Cryo electron microscopy to determine the structure of macromolecular complexes

Cryo-electron microscopy (cryo-EM) is a structural molecular and cellular biology technique that has experienced major advances in recent years. Technological developments in image recording as well as in processing software make it possible to obtain three-dimensional reconstructions of macromolecular assemblies at near-atomic resolution that were formerly obtained only by X-ray crystallography or NMR spectroscopy. In parallel, cryo-electron tomography has also benefitted from these technological advances, so that visualization of irregular complexes, organelles or whole cells with their molecular machines in situ has reached subnanometre resolution. Cryo-EM can therefore address a broad range of biological questions. The aim of this review is to provide a brief overview of the principles and current state of the cryo-EM field

Advances in xmipp for cryo-electron microscopy: From xmipp to scipion

Xmipp is an open-source software package consisting of multiple programs for processing data originating from electron microscopy and electron tomography, designed and managed by the Biocomputing Unit of the Spanish National Center for Biotechnology, although with contributions from many other developers over the world. During its 25 years of existence, Xmipp underwent multiple changes and updates. While there were many publications related to new programs and functionality added to Xmipp, there is no single publication on the Xmipp as a package since 2013. In this article, we give an overview of the changes and new work since 2013, describe technologies and techniques used during the development, and take a peek at the future of the package

Structural and mechanistic insights into pore formation by proteins of the membrane attack complex/perforin superfamily

Members of the membrane attack complex/perforin (MACPF) superfamily of pore-forming proteins are characterised by a common three-dimensional fold able to puncture lipid membranes. They are found in bacterial and eukaryotes, and include immune effectors, toxins and pathogenic virulence factors. Their conserved pore-forming domain follows the same mechanism whereby two bundles of α-helices unfurl into membrane-spanning β-hairpins. This thesis provides insights into the effects of MACPF proteins on biological membranes. Coarse-grain molecular dynamics simulations of the membrane attack complex (MAC) bound to its inhibitor CD59 reveal protein-lipid interactions and local changes in membrane thickness. These may serve as signals to recruit CD59 or the molecular machinery for MAC clearance. Some bacterial MACPF proteins called cholesterol-dependent cytolysins (CDCs) hijack CD59 on human cells as part of their pore formation pathway. Atomistic simulations of CD59 in a lipid bilayer show that it samples various orientations relative to the membrane, dictating whether its binding site is available for engaging MAC or CDCs, and thus for inhibiting or promoting pore formation. CDCs assemble on cholesterol-rich lipid membranes and undergo sequential conformational changes to puncture bilayers. Site-directed mutagenesis of two CDCs reveals that an amphipathic helix in the pore-forming helical bundles is responsible for tuning the lytic activity of these proteins. Understanding the molecular basis for the function of this helix will require the high-resolution structure of a CDC late prepore intermediate. The first steps towards solving this structure by cryo-electron microscopy are presented in this thesis.Open Acces

Structure

The nuclear pore complex, composed of proteins termed nucleoporins (Nups), is responsible for nucleocytoplasmic transport in eukaryotes. Nuclear pore complexes (NPCs) form an annular structure composed of the nuclear ring, cytoplasmic ring,\uc2\ua0a membrane ring, and two inner rings. Nup192 is\uc2\ua0a major component of the NPC's inner ring. We report the crystal structure of Saccharomyces cerevisiae Nup192 residues 2-960 [ScNup192(2-960)], which adopts an \uce\ub1-helical fold with three domains (i.e., D1, D2, and D3). Small angle X-ray scattering and electron microscopy (EM) studies reveal that ScNup192(2-960) could undergo long-range transition between "open" and "closed" conformations. We obtained a structural model of full-length ScNup192 based on EM, the structure of ScNup192(2-960), and homology modeling. Evolutionary analyses using the ScNup192(2-960) structure suggest that NPCs and vesicle-coating complexes are descended from a common membrane-coating ancestral complex. We show that suppression of Nup192 expression leads to compromised nuclear transport and hypothesize a role for Nup192 in modulating the permeability of the NPC central channel.C06 RR017528-01-CEM/CE/NCIPC CDC HHS/United StatesP30-EB-009998/EB/NIBIB NIH HHS/United StatesP41GM103393/GM/NIGMS NIH HHS/United StatesP41RR001209/RR/NCRR NIH HHS/United StatesR01 GM062427/GM/NIGMS NIH HHS/United StatesR01 GM071329/GM/NIGMS NIH HHS/United StatesR01 GM083960/GM/NIGMS NIH HHS/United StatesR01 GM083960/GM/NIGMS NIH HHS/United StatesU01 GM098256/GM/NIGMS NIH HHS/United StatesU01 GM098256/GM/NIGMS NIH HHS/United StatesU54 GM074945/GM/NIGMS NIH HHS/United StatesU54 GM094662/GM/NIGMS NIH HHS/United StatesU54 GM103511/GM/NIGMS NIH HHS/United StatesU54 GM103511/GM/NIGMS NIH HHS/United States2014-04-02T00:00:00Z23499021PMC375562

