Cover of the FEBS Journal, 2007, Vol 274(8)
Cover of Acta Crystallographica Section D, 2007, Vol 63(10)
Cover of the Journal of Molecular Biology, 2006, Vol 361(4)
Cyanide dihydratase (CynDpum)
Mycobacterium tuberculosis MshB
The fold of G. pallidus RAPc8 amidase: alpha-Helix and beta-sheet topology of G. pallidus RAPc8 amidase. beta-Sheets (labelled 1–14) are shown as purple arrows, while alpha-helices are shown as blue cylinders. The cyan cylinder in the topology diagram is the 3(10)-helix on which the active-site Cys166 resides. The secondary-structure elements are numbered in sequence from the N-terminus. The topology diagram was generated using the program TOPS (Westhead et al., 1999) and manually redrawn in TOPDRAW (Bond, 2003) for simplification. — Kimani et al., 2007
The fold of G. pallidus RAPc8 amidase: Stereoview of a cartoon representation of a G. pallidus RAPc8 amidase monomer. The bars indicate the locations of the interacting surfaces seen in the spiral-forming homologues. The green bar indicates the “A surface”, the red bar the “C surface” and the grey bar the “D surface”, following the nomenclature of Sewell et al. (2005). — See Kimani et al., 2007
G.pallidus RAPc8 amidase homohexameric structure: A cartoon representation of the biological complex viewed down the crystallographic threefold axis. The hexamer is composed of a trimer of dimers. The three twofold axes are perpendicular to the threefold axis, giving rise to a complex with D3 point-group symmetry having a projection that closely resembles that observed by negative-stain electron microscopy (Makhongela et al., 2007: insert) — See Kimani et al., 2007
G.pallidus RAPc8 amidase homohexameric structure: A cylindrical projection of the density of the hexamer, with the cylinder axis and the threefold axis aligned. The density of each monomer was projected separately, coloured and then combined to form the composite image. The monomers are labelled and coloured in a manner consistent with the previous image. The value of the projected density is higher in lighter areas. The conserved “A surfaces” link 1–2, 3–4 and 5–6. A second twofold interacting surface links 1–6, 2–3 and 4–5. — See Kimani et al., 2007
Electron-density maps around the active-site. A stereo surface rendering of the active-site pocket lying behind Trp138. The surface is coloured in CPK colours corresponding to the exposed surface atoms. The two magenta patches directly behind the wire rendition of Trp138r epresent the carboxyl O atoms of Glu142. The acyl intermediate formed after reaction with D-lactamide has been modelled and is also illustrated as a wire rendition. The location of the sp2 acyl carbon is indicated by the black arrow. The yellow surface behind the acyl carbon represents the sulfur of Cys166. The acyl oxygen is located in a pocket near Lys134. Glu59 is located directly behind the acyl oxygen and is inaccessible to solvent. It can be seen clearly that the L-actamide enantiomer would produce a clash between the hydroxyl and the carboxyl of Glu142. The image was drawn with UCSF Chimera (Pettersen et al., 2004). — See Kimani et al., 2007
Details of the interactions at the interfaces. A stereo diagram of the “A surface” interactions viewed perpendicular to the twofold axis. Helices from monomer 1 are coloured green, while those from monomer 2 are coloured blue. The figure shows the methionine packing in α6 and the connection between the interface and the active site through the hydrogen bond between Trp209 and Asn170, which stabilizes the 310-helix on which Cys166 is located. — See Kimani et al., 2007
G. sorghi nitrilase helical symmetries. By computing the amplitudes and phases of the power spectrum from a single cryo-filament, the correct indexing scheme can be determined. The amplitudes of the Fourier transform are indicated by intensity, the phases by hue. The scale-bar indicates the relative phase difference between the layer lines and represents a total of 2π radians. In the case of the uncertain layer-line: the helix arising from the front is in phase with that arising from the back and therefore the order of the layer-line is even. According to the three alternate indexing schemes in a previous figure, this layer-line is of order 3, 4 or 5 and since it is even, it must be of order 4. FFT calculated with 2D FFT/iFFT Adobe plugin (http://www.pages.drexel.edu/avc25/archive.htm#FFT) by Alex Chirokov. — See Woodward et al., 2008
Details of the interactions at the interfaces. Interactions of helices α7 and the N-terminal loops in monomers 2 (blue) and 3 (cyan) on the second twofold-related interface. This interface is stabilized mainly by electrostatic interactions, with two salt bridges between Asp265 and Arg2 of both subunits. — See Kimani et al., 2007
