Keywords: Selenocysteine synthase tRNA sec Stoichiometry a b s t r a c t In bacteria selenocystey... more Keywords: Selenocysteine synthase tRNA sec Stoichiometry a b s t r a c t In bacteria selenocysteyl-tRNA sec (SelC) is synthesized by selenocysteine synthase (SelA). Here we show by fluorescence anisotropy binding assays and electron microscopical symmetry analysis that the SelA-tRNA sec binding stoichiometry is of one tRNA sec molecule per SelA monomer (1:1) rather than the 1:2 value proposed previously. Negative stain transmission electron microscopy revealed a D5 pointgroup symmetry for the SelA-tRNA sec assembly both with and without tRNA sec bound. Furthermore, SelA can associate forming a supramolecular complex of stacked decamer rings, which does not occur in the presence of tRNA sec . We discuss the structure-function relationships of these assemblies and their regulatory role in bacterial selenocysteyl-tRNA sec synthesis.
The delivery of a specific amino acid to the translating ribosome is fundamental to protein synth... more The delivery of a specific amino acid to the translating ribosome is fundamental to protein synthesis. The binding of aminoacyl-transfer RNA to the ribosome is catalysed by the elongation factor Tu (EF-Tu). The elongation factor, the aminoacyl-tRNA and GTP form a stable 'ternary' complex that binds to the ribosome. We have used electron cryomicroscopy and angular reconstitution to visualize directly the kirromycin-stalled ternary complex in the A site of the 70S ribosome of Escherichia coli. Electron cryomicroscopy had previously given detailed ribosomal structures at 25 and 23 A resolution, and was used to determine the position of tRNAs on the ribosome. In particular, the structures of pre-translocational (tRNAs in A and P sites) and post-translocational ribosomes (P and E sites occupied) were both visualized at a resolution of approximately 20 A. Our three-dimensional reconstruction at 18 A resolution shows the ternary complex spanning the inter-subunit space with the acc...
... Brough, Kevin Buzzard, Jeff Cash, Rodney Coleman, Simon Colton, Dan Crisan, Darren Crowdy, Ma... more ... Brough, Kevin Buzzard, Jeff Cash, Rodney Coleman, Simon Colton, Dan Crisan, Darren Crowdy, Margaret Cunningham, Naranker Du-lay, Abbas Edalat, Susan Eisenbach, Tony Field, Duncan Gillies, Chris Hankin, Ian Hodkinson, Michael Huth, Sasha Ivanov, Gordon James ...
Multivariate statistical analysis and classification techniques are powerful tools in sorting noi... more Multivariate statistical analysis and classification techniques are powerful tools in sorting noisy electron micrographs of single particles according to their principal features, enabling one to form average images with an enhanced signal-tonoise ratio and a better reproducible resolution. We apply this methodology here to determining the characteristic views of the large (50S) ribosomal subunits from the eubacterium Escherichia coli and the archaebacteria Methanococcus vannielii, Sulfolobus solfataricus, and Halobacterium marismortuL Average images were obtained of the subunit in the common crown and kidney projections, but views of the particle in orientations intermediate between these two extremes were also elucidated for all species. These averages show reproducible detail of up to 2.0 nm resolution, thus enabling the visualization and interspecies comparison of many structural features as a first step toward comparing the actual three-dimensional structures. Our results disprove evolutionary lineages recently postulated on the basis of electron microscopical images of ribosomal subunits.
The full exploitation of single-molecule spectroscopy in disordered systems is often hampered by ... more The full exploitation of single-molecule spectroscopy in disordered systems is often hampered by spectral diffusion processes of the optical transitions due to structural fluctuations in the local environment of the probe molecule which leads to temporal averaging of the signal. Multivariate statistical pattern recognition techniques, originally developed for single-molecule cryoelectron microscopy, allow us to retrieve detailed information from optical single-molecule spectra. As an example, we present the phonon side band of the B800 excitations of the light-harvesting 2 (LH2) complex from Rhodospirillum molischianum, revealing the electron-phonon coupling strength for these transitions. The measured Debye-Waller factors, ranging from 0.4 to 0.9, fall in the regime of weak electron-phonon coupling.
