Structure determination of clathrin coats to subnanometer resolution by single particle cryo-electron microscopy

Clathrin triskelions can assemble into lattices of different shapes, sizes and symmetries. For many years, the structures of clathrin lattices have been studied by single particle cryo-electron microscopy, which probed the architecture of the D6 hexagonal barrel clathrin coat at the molecular level. By introducing additional image processing steps we have recently produced a density map for the D6 barrel clathrin coat at subnanometer resolution, enabling us to generate an atomic model for this lattice [Fotin, A., Cheng, Y., Sliz, P., Grigorieff, N., Harrison, S.C., Kirchhausen, T., Walz, T., 2004. Molecular model for a complete clathrin lattice from electron cryomicroscopy. Nature 432, 573-579]. We describe in detail here the image processing steps that we have added to produce a density map at this high resolution. These procedures should be generally applicable and may thus help determine the structures of other large protein assemblies to higher resolution by single particle cryo-electron microscopy.     

Automated cryoEM data acquisition and analysis of 284742 particles of GroEL

One of the goals in developing our automated electron microscopy data acquisition system, Leginon, was to improve both the ease of use and the throughput of the process of acquiring low dose images of macromolecular specimens embedded in vitreous ice. In this article, we demonstrate the potential of the Leginon system for high-throughput data acquisition by describing an experiment in which we acquired images of more than 280,000 particles of GroEL in a single 25 h session at the microscope. We also demonstrate the potential for an automated pipeline for molecular microscopy by showing that these particles can be subjected to completely automated procedures to reconstruct a three-dimensional (3D) density map to a resolution better than 8 A. In generating the 3D maps, we used a variety of metadata associated with the data acquisition and processing steps to sort and select the particles. These metadata provide a number of insights into factors that affect the quality of the acquired images and the resulting reconstructions. In particular, we show that the resolution of the reconstructed 3D density maps improves with decreasing ice thickness. These data provide a basis for assessing the capabilities of high-throughput macromolecular microscopy.     

The three-dimensional structure of complex I from Yarrowia lipolytica: a highly dynamic enzyme

The structure of complex I from Yarrowia lipolytica was determined by three-dimensional electron microscopy. A random conical data set was collected from deep stain embedded particles. More than 14000 image pairs were analyzed. Through extensive classification combined with three-dimensional reconstruction, it was possible for the first time to show a much more detailed substructure of the complex. The peripheral arm is subdivided in at least six domains. The membrane arm shows two major protrusions on its matrix facing side and exhibits a channel like feature on the side facing the cytoplasm. Structures resembling a tether connecting the subunits near the catalytic center with the protrusions of the membrane arm provide a second connection between matrix and membrane domain.     

Stature in archeological samples from central Italy: methodological issues and diachronic changes

Stature reconstructions from skeletal remains are usually obtained through regression equations based on the relationship between height and limb bone length. Different equations have been employed to reconstruct stature in skeletal samples, but this is the first study to provide a systematic analysis of the reliability of the different methods for Italian historical samples. Aims of this article are: 1) to analyze the reliability of different regression methods to estimate stature for populations living in Central Italy from the Iron Age to Medieval times; 2) to search for trends in stature over this time period by applying the most reliable regression method. Long bone measurements were collected from 1,021 individuals (560 males, 461 females), from 66 archeological sites for males and 54 for females. Three time periods were identified: Iron Age, Roman period, and Medieval period. To determine the most appropriate equation to reconstruct stature the Delta parameter of Gini (Memorie di metodologia statistica. Milano: Giuffre A. 1939), in which stature estimates derived from different limb bones are compared, was employed. The equations proposed by Pearson (Philos Trans R Soc London 192 (1899) 169-244) and Trotter and Gleser for Afro-Americans (Am J Phys Anthropol 10 (1952) 463-514; Am J Phys Anthropol 47 (1977) 355-356) provided the most consistent estimates when applied to our sample. We then used the equation by Pearson for further analyses. Results indicate a reduction in stature in the transition from the Iron Age to the Roman period, and a subsequent increase in the transition from the Roman period to the Medieval period. Changes of limb lengths over time were more pronounced in the distal than in the proximal elements in both limbs.     

Femoral dimensions and mid-thigh circumference in Alligator mississippiensis

The mid-thigh circumference of the intact hindlimb in Alligator mississippiensis is tightly correlated with transverse dimensions of the proximal and distal articular surfaces of the femur, and with minimum midshaft femoral circumference. Maximum diameter of the proximal articular end, width across the distal articular end, and midshaft circumference are the best femoral predictors of circumference of the intact thigh. Regression equations of mid-thigh circumference against these femoral dimensions can be used to estimate the transverse dimensions of the intact hindlimb in extinct crocodylian-like archosaurs.     

