3D Cryo-Electron Microscopy Reveals the Structure of a 3-Fluorenylmethyloxycarbonyl Zipper Motif Ensuring the Self-Assembly of Tripeptide Nanofibers.

Short peptide-based supramolecular hydrogels appeared as highly interesting materials for applications in many fields. The optimization of their properties relies mainly on the design of a suitable hydrogelator through an empirical trial-and-error strategy based on the synthesis of various types of peptides. This approach is in part due to the lack of prior structural knowledge of the molecular architecture of the various families of nanofibers.

Cryo-EM architecture of a near-native stretch-sensitive membrane microdomain.

Biological membranes are partitioned into functional zones termed membrane microdomains, which contain specific lipids and proteins. The composition and organization of membrane microdomains remain controversial because few techniques are available that allow the visualization of lipids in situ without disrupting their native behaviour. The yeast eisosome, composed of the BAR-domain proteins Pil1 and Lsp1 (hereafter, Pil1/Lsp1), scaffolds a membrane compartment that senses and responds to mechanical stress by flattening and releasing sequestered factors.

Cryo-EM structure of influenza helical nucleocapsid reveals NP-NP and NP-RNA interactions as a model for the genome encapsidation.

Influenza virus genome encapsidation is essential for the formation of a helical viral ribonucleoprotein (vRNP) complex composed of nucleoproteins (NP), the trimeric polymerase, and the viral genome. Although low-resolution vRNP structures are available, it remains unclear how the viral RNA is encapsidated and how NPs assemble into the helical filament specific of influenza vRNPs. In this study, we established a biological tool, the RNP-like particles assembled from recombinant influenza A virus NP and synthetic RNA, and we present the first subnanometric cryo-electron microscopy structure of the helical NP-RNA complex (8.

The giant mimivirus 1.2 Mb genome is elegantly organized into a 30-nm diameter helical protein shield.

Mimivirus is the prototype of the family of giant dsDNA viruses. Little is known about the organization of the 1.2 Mb genome inside the membrane-limited nucleoid filling the ~0.

Self-association of MreC as a regulatory signal in bacterial cell wall elongation.

The elongasome, or Rod system, is a protein complex that controls cell wall formation in rod-shaped bacteria. MreC is a membrane-associated elongasome component that co-localizes with the cytoskeletal element MreB and regulates the activity of cell wall biosynthesis enzymes, in a process that may be dependent on MreC self-association. Here, we use electron cryo-microscopy and X-ray crystallography to determine the structure of a self-associated form of MreC from Pseudomonas aeruginosa in atomic detail.

3D structure of three jumbo phage heads.

Jumbo phages are bacteriophages that carry more than 200 kbp of DNA. In this study we characterized two jumbo phages (ΦRSL2 and ΦXacN1) and one semi-jumbo phage (ΦRP13) at the structural level by cryo-electron microscopy. Focusing on their capsids, three-dimensional structures of the heads at resolutions ranging from 16 to 9 Å were calculated.

Structural Analysis of Jumbo Coliphage phAPEC6.

The phAPEC6 genome encodes 551 predicted gene products, with the vast majority (83%) of unknown function. Of these, 62 have been identified as virion-associated proteins by mass spectrometry (ESI-MS/MS), including the major capsid protein (Gp225; present in 1620 copies), which shows a HK97 capsid protein-based fold. Cryo-electron microscopy experiments showed that the 350-kbp DNA molecule of virus phAPEC6 is packaged in at least 15 concentric layers in the phage capsid.

In situ Structure of Rotavirus VP1 RNA-Dependent RNA Polymerase.

Rotaviruses, like other non-enveloped, double-strand RNA viruses, package an RNA-dependent RNA polymerase (RdRp) with each duplex of their segmented genomes. Rotavirus cell entry results in loss of an outer protein layer and delivery into the cytosol of an intact, inner capsid particle (the "double-layer particle," or DLP). The RdRp, designated VP1, is active inside the DLP; each VP1 achieves many rounds of mRNA transcription from its associated genome segment.

Integrated NMR and cryo-EM atomic-resolution structure determination of a half-megadalton enzyme complex.

