget Carragher

Abstract: Angular reconstitution is a methodology used to assign Euler angles to 2-D projections in order to obtain a 3-D model without the requirement of physical specimen tilting. Its general reliance on well-aligned class averages and manual user input leads to the established difficulty of accurately reconstructing an initial volume, particularly one without symmetry. To address this problem a highly automated and iterative approach has been developed, the input to which consists only of a set of class averages and a raw particle stack. The overall approach involves (1) the generation of a similarity matrix between initial class averages, (2) the construction of N different models (specified by the user) through angular reconstitution, (3) maximum-likelihood alignment of the N resulting models, (4) a novel method of model clustering with affinity propagation, and (5) an iterative batch refinement of the best 3-D average using a raw-particle stack. The method is demonstrated with several real datasets. 

Automated tomography and cryo-EM using a Direct Detection Device (DDD)

Anna-Clare Milazzo, UCSD

Co-Authors: Jason Lanman, Tomas Molina, Masako Terada, James Bouwer, Steve Peltier, Mark Ellisman, Nguyen-Huu Xuong

Abstract : New imaging systems are crucial in improving transmission electron microscopy (TEM) for the investigation of the structure and function of biological systems from the molecular to the cellular scale. The DDD (Direct Detection Device), a prototype camera developed at the National Center for Microscopy and Imaging Research (NCMIR), is a monolithic active pixel sensor that directly records the energy deposited by an incident electron. Due to the small imaging area of the 1k x 1k, 5 micron pixel pitch DDD prototype (5mm x 5mm) and 1.45 post magnification, manual image acquisition has been challenging. Integration with automated microscopy package allows for acquisition of higher resolution tilt series and increased throughput for cryo-EM data collection. The integration of the DDD with SerialEM on an FEI 120keV Tecnai Spirit TEM has resulted in higher quality data collection. We show the results from a double tilt tomogram of an epoxy embedded specimen of a mitochondria infected with Flock House virus at a magnification of 0.6nm/pixel. Due to the small size of the sensor, the integration with SerialEM also allows for the precise acquisition of large field of view montage images with image shift. SerialEM Navigator and low dose capabilities have allowed for preliminary experiments with single particle cryo-EM data collection with this prototype.

Molluscan Hemocyanins – from structure to function

Arne Moeller, Johannes Gutenberg University and NRAMM

Co-authors: Christos Gatogiannis, Frank Depoix &Juergen Markl

Abstract: Hemocyanins are the blue copper-containing respiratory proteins of many molluscs. Nautiluspompiliushemocyanin (NpH) is a cylindrical decamer composed of ten copies of a 350 kDapolypeptide subunit, in turn consisting of seven O2-binding functional units. Hemocyanins are highly allosteric, and the cooperativity ofoxygen binding should be transferred between functional units by conformational changes. Inthis context, we try to monitor structural changes caused by the reversible oxygen binding process. Our approach is to prepare the specimens in their fully oxygenated and deoxygenated state, respectively, and to perform independent 3D reconstructions of both states. To achieve this, we developed an automated plunge-freeze apparatus capable of flash freezing the specimen in liquid ethane under controlled atmospheric conditions. Prior to fixation, the molecules are either incubated in an atmosphere with 25 % O2 + 75% N2 (oxy-state), or in an atmosphere with 100 % N2 (deoxy-state). This led to two independent NpH datasets resulting in two 3D reconstructions with resolutions of ~8 Å. Their correlative analysis shows significant structural differences especially concerning the spatial orientation of certain FU types. On the basis of the two resulting molecular models it is now the task to interpret these differences in terms of allosteric interaction mechanisms.

Expansion of Bacteriophage phi6 Procapsid Revealed by cryoEM 

Daniel Nemecek, National Institute of Arthritis and Musculoskeletal and Skin Diseases, National Institutes of Health, Bethesda, MD 2

Co-Authors: J. Bernard Heymann, Naiqian Cheng, Jian Qiao, Leonard Mindich, and Alasdair C. Steven

Abstract: In maturation, the dsRNA bacteriophage phi6 undergoes dramatic conformational changes from the initial procapsid with its deeply recessed vertices to the near-spherical shell of the mature fully packaged capsid. We are studying these particles with particular focus on the hypothesis that conformational switches of the maturing procapsid control the sequential packaging of the three RNA segments. To shed light on the mechanics of the procapsid expansion, we have imaged unexpanded RNA-free procapsids; partially expanded RNA-free intermediates produced by in vitro manipulation of procapsids; and RNA-filled mature capsids by cryo-electron microscopy.  In addition, cryo-electron tomography revealed procapsids with non-uniformly expanded vertices.