Long-term retrospection on mangrove development using transdisciplinary approaches: A review

Large ecosystem processes often take place beyond the observation time of a researcher. Yet, through retrospective research scientists can approach and understand ecosystem changes. This contributes to the fundamental understanding of both human-induced and natural dynamics in ecosystems world-wide. This also holds for fast changing coastal areas with mangrove ecosystems, which are important for biodiversity, for coastal protection, and for the daily livelihood of millions of people in tropical coastal developing countries. In addition, retrospective research generates a basis for predictions that can be used early on to protect an ecosystem. In attempting to protect ecosystems from adverse human-induced change and destruction, and to manage them for sustainability, scientists are only beginning to investigate and understand natural ecosystem dynamics. It is important and advisable to gather, combine and analyse all possible data that allow a researcher to look back in time. This paper reviews the available retrospective methods, and highlights the transdisciplinary way (i.e. combination between basic and applied sciences on one hand, and social and human sciences on the other) in which retrospective research on a scale between months and centuries can be carried out, but it also includes methods on larger scales that may be marginally relevant. The paper particularly emphasizes the lack of transdisciplinary (not interdisciplinary) integration between sciences in retrospective research on mangrove forests in the past.     

Localization of the N-terminus of minor coat protein IIIa in the adenovirus capsid

Minor coat protein IIIa is conserved in all adenoviruses (Ads) and is required for correct viral assembly, but its precise function in capsid organization is unknown. The latest Ad capsid model proposes that IIIa is located underneath the vertex region. To obtain experimental evidence on the location of IIIa and to further define its role, we engineered the IIIa gene to encode heterologous N-terminal peptide extensions. Recombinant Ad variants with IIIa encoding six-histidine (6His) tag, 6His, and FLAG peptides, or with 6His linked to FLAG with a (Gly₄Ser)₃ linker were rescued and analyzed for virus yield, capsid incorporation of heterologous peptides, and capsid stability. Longer extensions could not be rescued. Western blot analysis confirmed that the modified IIIa proteins were expressed in infected cells and incorporated into virions. In the Ad encoding the 6His-linker-FLAG-IIIa gene, the 6His tag was present in light particles, but not in mature virions. Immunoelectron microscopy of this virus showed that the FLAG epitope is not accessible to antibodies on the viral particles. Three-dimensional electron microscopy and difference mapping located the IIIa N-terminal extension beneath the vertex complex, wedged at the interface between the penton base and peripentonal hexons, therefore supporting the latest proposed model. The position of the IIIa N-terminus and its low tolerance for modification provide new clues for understanding the role of this minor coat protein in Ad capsid assembly and disassembly.     

Reconstructing pedigrees: a stochastic perspective

A pedigree is a directed graph that describes how individuals are related through ancestry in a sexually-reproducing population. In this paper we explore the question of whether one can reconstruct a pedigree by just observing sequence data for present day individuals. This is motivated by the increasing availability of genomic sequences, but in this paper we take a more theoretical approach and consider what models of sequence evolution might allow pedigree reconstruction (given sufficiently long sequences). Our results complement recent work that showed that pedigree reconstruction may be fundamentally impossible if one uses just the degrees of relatedness between different extant individuals. We find that for certain stochastic processes, pedigrees can be recovered up to isomorphism from sufficiently long sequences.     

“Ontellicke boomen” or “countless trees”: reconstructing the late medieval vegetation surrounding the 16th century St Margaretha Convent, Leiden, The Netherlands

At an excavation of the late medieval St Margaretha Convent in Leiden (The Netherlands), archaeobotanical results could be compared with historical data. Both wood and macroremains were analysed to reconstruct the local vegetation and seek evidence of the cultivation of plants. The historical sources available for this estate are a charter prepared in 1572, which listed all trees present just after the abandonment of the convent, and an illustration from 1574 of the convent and its grounds. The charter mentions Salix and Alnus as the most numerous trees present, followed by several other taxa. The archaeological evidence from the wood remains, mostly construction timber with a dominance of Quercus, shows the use of indigenous taxa and some non-indigenous material, which was partly re-used. It is possible that some of the Coniferae were very early home-grown specimens. The trees represented in the macroremains were most probably growing in the immediate vicinity. They specify some of the taxa found as wood or mentioned in the historical text. The identified cultivated plants could all have been grown locally. The vegetation is in general represented by ruderal taxa and plants growing in wet conditions, which form an assemblage typical of an abandoned rural area.     

A genetic assessment of polyandry and breeding-site fidelity in lemon sharks

We here employ 11 microsatellite markers and recently developed litter reconstruction methods to infer mating system parameters (i.e. polyandry and breeding-site fidelity) at a lemon shark nursery site in Marquesas Key, Florida. Four hundred and eight juvenile or subadult sharks were genotyped over eight complete breeding seasons. Using this information, we were able to infer family structure, as well as fully or partially reconstruct genotypes of 46 mothers and 163 fathers. Multiple litter reconstruction methods were used, and novel simulations helped define apparent bias and precision of at least some mating system parameters. For Marquesas Key, we find that adult female lemon sharks display high levels of polyandry (81% of all litters sampled) and stronger fidelity to the nursery site than do males. Indeed, few male sharks sired offspring from more than one litter during the course of the study. These findings were quite similar to previous results from another lemon shark nursery site (Bimini, Bahamas), suggesting conserved mating system parameters despite significant variation in early life-history traits (i.e. body size and growth) among sites. The finding of at least some site fidelity in females also supports the need for careful conservation of each nursery.