Structure of the Mammalian Ribosome-Sec61 Complex to 3.4 Å Resolution

Cotranslational protein translocation is a universally conserved process for secretory and membrane protein biosynthesis. Nascent polypeptides emerging from a translating ribosome are either transported across or inserted into the membrane via the ribosome-bound Sec61 channel. Here, we report structures of a mammalian ribosome-Sec61 complex in both idle and translating states, determined to 3.4 and 3.9 Å resolution. The data sets permit building of a near-complete atomic model of the mammalian ribosome, visualization of A/P and P/E hybrid-state tRNAs, and analysis of a nascent polypeptide in the exit tunnel. Unprecedented chemical detail is observed for both the ribosome-Sec61 interaction and the conformational state of Sec61 upon ribosome binding. Comparison of the maps from idle and translating complexes suggests how conformational changes to the Sec61 channel could facilitate translocation of a secreted polypeptide. The high-resolution structure of the mammalian ribosome-Sec61 complex provides a valuable reference for future functional and structural studies

Optimization problems in electron microscopy of single particles

The final publication is available at Springer via http://dx.doi.org/10.1007/s10479-006-0078-8Electron Microscopy is a valuable tool for the elucidation of the three-dimensional structure of macromolecular complexes. Knowledge about the macromolecular structure provides important information about its function and how it is carried out. This work addresses the issue of three-dimensional reconstruction of biological macromolecules from electron microscopy images. In particular, it focuses on a methodology known as “single-particles” and makes a thorough review of all those steps that can be expressed as an optimization problem. In spite of important advances in recent years, there are still unresolved challenges in the field that offer an excellent testbed for new and more powerful optimization techniques.We acknowledge partial support from the “Comunidad Autónoma de Madrid” through grants CAM-07B-0032-2002, GR/SAL/0653/2004 and GR/SAL/0342/2004, the “Comisión Interministerial de Ciencia yTecnologia” of Spain through grants BIO2001-1237, BIO2001-4253-E, BIO2001-4339-E, BIO2002- 10855-E, BFU2004-00217/BMC, the Spanish FIS grant (G03/185), the European Union through grants QLK2- 2000-00634, QLRI-2000-31237, QLRT-2000-0136, QLRI-2001-00015, FP6-502828 and the NIH through grant HL70472. Alberto Pascual and Roberto Marabini acknowledge support by the Spanish Ramon y Cajal Program

Structure and uncoating of immature adenovirus

Maturation via proteolytical processing is a common trait in the viral world, and is often accompanied by large conformational changes and rearrangements in the capsid. The adenovirus protease has been shown to play a dual role in the viral infectious cycle: (a) in maturation, as viral assembly starts with precursors to several of the structural proteins, but ends with proteolytically processed versions in the mature virion; and (b) in entry, because protease-impaired viruses have difficulties in endosome escape and uncoating. Indeed, viruses that have not undergone proteolytical processing are not infectious. We present the 3D structure of immature adenovirus particles, as represented by the thermosensitive mutant Ad2 ts1 grown under nonpermissive conditions, and compare it with the mature capsid. Our 3DEM maps at subnanometer resolution indicate that adenovirus maturation does not involve large scale conformational changes in the capsid. Difference maps reveal the location of unprocessed peptides pIIIa and pVI and help to define their role in capsid assembly and maturation. An intriguing difference appears in the core, indicating a more compact organization and increased stability of the immature cores. We have further investigated these properties by in vitro disassembly assays. Fluorescence and electron microscopy experiments reveal differences in the stability and uncoating of immature viruses, both at the capsid and core levels, as well as disassembly intermediates not previously imaged.This work was supported by grants from the Ministerio de Ciencia e Innovación of Spain (BFU2007-60228 to C.S.M. and BIO2007-67150-C03-03 to R.M.), the Comunidad Autónoma de Madrid and Consejo Superior de Investigaciones Científicas (CCG08-CSIC/SAL-3442 to C.S.M.) and the National Institutes of Health (5R01CA111569 to D.T.C., R0141599 to W.F.M. and GM037705 to S.J.F.). R.M.-C. is a recipient of a PFIS fellowship from the Instituto de Salud Carlos III of Spain. A.J.P.-B. holds a CSIC JAE-Doc postdoctoral position, partially funded by the European Social FundPeer reviewe

Architecture of Pol II(G) and molecular mechanism of transcription regulation by Gdown1.