Graphene Oxide: a substrate for imaging biological materials

Radosav S. Pantelic, Max Planck Institute of Biochemistry, Department of Molecular Structural Biology, Martinsried, Germany

Co-authors: Jannik C. Meyer, Ute Kaiser, Wolfgang Baumeister, Jürgen M. Plitzko

Abstract: Graphene oxide is a hydrophilic derivative of graphene to which biological macromolecules readily attach, with properties superior to those of the amorphous carbon films commonly used in electron microscopy. The single layered crystalline lattice of carbon is highly electron transparent, and exhibits a remarkable mechanical and elastic strength. Hence, graphene oxide is a particularly promising substrate for the examination of biological materials by electron microscopy. In this communication we describe the use of graphene oxide substrates in imaging unstained and vitrified biological macromolecules.

Remodelling the small ribosomal subunit: Structural studies of canonical translation initiation in eukaryotes

Lori A Passmore, MRC Laboratory of Molecular Biology, Cambridge UK

Co-Authors: T Martin Schmeing, Jon R Lorsch, V Ramakrishnan

Abstract: During initiation of translation, initiator tRNA and the start codon of mRNA are positioned in the ribosomal P site to form a functional 80S ribosome.  In eukaryotes, this process requires at least 12 eukaryotic initiation factors (eIFs), composed of 28 different polypeptides.  However, the functions of many eIFs and the molecular mechanisms of translation initiation remain unclear.  We are using cryo-EM and biochemical studies to understand the roles of some of the initiation factors. We have shown that eIF1 and eIF1A stabilise a conformational change that opens the mRNA binding channel.  Other factors appear to bind more flexibly and this may have functional implications.

Electron Microscopy of XRCC4 and the DNA Ligase IV-XRCC4 DNA Repair Complex

Maria Angeles Recuero-Checa and Ernesto Arias-Palomo, Biological Research Center. Madrid, Spain

Co-Authors: Doré AS, Rivera-Calzada A, Scheres SHW, Maman JD, Pearl LH, Lloca O

Abstract: DSBs are the most toxic form of DNA damage, and are repaired by an NHEJ process where no sister chromatids are available during the cell cycle. There are several proteins that form macromolecular complexes on DNA implicated in the process. The DNA Ligase IV – Xrcc4 complex has a molecular weight of 180 kDa and it's responsible for ligating broken DNA ends in this pathway. Mutations in DNA ligase IV (Lig4) lead to immunodeficiency and radiosensitivity in humans. We have purified full-length Xrcc4, and the Lig4 - Xrcc4 complex, and are trying to determine their 3-dimensional structures by single-particle electron microscopy. Partial structural information for Lig4 and Xrcc4 are available, however the arrangement of the proteins within the full complex is still unknown.

Tomography: A Tool for Exploring C. elegans Anatomy

William J. Rice, New York Structural Biology Center

Co-Authors: David H. Hall, Kristin A. Politi, KD Derr, Kevin Fisher, Chris Crocker, Leslie Gunther, Ashleigh Bouchelion and Ken C.Q. Nguyen

Abstract: The wiring diagram of C. elegans has been studied intensively using serial section reconstruction for several decades, so that every neuron is now known in considerable detail, including virtually all chemical synapses and many gap junctions. Nevertheless, limitations of the serial section technique (poor resolution of detail within the depth of each 50 nm section) have made it difficult to model the fine details in some neurons, including finer branches of sensory neurons (FLP, PVD), many organelles, and the exact constituents at a chemical synapse. Over the past year, we have been exploring the potential of electron tomography across a wide variety of tissues and organelles. Here we show examples of the type of details that can now be seen better, both in neurons, muscles, and other tissues.