We have investigated the spectral diffusion and the electron-phonon coupling of B800 bacteriochlo... more We have investigated the spectral diffusion and the electron-phonon coupling of B800 bacteriochlorophyll a molecules in the peripheral light-harvesting complex LH2 for three different species of purple bacteria, Rhodobacter sphaeroides, Rhodospirillum molischianum, and Rhodopseudomonas acidophila. We come to the conclusion that B800 binding pockets for Rhodobacter sphaeroides and Rhodopseudomonas acidophila are rather similar with respect to the polarity of the protein environment but that the packaging of the ab-polypeptides seems to be less tight in Rb. sphaeroides with respect to the other two species.
Angular reconstitution in combination with multivariate statistical techniques to classify and av... more Angular reconstitution in combination with multivariate statistical techniques to classify and average the characteristic views of a molecule form a complete, selfcontained methodology for the high-resolution threedimensional (3D) structure analysis of uncrystallized macromolecules by electron microscopy. The angular reconstitution approach is based on the fact that two different two-dimensional (2D) projections of a 3D object always have a one-dimensional (1D) line projection in common. From the angles between such common-line projections, the relative Euler angle orientations of projections can be determined. Our single-particle electron microscopical approach has already yielded structures to resolution levels of ~10Å and no theoretical resolution limits are yet in sight.
Large datasets are emerging in many fields of image processing including: electron microscopy, li... more Large datasets are emerging in many fields of image processing including: electron microscopy, light microscopy, medical X-ray imaging, astronomy, etc. Novel computer-controlled instrumentation facilitates the collection of very large datasets containing thousands of individual digital images. In single-particle cryogenic electron microscopy ("cryo-EM"), for example, large datasets are required for achieving quasi-atomic resolution structures of biological complexes. Based on the collected data alone, large datasets allow us to precisely determine the statistical properties of the imaging sensor on a pixel-by-pixel basis, independent of any "a priori" normalization routinely applied to the raw image data during collection ("flat field correction"). Our straightforward "a posteriori" correction yields clean linear images as can be verified by Fourier Ring Correlation (FRC), illustrating the statistical independence of the corrected images over ...
Here we describe the first 3D structure of the photosystem II (PSII) supercomplex of higher plant... more Here we describe the first 3D structure of the photosystem II (PSII) supercomplex of higher plants, constructed by single particle analysis of images obtained by cryoelectron microscopy. This large multisubunit membrane protein complex functions to absorb light energy and catalyze the oxidation of water and reduction of plastoquinone. The resolution of the 3D structure is 24 A and emphasizes the dimeric nature of the supercomplex. The extrinsic proteins of the oxygen-evolving complex (OEC) are readily observed as a tetrameric cluster bound to the lumenal surface. By considering higher resolution data, obtained from electron crystallography, it has been possible to relate the binding sites of the OEC proteins with the underlying intrinsic membrane subunits of the photochemical reaction center core. The model suggests that the 33 kDa OEC protein is located towards the CP47/D2 side of the reaction center but is also positioned over the C-terminal helices of the D1 protein including its...
Biology is a challenging and complicated mess. Understanding this challenging complexity is the r... more Biology is a challenging and complicated mess. Understanding this challenging complexity is the realm of the biological sciences: trying to make sense of the massive, messy data in terms of discovering patterns and uncovering underlying general rules. Among the most powerful mathematical tools for organising and helping to structure complex, heterogeneous and noisy data are the tools provided by the family of multivariate statistical analysis (MSA) approaches. In electron microscopy (EM), MSA was first introduced to in 1980, to help sort out different views of macromolecules in a micrograph. After almost 30 years of continuous use and developments, new MSA applications are still being proposed regularly. The speed of general computing has increased dramatically in the three decades since the first use of MSA in electron microscopy. However, we have seen an even more rapid increase in the size and the complexity of the EM data sets we wish to analyse. Therefore, the speed of the MSA computations has become a very serious bottleneck limiting its use. The recent parallelisation of MSA programs open whole new possibilities by making the process to run orders of magnitudes faster exploiting the capacity of hundreds of CPUs simultaneously. The purpose of this paper is to not only intuitively explain the basic principles of multivariate statistical eigenvector-eigenvalue data compression to the novice in the field, but also to provide the more experienced researcher in structural biology with the formulas associated with the various MSA approaches.
