Biomedical subjects

Ardan Patwardhan

Publications and source records attributed to Ardan Patwardhan.

9 recordsLinked to original sources

PubMed2023

EMPIAR: the Electron Microscopy Public Image Archive.

Public archiving in structural biology is well established with the Protein Data Bank (PDB; wwPDB.org) catering for atomic models and the Electron Microscopy Data Bank (EMDB; emdb-empiar.org) for 3D reconstructions from cryo-EM experiments. Even before the recent rapid growth in cryo-EM, there was an expressed community need for a public archive of image data from cryo-EM experiments for validation, software development, testing and training. Concomitantly, the proliferation of 3D imaging techniques for cells, tissues and organisms using volume EM (vEM) and X-ray tomography (XT) led to calls from these communities to publicly archive such data as well. EMPIAR (empiar.org) was developed as a public archive for raw cryo-EM image data and for 3D reconstructions from vEM and XT experiments and now comprises over a thousand entries totalling over 2 petabytes of data. EMPIAR resources include a deposition system, entry pages, facilities to search, visualize and download datasets, and a REST API for programmatic access to entry metadata. The success of EMPIAR also poses significant challenges for the future in dealing with the very fast growth in the volume of data and in enhancing its reusability.

Imaging, Three-Dimensional↗

PubMed2007

Different quaternary structures of human RECQ1 are associated with its dual enzymatic activity.

RecQ helicases are essential for the maintenance of chromosome stability. In addition to DNA unwinding, some RecQ enzymes have an intrinsic DNA strand annealing activity. The function of this dual enzymatic activity and the mechanism that regulates it is, however, unknown. Here, we describe two quaternary forms of the human RECQ1 helicase, higher-order oligomers consistent with pentamers or hexamers, and smaller oligomers consistent with monomers or dimers. Size exclusion chromatography and transmission electron microscopy show that the equilibrium between the two assembly states is affected by single-stranded DNA (ssDNA) and ATP binding, where ATP or ATPgammaS favors the smaller oligomeric form. Our three-dimensional electron microscopy reconstructions of human RECQ1 reveal a complex cage-like structure of approximately 120 A x 130 A with a central pore. This oligomeric structure is stabilized under conditions in which RECQ1 is proficient in strand annealing. In contrast, competition experiments with the ATPase-deficient K119R and E220Q mutants indicate that RECQ1 monomers, or tight binding dimers, are required for DNA unwinding. Collectively, our findings suggest that higher-order oligomers are associated with DNA strand annealing, and lower-order oligomers with DNA unwinding.

Binding, Competitive↗

PubMed2006

Structural basis of the Methanothermobacter thermautotrophicus MCM helicase activity.

The MCM complex from the archaeon Methanother-mobacter thermautotrophicus is a model for the eukaryotic MCM2-7 helicase. We present electron-microscopy single-particle reconstructions of a DNA treated M.thermautotrophicus MCM sample and a ADP.AlF(x) treated sample, respectively assembling as double hexamers and double heptamers. The electron-density maps display an unexpected asymmetry between the two rings, suggesting that large conformational changes can occur within the complex. The structure of the MCM N-terminal domain, as well as the AAA+ and the C-terminal HTH dom-ains of ZraR can be fitted into the reconstructions. Distinct configurations can be modelled for the AAA+ and the HTH domains, suggesting the nature of the conformational change within the complex. The pre-sensor 1 and the helix 2 insertions, important for the activity, can be located pointing towards the centre of the channel in the presence of DNA. We propose a mechanistic model for the helicase activity, based on a ligand-controlled rotation of the AAA+ subunits.

Adenosine Triphosphatases↗

PubMed2006

Structural studies of the archaeal MCM complex in different functional states.

The primary candidate for the eukaryotic replicative helicase is the MCM2-7 complex, a hetero-oligomer formed by six AAA+ paralogous polypeptides. A simplified model for structure-function studies is the homo-oligomeric orthologue from the archaeon Methanothermobacter thermoautotrophicus. The crystal structure of the DNA-interacting N-terminal domain of this homo-oligomer revealed a double hexamer in a head-to-head configuration; single-particle electron microscopy studies have shown that the full-length protein complex can form both single and double rings, in which each ring can consist of a cyclical arrangement of six or seven subunits. Using single-particle techniques and especially multivariate statistical symmetry analysis, we have assessed the changes in stoichiometry that the complex undergoes when treated with various nucleotide analogues or when binding a double-stranded DNA fragment. We found that the binding of nucleotides or of double-stranded DNA leads to the preferred formation of double-ring structures. Specifically, the protein complex is present as a double heptamer when treated with a nucleotide analogue, but it is rather found as a double hexamer when complexed with double-stranded DNA. The possible physiological role of the various stoichiometries of the complex is discussed in the light of the proposed mechanisms of helicase activity.

Adenosine Diphosphate↗

PubMed2005

Conformational changes of Escherichia coli sigma54-RNA-polymerase upon closed-promoter complex formation.