Determining the structures of COPII coated lipid tubes generated in vitro

Chris Russell, Florida State University

Co-Author: Scott Stagg

Abstract:  COPII vesicles play a critical role in eukaryotic cells by transporting proteins and lipids from the endoplasmic reticulum to the Golgi apparatus.  COPII cages consist of Sar1 (a regulatory GTPase), Sec23/24 (Sar1's GAP and cargo recognition activities), and Sec13/31 (promotes coat assembly).  Active Sar1 in the GTP-bound conformation will form membrane tubules.  We have also demonstrated that when Sec23/24 is added to preformed Sar1 tubes, they expand from ~70Å in diameter to almost 1000Å in diameter which matches the diameter of COPII cages.  Determining the structure of these membrane tubules provides valuable insights into the mechanisms by which membrane curvature and cargo recruitment are achieved in typical COPII vesicles.

The random-model method enables ab initio three-dimensional reconstruction of asymmetric particles and determination of particle symmetry

Eduardo Sanz-García, Department of Chemistry and Biochemistry, Brigham Young University

Co-authors: Aaron B. Stewart, David M. Belnap

Abstract: Model-based three-dimensional reconstruction techniques are powerful technique for structural determination of macromolecular complexes. However, they depend on starting models. We present an asymmetric extension to the random-model (RM) method that allows the ab initio generation of starting models directly from the imaged particles. The procedure is suitable for asymmetric particles as well as for other point-group symmetries. The RM method can be used for symmetry determination when no other type of structural or biochemical data is available. Currently, the asymmetric RM method fails to reconstruct images of smooth, spherical structures.  

Two-Dimensional, One Degree-of-Freedom Analysis of Human Dicer Protein EM Images

Hemant D. Tagare, Yale University

Co-Authors: Hong-Wei Wang, Frederick Sigworth

Abstract: We present a technique for finding one-degree-of-freedom flexible structures in Cryo-EM images. A preliminary two-dimensional algorithm is presented and used to analyze Human Dicer Protein EM images. The results suggest possible non-rigid flexibility in the Human Dicer protein.

Structural studies of Sulfolobus Islandicus Rod-shaped Virus

Rebecca E. Taurog, Department of Molecular Biology, The Scripps Research Institute

Co-Authors: David Prangishivili , Jennifer Fulton, Mark Young , Edward H. Egelman , John E. Johnson

Abstract: The Sulfolobus Islandicus rod-shaped virus (SIRV) infects thermophilic, acidophilic archaea that thrive at 80ºC and pH ~2. The viral rod is composed of a single coat protein that is binds and compacts the linear dsDNA genome. We have undertaken structural studies of SIRV to understand how the coat protein assembles to form a helical virion that can withstand such a harsh environment.  Cryoelectron microscopy of the virion reveals it to be a highly irregular helix, with substantial variability in the helical twist. Preliminary reconstructions have been calculated based on virus segments that show a more consistent twist and pitch, using iterative helical real space reconstruction algorithms.

Structural Insights into RNA Processing by the Human RISC-Loading Complex

David Taylor, Department of Molecular Biophysics and Biochemistry, Yale University

Co-authors: Hong-Wei Wang, Cameron Noland, Bunpote Siridechadilok, Enbo Ma, Karin Felderer, Jennfier A. Doudna, and Eva Nogales

Abstract: Targeted gene silencing by RNA interference (RNAi) requires loading of a short guide RNA (small interfering RNA (siRNA) or microRNA (miRNA)) onto an Argonaute protein to form the functional center of an RNA-induced silencing complex (RISC). In humans, Argonaute2 (AGO2) assembles with the guide RNA–generating enzyme Dicer and the RNA-binding protein TRBP to form a RISC-loading complex (RLC), which is necessary for efficient transfer of nascent siRNAs and miRNAs from Dicer to AGO2. Here, using single-particle EM analysis, we show that human Dicer has an L-shaped structure. The RLC Dicer's N-terminal DExH/D domain, located in a short 'base branch', interacts with TRBP, whereas its C-terminal catalytic domains in the main body are proximal to AGO2. A model generated by docking the available atomic structures of Dicer and Argonaute homologs into the RLC reconstruction suggests a mechanism for siRNA transfer from Dicer to AGO2.