Tight binding of Gdown1 represses RNA polymerase II (Pol II) function in a manner that is reversed by Mediator, but the structural basis of these processes is unclear. Although Gdown1 is intrinsically disordered, its Pol II interacting domains were localized and shown to occlude transcription factor IIF (TFIIF) and transcription factor IIB (TFIIB) binding by perfect positioning on their Pol II interaction sites. Robust binding of Gdown1 to Pol II is established by cooperative interactions of a strong Pol II binding region and two weaker binding modulatory regions, thus providing a mechanism both for tight Pol II binding and transcription inhibition and for its reversal. In support of a physiological function for Gdown1 in transcription repression, Gdown1 co-localizes with Pol II in transcriptionally silent nuclei of early Drosophila embryos but re-localizes to the cytoplasm during zygotic genome activation. Our study reveals a self-inactivation through Gdown1 binding as a unique mode of repression in Pol II function

Structure of the fanconi anaemia monoubiquitin ligase complex

The Fanconi anaemia (FA) pathway repairs DNA damage caused by endogenous and chemotherapy-induced DNA crosslinks, and responds to replication stress1,2. Genetic inactivation of this pathway by mutation of genes encoding FA complementation group (FANC) proteins impairs development, prevents blood production and promotes cancer1,3. The key molecular step in the FA pathway is the monoubiquitination of a pseudosymmetric heterodimer of FANCD2-FANCI4,5 by the FA core complex-a megadalton multiprotein E3 ubiquitin ligase6,7. Monoubiquitinated FANCD2 then recruits additional protein factors to remove the DNA crosslink or to stabilize the stalled replication fork. A molecular structure of the FA core complex would explain how it acts to maintain genome stability. Here we reconstituted an active, recombinant FA core complex, and used cryo-electron microscopy and mass spectrometry to determine its structure. The FA core complex comprises two central dimers of the FANCB and FA-associated protein of 100 kDa (FAAP100) subunits, flanked by two copies of the RING finger subunit, FANCL. These two heterotrimers act as a scaffold to assemble the remaining five subunits, resulting in an extended asymmetric structure. Destabilization of the scaffold would disrupt the entire complex, resulting in a non-functional FA pathway. Thus, the structure provides a mechanistic basis for the low numbers of patients with mutations in FANCB, FANCL and FAAP100. Despite a lack of sequence homology, FANCB and FAAP100 adopt similar structures. The two FANCL subunits are in different conformations at opposite ends of the complex, suggesting that each FANCL has a distinct role. This structural and functional asymmetry of dimeric RING finger domains may be a general feature of E3 ligases. The cryo-electron microscopy structure of the FA core complex provides a foundation for a detailed understanding of its E3 ubiquitin ligase activity and DNA interstrand crosslink repair

The Cryo-EM Structure of a Complete 30S Translation Initiation Complex from Escherichia coli

Formation of the 30S initiation complex (30S IC) is an important checkpoint in regulation of gene expression. The selection of mRNA, correct start codon, and the initiator fMet-tRNAfMet requires the presence of three initiation factors (IF1, IF2, IF3) of which IF3 and IF1 control the fidelity of the process, while IF2 recruits fMet-tRNAfMet. Here we present a cryo-EM reconstruction of the complete 30S IC, containing mRNA, fMet-tRNAfMet, IF1, IF2, and IF3. In the 30S IC, IF2 contacts IF1, the 30S subunit shoulder, and the CCA end of fMet-tRNAfMet, which occupies a novel P/I position (P/I1). The N-terminal domain of IF3 contacts the tRNA, whereas the C-terminal domain is bound to the platform of the 30S subunit. Binding of initiation factors and fMet-tRNAfMet induces a rotation of the head relative to the body of the 30S subunit, which is likely to prevail through 50S subunit joining until GTP hydrolysis and dissociation of IF2 take place. The structure provides insights into the mechanism of mRNA selection during translation initiation

Which executive functioning deficits are associated with AD/HD, ODD/CD and comorbid AD/HD+ODD/CD?