De Novo Protein Structure Modeling from Cryoem Data Through a Dynamic Programming Algorithm in the Secondary Structure Topology Graph

Proteins are the molecules carry out the vital functions and make more than the half of dry weight in every cell. Protein in nature folds into a unique and energetically favorable 3-Dimensional (3-D) structure which is critical and unique to its biological function. In contrast to other methods for protein structure determination, Electron Cryorricroscopy (CryoEM) is able to produce volumetric maps of proteins that are poorly soluble, large and hard to crystallize. Furthermore, it studies the proteins in their native environment. Unfortunately, the volumetric maps generated by current advances in CryoEM technique produces protein maps at medium resolution about (~5 to 10Å) in which it is hard to determine the atomic-structure of the protein. However, the resolution of the volumetric maps is improving steadily, and recent works could obtain atomic models at higher resolutions (~3Å). De novo protein modeling is the process of building the structure of the protein using its CryoEM volumetric map. Thereupon, the volumetric maps at medium resolution generated by CryoEM technique proposed a new challenge. At the medium resolution, the location and orientation of secondary structure elements (SSE) can be visually and computationally identified. However, the order and direction (called protein topology) of the SSEs detected from the CryoEM volumetric map are not visible. In order to determine the protein structure, the topology of the SSEs has to be figured out and then the backbone can be built. Consequently, the topology problem has become a bottle neck for protein modeling using CryoEM In this dissertation, we focus to establish an effective computational framework to derive the atomic structure of a protein from the medium resolution CryoEM volumetric maps. This framework includes a topology graph component to rank effectively the topologies of the SSEs and a model building component. In order to generate the small subset of candidate topologies, the problem is translated into a layered graph representation. We developed a dynamic programming algorithm (TopoDP) for the new representation to overcome the problem of large search space. Our approach shows the improved accuracy, speed and memory use when compared with existing methods. However, the generating of such set was infeasible using a brute force method. Therefore, the topology graph component effectively reduces the topological space using the geometrical features of the secondary structures through a constrained K-shortest paths method in our layered graph. The model building component involves the bending of a helix and the loop construction using skeleton of the volumetric map. The forward-backward CCD is applied to bend the helices and model the loops

Understanding the Molecular Mechanism of Single-Strand Annealing Homologous DNA Recombination in Viruses, by Cryo-Electron Microscopy