Keywords: Selenocysteine synthase tRNA sec Stoichiometry a b s t r a c t In bacteria selenocystey... more Keywords: Selenocysteine synthase tRNA sec Stoichiometry a b s t r a c t In bacteria selenocysteyl-tRNA sec (SelC) is synthesized by selenocysteine synthase (SelA). Here we show by fluorescence anisotropy binding assays and electron microscopical symmetry analysis that the SelA-tRNA sec binding stoichiometry is of one tRNA sec molecule per SelA monomer (1:1) rather than the 1:2 value proposed previously. Negative stain transmission electron microscopy revealed a D5 pointgroup symmetry for the SelA-tRNA sec assembly both with and without tRNA sec bound. Furthermore, SelA can associate forming a supramolecular complex of stacked decamer rings, which does not occur in the presence of tRNA sec . We discuss the structure-function relationships of these assemblies and their regulatory role in bacterial selenocysteyl-tRNA sec synthesis.
The delivery of a specific amino acid to the translating ribosome is fundamental to protein synth... more The delivery of a specific amino acid to the translating ribosome is fundamental to protein synthesis. The binding of aminoacyl-transfer RNA to the ribosome is catalysed by the elongation factor Tu (EF-Tu). The elongation factor, the aminoacyl-tRNA and GTP form a stable 'ternary' complex that binds to the ribosome. We have used electron cryomicroscopy and angular reconstitution to visualize directly the kirromycin-stalled ternary complex in the A site of the 70S ribosome of Escherichia coli. Electron cryomicroscopy had previously given detailed ribosomal structures at 25 and 23 A resolution, and was used to determine the position of tRNAs on the ribosome. In particular, the structures of pre-translocational (tRNAs in A and P sites) and post-translocational ribosomes (P and E sites occupied) were both visualized at a resolution of approximately 20 A. Our three-dimensional reconstruction at 18 A resolution shows the ternary complex spanning the inter-subunit space with the acc...
... Brough, Kevin Buzzard, Jeff Cash, Rodney Coleman, Simon Colton, Dan Crisan, Darren Crowdy, Ma... more ... Brough, Kevin Buzzard, Jeff Cash, Rodney Coleman, Simon Colton, Dan Crisan, Darren Crowdy, Margaret Cunningham, Naranker Du-lay, Abbas Edalat, Susan Eisenbach, Tony Field, Duncan Gillies, Chris Hankin, Ian Hodkinson, Michael Huth, Sasha Ivanov, Gordon James ...
Multivariate statistical analysis and classification techniques are powerful tools in sorting noi... more Multivariate statistical analysis and classification techniques are powerful tools in sorting noisy electron micrographs of single particles according to their principal features, enabling one to form average images with an enhanced signal-tonoise ratio and a better reproducible resolution. We apply this methodology here to determining the characteristic views of the large (50S) ribosomal subunits from the eubacterium Escherichia coli and the archaebacteria Methanococcus vannielii, Sulfolobus solfataricus, and Halobacterium marismortuL Average images were obtained of the subunit in the common crown and kidney projections, but views of the particle in orientations intermediate between these two extremes were also elucidated for all species. These averages show reproducible detail of up to 2.0 nm resolution, thus enabling the visualization and interspecies comparison of many structural features as a first step toward comparing the actual three-dimensional structures. Our results disprove evolutionary lineages recently postulated on the basis of electron microscopical images of ribosomal subunits.
The full exploitation of single-molecule spectroscopy in disordered systems is often hampered by ... more The full exploitation of single-molecule spectroscopy in disordered systems is often hampered by spectral diffusion processes of the optical transitions due to structural fluctuations in the local environment of the probe molecule which leads to temporal averaging of the signal. Multivariate statistical pattern recognition techniques, originally developed for single-molecule cryoelectron microscopy, allow us to retrieve detailed information from optical single-molecule spectra. As an example, we present the phonon side band of the B800 excitations of the light-harvesting 2 (LH2) complex from Rhodospirillum molischianum, revealing the electron-phonon coupling strength for these transitions. The measured Debye-Waller factors, ranging from 0.4 to 0.9, fall in the regime of weak electron-phonon coupling.