More than the sum of its parts: Physical and mechanistic coupling in the viral two-component recombinases

Gökhan Tolun, University of North Carolina, Chapel Hill, NC.

Co-Authors: Richard S. Myers, Jack D. Griffith.

Abstract: Single-strand-annealing homologous recombination is catalyzed by two-component recombinases in dsDNA viruses which span a diverse taxonomy. The two components are an exonuclease that resects the dsDNA break, exposing a 3’-overhang which is then coated by the second component, a ssDNA-binding and an annealase. It was known that in λ phage, λExo (exonuclease) and Redβ (annealase) form a 1:1 complex. We showed that this is a very large complex, likely composed of 12 subunits of each protein, and proposed a model. Moreover, our data suggests that λExo loads Redβ onto the nascent ssDNA generated by λExo, and the dwell-time of λExo is increased significantly by Redβ. Therefore, this protein complex appears to be more than the sum of its parts.

Appion: a general use toolbox for electron microscopy

Neil R Voss, National Resource for Automated Molecular Microscopy, The Scripps Research Institute

Co-authors: NR Voss, D Lyumkis, EJ Brignole, A Cheng, PW Lau, A Mulder, GC Lander, EL Jacovetty, JD Quispe, J Pulokas, C Yoshioka, C Irving, CS Potter, and B Carragher

Abstract: The use of cryoEM and 3D image reconstruction is becoming increasingly common. Our vision for this technique is to provide a straightforward manner for users to start from 2D images and produce a reliable 3D reconstruction through a system that both facilitates the processing steps and transparently displays the results. The user interacts with the Appion, a modular pipeline that extends existing software applications and procedures,  via web-based forms directly linked to an underlying database. Presented here is the working Appion pipeline with examples demonstrating its ability to facilitate many different projects simultaneously.

The structure of RNA polymerase in complex with the essential transcription elongation factor NusA

Xiao Yang and Peter J. Lewis, School of Environmental and Life Sciences, University of Newcastle, Callaghan, Australia

Co-authors: Seeseei Molimau, Geoffrey P. Doherty, Elecia Johnston Jon Marles-Wright, Rosalba Rothnagel, Ben Hankamer³, Richard J. Lewis,

Abstract: There are three stages of transcribing DNA into RNA. These stages are initiation, elongation and termination, and they are well understood biochemically. However, partly due to the difficulties in obtaining diffracting crystals, very little structural information is available on transcription complexes. In order to understand the mechanisms of transcription regulation, we describe the first structure of a bacterial RNA polymerase in complex with the essential transcription elongation factor NusA using a combination of electron microscopy and single particle analysis. NusA is required to enhance the pausing (and termination) activity of RNA polymerase which is important in coupling transcription and translation, and is also required for the formation of the highly processive pause-resistant antitermination complexes involved in rRNA synthesis. The resulting structure between the polymerase and NusA from Bacillus subtilis provides important insights into the transition from an initiation to an elongation complex, and how NusA is able to modulate transcription elongation and termination.

The structure of acetylcholine receptor clusters at the neuromuscular junction

Benoît Zuber, Medical Research Council Laboratory of Molecular Biology, Cambridge

Abstract: Clustering of acetylcholine receptors (AChR) at the neuromuscular junction is essential to muscle function. Rapsyn, a 43 kDa cytoplasmic protein, is necessary for the onset and the stability of AChR clusters and is believed to act as a scaffold. The stoechiometry and topology of AChR:rapsyn complex are debated. Previous studies could not resolve single molecules and only observed average behaviours of large populations of molecules, often in non-muscle expression systems.

We addressed these issues with cryoelectron tomography of Torpedo marmorata electric organ, a model system of the neuromuscular junction, prepared by various ways. Subtomogram averaging was used to enhance the signal-to-noise ratio and revealed radial densities bound to AChR cytoplasmic tip. Subtomogram classification showed that a variable number of densities (most likely rapsyn) are bound to AChR. Each radial density acts as a bridge between adjacent AChRs.