Power spectrum calculated from vertically aligned helical segments. Layer lines arise from the front surface of the filament only, therefore phase information usually used to eliminate the ambiguity associated with indexing helical patterns is unavailable. Layer lines occur at 1/74 å, 1/49 å and 1/37 å. Three indexing schemes are compatible with the data at the resolution available. Red spots indicate the resulting indexing scheme if the unknown layer-line is of order 5 (hence 13 dimers in 2 turns or 13:2), blue spots indicate the indexing scheme that results from the unknown layer-line being order 4 (hence 11:2) and greenspots indicate the resulting indexing scheme if the layer-line is of order 3 (hence 9:2). The pitch is 7nm. — See Woodward et al., 2008
G. sorghi nitrilase helical symmetries. By computing the amplitudes and phases of the power spectrum from a single cryo-filament, the correct indexing scheme can be determined. The amplitudes of the Fourier transform are indicated by intensity, the phases by hue. The scale-bar indicates the relative phase difference between the layer lines and represents a total of 2π radians. In the case of the uncertain layer-line: the helix arising from the front is in phase with that arising from the back and therefore the order of the layer-line is even. According to the three alternate indexing schemes in a previous figure, this layer-line is of order 3, 4 or 5 and since it is even, it must be of order 4. FFT calculated with 2D FFT/iFFT Adobe plugin (http://www.pages.drexel.edu/avc25/archive.htm#FFT) by Alex Chirokov. — See Woodward et al., 2008
G. sorghi nitrilase helical symmetries. The IHRSR algorithm was initiated at the three helical symmetries, predicted by indexing and converged on three stable reconstructions. All the reconstructions are plausible in the sense that the dimeric structure of the repeating unit can be visualised and the interactions appear reasonable based on our limited biochemical knowledge. — See Woodward et al., 2008
Electron-density maps around the active-site. A stereoview of the electron-density maps around the active-site residues Glu59, Lys134 and Cys166. The 2Fobs – Fcalc map contoured at 1.3 is shown in blue, while the positive Fobs – Fcalc difference map contoured at 3.0σ is shown in red. The positive difference electron density around the Sγ atom of Cys166 suggests oxidative modification, probably to a mixture of species including sulfinic acid. — See Kimani et al., 2007
The catalytic triad. Stereoview of the active site, showing the network of probable electrostatic interactions. The catalytic triad residues are shown in blue. — See Kimani et al., 2007
Details of the interactions at the interfaces. The details of the “A surface” interaction between helices α5 viewed parallel to the twofold axis. Arg176 and Glu173 form salt bridges linking the subunits. Trp209 and Asn170 contribute a hydrogen bond to the interface. — See Kimani et al., 2007
G.pallidus RAPc8 amidase homohexameric structure: Cartoon image of the second twofold interface. Interaction between monomer 2 (green) and monomer 3 (magenta) viewed down a twofold axis. Helices 7 and the N-terminal loops are labelled. The conserved dimer interface (“A surface”) involves helices 5 and 6, which are labelled — See Kimani et al., 2007
G.pallidus RAPc8 amidase homohexameric structure: A transparent surface representation of the hexamer viewed down the twofold axis linking monomers 1 and 2 in the G. pallidus RAPc8 amidase hexameric complex. The monomers 1 (red) and 2 (green) are also shown as cartoons which together with the surface clearly illustrate the interlock formed by the C-terminal sequences in the hexamer as well as the contact of the tails with the core structure — See Kimani et al., 2007
Filtered and contrast-enhanced cryo-micrograph of G. sorghi nitrilase. Asterisk indicates an end-on view of a short filament. Scale-bar indicates 50nm. — See Woodward et al., 2008
The catalytic triad. The arrangement of the established catalytic triad residues and Glu142 in G. pallidus RAPc8 amidase compared (Cohen, 1997) with homologous nitrilase-superfamily structures (PDB codes 1j31, 1f89, 1erz and 1ems). 1j31 is in yellow, 1ems in green, 1erz in cyan, 1f89 in magenta and G. pallidus RAPc8 amidase in blue. The catalytic triad residues (Glu59, Lys134, Cys166) and Glu142 have similar positions to those of the corresponding residues in the four non-amidase structures, indicating their role in driving catalysis in the superfamily. — See Kimani et al., 2007
Unprocessed micrograph of cryo-metal-shadowed G. sorghi nitrilase. The shadowing is at an elevation of 45°. The majority of the metal falls onto the front surface of the helix, allowing unambiguous determination of handedness (left-handed). Arrow indicates approximate shadowing direction. Scale-bar indicates 50nm. — See Woodward et al., 2008