We have investigated the spectral diffusion and the electron-phonon coupling of B800 bacteriochlo... more We have investigated the spectral diffusion and the electron-phonon coupling of B800 bacteriochlorophyll a molecules in the peripheral light-harvesting complex LH2 for three different species of purple bacteria, Rhodobacter sphaeroides, Rhodospirillum molischianum, and Rhodopseudomonas acidophila. We come to the conclusion that B800 binding pockets for Rhodobacter sphaeroides and Rhodopseudomonas acidophila are rather similar with respect to the polarity of the protein environment but that the packaging of the ab-polypeptides seems to be less tight in Rb. sphaeroides with respect to the other two species.
Angular reconstitution in combination with multivariate statistical techniques to classify and av... more Angular reconstitution in combination with multivariate statistical techniques to classify and average the characteristic views of a molecule form a complete, selfcontained methodology for the high-resolution threedimensional (3D) structure analysis of uncrystallized macromolecules by electron microscopy. The angular reconstitution approach is based on the fact that two different two-dimensional (2D) projections of a 3D object always have a one-dimensional (1D) line projection in common. From the angles between such common-line projections, the relative Euler angle orientations of projections can be determined. Our single-particle electron microscopical approach has already yielded structures to resolution levels of ~10Å and no theoretical resolution limits are yet in sight.
Large datasets are emerging in many fields of image processing including: electron microscopy, li... more Large datasets are emerging in many fields of image processing including: electron microscopy, light microscopy, medical X-ray imaging, astronomy, etc. Novel computer-controlled instrumentation facilitates the collection of very large datasets containing thousands of individual digital images. In single-particle cryogenic electron microscopy ("cryo-EM"), for example, large datasets are required for achieving quasi-atomic resolution structures of biological complexes. Based on the collected data alone, large datasets allow us to precisely determine the statistical properties of the imaging sensor on a pixel-by-pixel basis, independent of any "a priori" normalization routinely applied to the raw image data during collection ("flat field correction"). Our straightforward "a posteriori" correction yields clean linear images as can be verified by Fourier Ring Correlation (FRC), illustrating the statistical independence of the corrected images over ...
Here we describe the first 3D structure of the photosystem II (PSII) supercomplex of higher plant... more Here we describe the first 3D structure of the photosystem II (PSII) supercomplex of higher plants, constructed by single particle analysis of images obtained by cryoelectron microscopy. This large multisubunit membrane protein complex functions to absorb light energy and catalyze the oxidation of water and reduction of plastoquinone. The resolution of the 3D structure is 24 A and emphasizes the dimeric nature of the supercomplex. The extrinsic proteins of the oxygen-evolving complex (OEC) are readily observed as a tetrameric cluster bound to the lumenal surface. By considering higher resolution data, obtained from electron crystallography, it has been possible to relate the binding sites of the OEC proteins with the underlying intrinsic membrane subunits of the photochemical reaction center core. The model suggests that the 33 kDa OEC protein is located towards the CP47/D2 side of the reaction center but is also positioned over the C-terminal helices of the D1 protein including its...
Biology is a challenging and complicated mess. Understanding this challenging complexity is the r... more Biology is a challenging and complicated mess. Understanding this challenging complexity is the realm of the biological sciences: trying to make sense of the massive, messy data in terms of discovering patterns and uncovering underlying general rules. Among the most powerful mathematical tools for organising and helping to structure complex, heterogeneous and noisy data are the tools provided by the family of multivariate statistical analysis (MSA) approaches. In electron microscopy (EM), MSA was first introduced to in 1980, to help sort out different views of macromolecules in a micrograph. After almost 30 years of continuous use and developments, new MSA applications are still being proposed regularly. The speed of general computing has increased dramatically in the three decades since the first use of MSA in electron microscopy. However, we have seen an even more rapid increase in the size and the complexity of the EM data sets we wish to analyse. Therefore, the speed of the MSA computations has become a very serious bottleneck limiting its use. The recent parallelisation of MSA programs open whole new possibilities by making the process to run orders of magnitudes faster exploiting the capacity of hundreds of CPUs simultaneously. The purpose of this paper is to not only intuitively explain the basic principles of multivariate statistical eigenvector-eigenvalue data compression to the novice in the field, but also to provide the more experienced researcher in structural biology with the formulas associated with the various MSA approaches.
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