Atomic-resolution structure determination is crucial for understanding protein function. Cryo-EM and NMR spectroscopy both provide structural information, but currently cryo-EM does not routinely give access to atomic-level structural data, and, generally, NMR structure determination is restricted to small (<30 kDa) proteins. We introduce an integrated structure determination approach that simultaneously uses NMR and EM data to overcome the limits of each of these methods.

Structure and assembly of pilotin-dependent and -independent secretins of the type II secretion system.

The type II secretion system (T2SS) is a cell envelope-spanning macromolecular complex that is prevalent in Gram-negative bacterial species. It serves as the predominant virulence mechanism of many bacteria including those of the emerging human pathogens Vibrio vulnificus and Aeromonas hydrophila. The system is composed of a core set of highly conserved proteins that assemble an inner membrane platform, a periplasmic pseudopilus and an outer membrane complex termed the secretin.

Imaging Plastids in 2D and 3D: Confocal and Electron Microscopy.

Internal chloroplast structures present complex and various characteristics, which are still largely undetermined due to insufficient imaging investigation. Information on chloroplast morphology has traditionally been collected using light microscopy (LM), confocal laser scanning microscopy (CLSM), transmission electron microscopy (TEM), and scanning electron microscopy (SEM) techniques. However, recent technological progresses in the field of microscopy have made it possible to visualize the internal structure of chloroplast in far greater detail and in 3D.

Nucleoprotein from the unique human infecting Orthobunyavirus of Simbu serogroup (Oropouche virus) forms higher order oligomers in complex with nucleic acids in vitro.

Oropouche virus (OROV) is the unique known human pathogen belonging to serogroup Simbu of Orthobunyavirus genus and Bunyaviridae family. OROV is transmitted by wild mosquitoes species to sloths, rodents, monkeys and birds in sylvatic environment, and by midges (Culicoides paraensis and Culex quinquefasciatus) to man causing explosive outbreaks in urban locations. OROV infection causes dengue fever-like symptoms and in few cases, can cause clinical symptoms of aseptic meningitis.

Plastid thylakoid architecture optimizes photosynthesis in diatoms.

Photosynthesis is a unique process that allows independent colonization of the land by plants and of the oceans by phytoplankton. Although the photosynthesis process is well understood in plants, we are still unlocking the mechanisms evolved by phytoplankton to achieve extremely efficient photosynthesis. Here, we combine biochemical, structural and in vivo physiological studies to unravel the structure of the plastid in diatoms, prominent marine eukaryotes.

Fusion to a homo-oligomeric scaffold allows cryo-EM analysis of a small protein.

Recent technical advances have revolutionized the field of cryo-electron microscopy (cryo-EM). However, most monomeric proteins remain too small (<100 kDa) for cryo-EM analysis. To overcome this limitation, we explored a strategy whereby a monomeric target protein is genetically fused to a homo-oligomeric scaffold protein and the junction optimized to allow the target to adopt the scaffold symmetry, thereby generating a chimeric particle suitable for cryo-EM.

Cryo-electron Microscopy Structure of the Native Prototype Foamy Virus Glycoprotein and Virus Architecture.

Foamy viruses (FV) belong to the genus Spumavirus, which forms a distinct lineage in the Retroviridae family. Although the infection in natural hosts and zoonotic transmission to humans is asymptomatic, FVs can replicate well in human cells making it an attractive gene therapy vector candidate. Here we present cryo-electron microscopy and (cryo-)electron tomography ultrastructural data on purified prototype FV (PFV) and PFV infected cells.

Structure determination of feline calicivirus virus-like particles in the context of a pseudo-octahedral arrangement.

The vesivirus feline calicivirus (FCV) is a positive strand RNA virus encapsidated by an icosahedral T=3 shell formed by the viral VP1 protein. Upon its expression in the insect cell - baculovirus system in the context of vaccine development, two types of virus-like particles (VLPs) were formed, a majority built of 60 subunits (T=1) and a minority probably built of 180 subunits (T=3). The structure of the small particles was determined by x-ray crystallography at 0.