RNA polymerase from the mesophile Escherichia coli exists in two forms, the core enzyme and the holoenzyme. Using cryo-electron microscopy and single-particle analysis, we have obtained the structure of the complete RNA polymerase from E.coli containing the sigma54 factor within the closed-promoter complex. Comparisons with earlier reconstructions of the core enzyme and the sigma54 holoenzyme reveal the behaviour of this major variant RNA polymerase in defined functional states. The binding of DNA leads to significant conformational changes in the enzyme's catalytic subunits, apparently a necessity for the initiation of enhancer-dependent promoter-specific transcription.

Binding Sites↗

PubMed2004

A measure for the angle between projections based on the extent of correlation between corresponding central sections.

A pre-condition for the ab initio assignment of Euler angles to a set of projections from an asymmetric object is that at least three of the available projections correspond to rotations about different axes. For symmetric objects this condition may be relaxed. There are some applications of single-particle electron microscopy, such as the reconstruction of filamentous macromolecular assemblies, where all available projections more-or-less correspond to rotations about a common rotation axis making it difficult to satisfy this condition. Here, a method has been developed to overcome this problem, based on the fact that the correlation between two central sections of the Fourier transform of a compact object will not be limited to an infinitesimal central line but will have a finite extent, which is related to the angle between the corresponding projections. Projections from model filaments, with different degrees of rotational symmetry about the long axis, have been used to test the methodology. The results show that angle determination is robust down to signal-to-noise ratios as low as 2 and that, in general, the error decreases as the degree of symmetry increases. The method has been used to assign angles to a set of negatively stained muscle thick filament projections to obtain an initial 3D reconstruction. The main features of the projections are seen to be faithfully reproduced in the reprojections from the reconstruction. A real-space adaptation of this method is also discussed.

Fourier Analysis↗

PubMed2004

Single particle analysis of filamentous and highly elongated macromolecular assemblies.

The application of single particle techniques to the three-dimensional analysis of electron microscope images of elongated or filamentous macromolecular assemblies is evaluated, taking as an example the muscle thin filament. Although the thin filament contains local helical symmetry, because of the inherent variable twist along it, the helical coherence does not extend for large enough distances to allow the symmetry to be used for full reconstruction of the tropomyosin/troponin repeat along the filament. The muscle thin filament therefore represents a general case of a filamentous object in that it is not possible to exploit symmetry in a full analysis. Due to the nature of the imaging process in the electron microscope, only projections of the thin filament around its long axis are available without tilting the grid. Crucially, projection images around a single axis do not provide enough information to assign Euler angles ab initio using current methods. Tests with a model thin filament structure indicated that an out-of-plane tilt of approximately 20 degrees was needed for ab initio angular assignment of sufficient accuracy to calculate a 3D structure to a resolution of approximately 25 A. If no out-of-plane views are available, an alternative approach is to use a prior 3D model as a reference for the initial angle assignment. Tests with the thin filament model indicated that reasonably accurate angular assignment can be made using a reference containing actin, but lacking the regulatory proteins tropomyosin and troponin. We also found that an adaptation of the exact filtered back projection method is required to allow the correct weighting of projection images in which the particle has a very large axial ratio. This adaptation resulted in significant improvements in the reconstruction.

Actin Cytoskeleton↗

PubMed2003

Transmission electron microscopy of weakly scattering objects described by operator algebra.

An operator algebra description of Fourier optics is used to examine the imaging properties of transmission electron microscopy when applied to the study of weak specimens. Effects due to the curvature of the incident beam, the finite extent of the source, beam tilt, and objective aperture shift are examined. An expression for the contrast transfer function is derived that can account for either beam tilt in conjunction with a centered aperture or a shifted aperture in conjunction with an aligned beam. It shows that high phase contrast over a broad spatial-frequency range can be achieved by laterally shifting the objective aperture rather than defocusing the specimen, as is normally done.

Journal Article↗

PubMedDate not supplied

A two step approach for semi-automated particle selection from low contrast cryo-electron micrographs.

Over recent years advances in cryo-electron microscopy for the study of macromolecular structure have resulted in resolutions in the range 10-15 A becoming routine. With this drive for increased resolution comes the need to collect larger datasets, commonly >10,000 particle images. Manual selection of particles from micrographs is often difficult and with such large numbers of particles now involved it is also laborious and a common bottleneck. Automated methods do exist but are normally restricted to specific samples or data, i.e., spherical particles, no aggregation, high contrast, and low noise. A two step approach has been developed that remains general and can be applied to low contrast, high noise micrographs of small molecules. Specifically, application of the approach is presented using micrographs of Escherichia coli RNA polymerase, which due to low contrast and the relatively small size of the molecule prove difficult to pick manually. To test the automated approach, independent reconstructions of RNA polymerase were carried out using manual and automatically picked data. The two reconstructions are shown to be comparable and the reconstruction from the automatically picked dataset is at a higher resolution, due to an increase in the number of particles picked.

Algorithms↗