Item does not contain fulltextThis study investigated (1) whether attention deficit/hyperactivity disorder (AD/HD) is associated with executive functioning (EF) deficits while controlling for oppositional defiant disorder/conduct disorder (ODD/CD), (2) whether ODD/CD is associated with EF deficits while controlling for AD/HD, and (3)~whether a combination of AD/HD and ODD/CD is associated with EF deficits (and the possibility that there is no association between EF deficits and AD/HD or ODD/CD in isolation). Subjects were 99~children ages 6–12 years. Three putative domains of EF were investigated using well-validated tests: verbal fluency, working memory, and planning. Independent of ODD/CD, AD/HD was associated with deficits in planning and working memory, but not in verbal fluency. Only teacher rated AD/HD, but not parent rated AD/HD, significantly contributed to the prediction of EF task performance. No EF deficits were associated with ODD/CD. The presence of comorbid AD/HD accounts for the EF deficits in children with comorbid AD/HD+ODD/CD. These results suggest that EF deficits are unique to AD/HD and support the model proposed by R. A. Barkley (1997).17 p

Marker-free image registration of electron tomography tilt-series

<p>Abstract</p> <p>Background</p> <p>Tilt series are commonly used in electron tomography as a means of collecting three-dimensional information from two-dimensional projections. A common problem encountered is the projection alignment prior to 3D reconstruction. Current alignment techniques usually employ gold particles or image derived markers to correctly align the images. When these markers are not present, correlation between adjacent views is used to align them. However, sequential pairwise correlation is prone to bias and the resulting alignment is not always optimal.</p> <p>Results</p> <p>In this paper we introduce an algorithm to find regions of the tilt series which can be tracked within a subseries of the tilt series. These regions act as landmarks allowing the determination of the alignment parameters. We show our results with synthetic data as well as experimental cryo electron tomography.</p> <p>Conclusion</p> <p>Our algorithm is able to correctly align a single-tilt tomographic series without the help of fiducial markers thanks to the detection of thousands of small image patches that can be tracked over a short number of images in the series.</p

Structure-based classification of tauopathies

The ordered assembly of tau protein into filaments characterizes several neurodegenerative diseases, which are called tauopathies. It was previously reported that, by cryo-electron microscopy, the structures of tau filaments from Alzheimer’s disease, Pick’s disease, chronic traumatic encephalopathy and corticobasal degeneration are distinct. Here we show that the structures of tau filaments from progressive supranuclear palsy (PSP) define a new three-layered fold. Moreover, the structures of tau filaments from globular glial tauopathy are similar to those from PSP. The tau filament fold of argyrophilic grain disease (AGD) differs, instead resembling the four-layered fold of corticobasal degeneration. The AGD fold is also observed in ageing-related tau astrogliopathy. Tau protofilament structures from inherited cases of mutations at positions +3 or +16 in intron 10 of MAPT (the microtubule-associated protein tau gene) are also identical to those from AGD, suggesting that relative overproduction of four-repeat tau can give rise to the AGD fold. Finally, the structures of tau filaments from cases of familial British dementia and familial Danish dementia are the same as those from cases of Alzheimer’s disease and primary age-related tauopathy. These findings suggest a hierarchical classification of tauopathies on the basis of their filament folds, which complements clinical diagnosis and neuropathology and also allows the identification of new entities—as we show for a case diagnosed as PSP, but with filament structures that are intermediate between those of globular glial tauopathy and PSP

Mechanism of eIF6 release from the nascent 60S ribosomal subunit.

SBDS protein (deficient in the inherited leukemia-predisposition disorder Shwachman-Diamond syndrome) and the GTPase EFL1 (an EF-G homolog) activate nascent 60S ribosomal subunits for translation by catalyzing eviction of the antiassociation factor eIF6 from nascent 60S ribosomal subunits. However, the mechanism is completely unknown. Here, we present cryo-EM structures of human SBDS and SBDS-EFL1 bound to Dictyostelium discoideum 60S ribosomal subunits with and without endogenous eIF6. SBDS assesses the integrity of the peptidyl (P) site, bridging uL16 (mutated in T-cell acute lymphoblastic leukemia) with uL11 at the P-stalk base and the sarcin-ricin loop. Upon EFL1 binding, SBDS is repositioned around helix 69, thus facilitating a conformational switch in EFL1 that displaces eIF6 by competing for an overlapping binding site on the 60S ribosomal subunit. Our data reveal the conserved mechanism of eIF6 release, which is corrupted in both inherited and sporadic leukemias.Supported by a Federation of European Biochemical Societies Long term Fellowship (to FW), Specialist Programme from Bloodwise [12048] (AJW), the Medical Research Council [MC_U105161083] (AJW) and [U105115237] (RRK), Wellcome Trust strategic award to the Cambridge Institute for Medal Research [100140], Tesni Parry Trust (AJW), Ted’s Gang (AJW) and the Cambridge NIHR Biomedical Research Centre.This is the author accepted manuscript. The final version is available from Nature Publishing Group via http://dx.doi.org/10.1038/nsmb.311