Structural similarity of secretins from type II and type III secretion systems.

Secretins, the outer membrane components of several secretion systems in Gram-negative bacteria, assemble into channels that allow exoproteins to traverse the membrane. The membrane-inserted, multimeric regions of PscC, the Pseudomonas aeruginosa type III secretion system secretin, and PulD, the Klebsiella oxytoca type II secretion system secretin, were purified after cell-free synthesis and their structures analyzed by single particle cryoelectron microscopy. Both homomultimeric, barrel-like structures display a "cup and saucer" architecture.

Monomeric nucleoprotein of influenza A virus.

Isolated influenza A virus nucleoprotein exists in an equilibrium between monomers and trimers. Samples containing only monomers or only trimers can be stabilized by respectively low and high salt. The trimers bind RNA with high affinity but remain trimmers, whereas the monomers polymerise onto RNA forming nucleoprotein-RNA complexes.

Location of the dsRNA-dependent polymerase, VP1, in rotavirus particles.

Double-stranded RNA (dsRNA) viruses transcribe and replicate RNA within an assembled, inner capsid particle; only plus-sense mRNA emerges into the intracellular milieu. During infectious entry of a rotavirus particle, the outer layer of its three-layer structure dissociates, delivering the inner double-layered particle (DLP) into the cytosol. DLP structures determined by X-ray crystallography and electron cryomicroscopy (cryoEM) show that the RNA coils uniformly into the particle interior, avoiding a "fivefold hub" of more structured density projecting inward from the VP2 shell of the DLP along each of the twelve 5-fold axes.

Conformational states of a bacterial α2-macroglobulin resemble those of human complement C3.

α(2) macroglobulins (α(2)Ms) are broad-spectrum protease inhibitors that play essential roles in the innate immune system of eukaryotic species. These large, multi-domain proteins are characterized by a broad-spectrum bait region and an internal thioester, which, upon cleavage, becomes covalently associated to the target protease, allowing its entrapment by a large conformational modification. Notably, α(2)Ms are part of a larger protein superfamily that includes proteins of the complement system, such as C3, a multi-domain macromolecule which is also characterized by an internal thioester-carrying domain and whose activation represents the pivotal step in the complement cascade.

Oligomerization paths of the nucleoprotein of influenza A virus.

The influenza viruses contain a segmented, negative strand RNA genome. Each RNA segment is covered by multiple copies of the nucleoprotein (NP) and is associated with the polymerase complex into ribonucleoprotein (RNP) particles. Despite its importance in the virus life cycle, the interactions between the NP and the genome are not well understood.

Cryo-EM structure of the E. coli translating ribosome in complex with SRP and its receptor.

We report the 'early' conformation of the Escherichia coli signal recognition particle (SRP) and its receptor FtsY bound to the translating ribosome, as determined by cryo-EM. FtsY binds to the tetraloop of the SRP RNA, whereas the NG domains of the SRP protein and FtsY interact weakly in this conformation. Our results suggest that optimal positioning of the SRP RNA tetraloop and the Ffh NG domain leads to FtsY recruitment.

Ab initio high-resolution single-particle 3D reconstructions: the symmetry adapted functions way.

A protocol to attain high-resolution single-particle reconstructions is presented. The protocol is the concatenation of two procedures: one to obtain an ab initio low-resolution reconstruction, the other to determine a fixed point of the consecutive applications of fast projection matching and 3D reconstruction. It is a reciprocal space formulation where the Fourier coefficients of the 3D scattering density are expressed in terms of symmetry adapted functions and the 2D particle images are represented by their Fourier-Bessel transforms.

Fast projection matching for cryo-electron microscopy image reconstruction.

A new FFT-accelerated projection matching method is presented and tested. The electron microscopy images are represented by their Fourier-Bessel transforms and the 3D model by its expansion in spherical harmonics, or more specifically in terms of symmetry-adapted functions. The rotational and translational properties of these representations are used to quickly access all the possible 2D projections of the 3D model, which allow an exhaustive inspection of the whole five-dimensional domain of parameters associated to each particle.