The single-strand annealing homologous recombination (SSA) is one of the dsDNA break repair pathways, and albeit its importance from bacteria to bacteriophages, its molecular function is still unknown. The SSA reaction is catalysed by the enzyme complexes known as Exonuclease Annealase Two-component Recombinase (EATRs). The RecT and ORF6 proteins are single-stranded DNA-binding and annealing proteins expressed in E. coli and Kaposi’s sarcoma-associated herpesvirus (KSHV), respectively. RecT has already been shown to catalyse the SSA reaction. Although ORF6 has been shown to bind to ssDNA, further experimental evidence is needed to solidify its annealase activity. Since structure can dictate the function, this thesis aimed to determine the structure of the annealases RecT and ORF6 using a state-in-art cryo-electron microscopy technique. Furthermore, the shadow-casting EM technique has been established by optimising it for the equipment available at UOW, which is helpful for imaging the substrate DNA intermediates and the nucleoprotein complexes formed during SSA to better understand the molecular mechanistic details of this reaction. This thesis includes the details about RecT and ORF6 proteins’ cloning, expression, and purification, which were further optimised for purity and homogeneity for cryo-electron microscopy with the help of negative staining electron microscopy (NSEM). Additionally, based on several NSEM analyses, the C-terminal His-tag containing RecT (RecTCH) oligomerisation on ssDNA was studied, and a general mechanism of its oligomerisation is described. Unfortunately, during the RecTCH protein’s cryo-EM sample optimisation, the LiRecT structure was published by another group. Therefore, work on that project was ceased at that point. Several novel findings on ORF6 are reported in this thesis. Primarily, the concentration of the purified protein was increased 3 times more than the reports in the literature. Based on the NSEM and preliminary cryo-EM map of ORF6, it is shown that the ORF6 structure overall resembles the HSV1-ICP8 protein. Further, based on the steady-state and time-resolved fluorescence resonance energy transfer (FRET) experiments, a model for the ORF6 annealing mechanism is suggested. Towards generating a high-resolution structure, ORF6 monomers and filaments were optimised and imaged by using cryo-EM. Processing a data set obtained from a monomeric ORF6 sample showed the presence of conformational heterogeneity in the particles, which was expected as the ORF6 AlphaFold model shows that the N-terminal and C-terminal domains are connected by an 18 amino acids long loop, allowing C-terminal domain to be relatively flexible to move around. Processing of another data set obtained from a sample containing ORF6 filaments generated 2-dimensional averages that look promising for generating a high-resolution structure. This thesis also shows the details related to the installation and optimisation of the shadowing technique using a modern material, graphene oxide (GO), as a support film. This technique involves optimising both sample preparation and instrumentation for metal evaporation and deposition. For sample preparation, GO was deposited on cryo-EM holey grids, on which the sample was mounted. For instrumentation optimisation, a DENTON brand evaporator was used. The grid stage was re-engineered using AutoCAD to achieve the finest metal evaporation, and parameters such as amperage, vacuum, metal thickness, and angles were optimised. The optimised parameters were used to shadow-cast different lengths of DNA and their complexes with proteins, and good contrast images were acquired for qualitative and quantitative analyses. Overall, this thesis presents two main novel findings. First, RecTCH monomers oligomerise into an open ring-shaped structure, which stacks together to generate short filaments. Second, to anneal two complementary ssDNA strands, ORF6 first forms filaments with both ssDNA, which then come in contact with each other rapidly to anneal the complementary strands. Once the annealing finishes, the annealed dsDNA is released from the filaments as the filaments fall apart into monomers. We also found that ORF6 monomers oligomerise to form the helical and non-helical filaments in the presence of DTT+Mg2+ and DTT-containing buffer, respectively

Acknowledging Errors: Advanced Molecular Replacement with Phaser.

Molecular replacement is a method for solving the crystallographic phase problem using an atomic model for the target structure. State-of-the-art methods have moved the field significantly from when it was first envisaged as a method for solving cases of high homology and completeness between a model and target structure. Improvements brought about by application of maximum likelihood statistics mean that various errors in the model and pathologies in the data can be accounted for, so that cases hitherto thought to be intractable are standardly solvable. As a result, molecular replacement phasing now accounts for the lion's share of structures deposited in the Protein Data Bank. However, there will always be cases at the fringes of solvability. I discuss here the approaches that will help tackle challenging molecular replacement cases.This work was supported by grant BB/L006014/1 from the BBSRC, UK.This is the author accepted manuscript. It is currently under an indefinite embargo pending publication by Springer

Advances in Xmipp for cryo-electron microscopy: from Xmipp to Scipion

Xmipp is an open-source software package consisting of multiple programs for processing data originating from electron microscopy and electron tomography, designed and managed by the Biocomputing Unit of the Spanish National Center for Biotechnology, although with contributions from many other developers over the world. During its 25 years of existence, Xmipp underwent multiple changes and updates. While there were many publications related to new programs and functionality added to Xmipp, there is no single publication on the Xmipp as a package since 2013. In this article, we give an overview of the changes and new work since 2013, describe technologies and techniques used during the development, and take a peek at the future of the package