Beamline BM14 Highlights 2014 beta

Highlight 1

BM14 counts 1117 structures when PDB reached 100000+ structures (on 14 May 2014)

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Highlight 2: Partnership with India

Since the beginning of year 2010, the BM14 facility provides the access to the MX groups from India.

Increase in PDB deposits: 100+ Structures per year since year 2010.

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Highlight 3: Use of Sulphur as a source of Anomalous Scattering

Abstract

Type IV pili are surface-exposed filaments and bacterial virulence factors, represented by the Tfpa and Tfpb types, which assemble via specific machineries. The Tfpb group is further divided into seven variants, linked to heterogeneity in the assembly machineries. Here we focus on PilO2Bp, a protein component of the Tfpb R64 thin pilus variant assembly machinery from the pathogen Burkholderia pseudomallei. PilO2Bp belongs to the PF06864 Pfam family, for which an improved definition is presented based on newly derived Hidden Markov Model (HMM) profiles. The 3D structure of the N-terminal domain of PilO2Bp (N-PilO2Bp), here reported, is the first structural representative of the PF06864 family. N-PilO2Bp presents an actin-like ATPase fold that is shown to be present in BfpC, a different variant assembly protein; the new HMM profiles classify BfpC as a PF06864 member. Our results provide structural insight into the PF06864 family and on the Type IV pili assembly machinery.

Successful S-SAD phasing often relies on the presence of additional/unexpected weak anomalous scattering species (a phosphate ion in this case) in addition to the protein Met/Cys sulphur atoms.

Crystallographic data collection statistics

The sulfur-SAD data sets were collected at 7keV (λ=1.7712Å) by exploiting the goniostat ĸ geometry (ĸ=0°, ĸ=35° and ĸ=70°), to limit systematic errors associated with X-ray absorption or radiation damage, and to achieve high multiplicity within the collected data.

The harmonic contamination from 21keV (for the 7keV set) was reduced by offsetting the second crystal of the beam.

The 1.55Å resolution data set was collected at energy of 12.7keV, and treated as a native dataset.

S-SAD phasing were successful when employing the merged ĸ=0° and ĸ=35° SSAD datasets, measured to 2.2Å resolution.

N-PilO2Bp 3D structure and intermolecular association

A. Overall fold of N-PilO2Bp composed of two α/β topology subdomains, each displaying a mixed β-sheet, separated by a (central) cleft. The bound phosphate ion is shown as spheres.

B. Topology diagram of N-PilO2Bp. This diagram was generated using PDBSum server (www.ebi.ac.uk/pdbsum/) [52].

C. Crystal packing of the phosphate-containing N-PilO2Bp structure, showing the three crystal packing dimers formed by alternative interactions between four symmetry-related monomers (green, blue, magenta and black). The three interfaces are highlighted by black, blue and red shading. The first ‘dimer’, is formed by the interaction between the green (or blue) and the magenta (or black) monomers and the light green (or light blue) phosphate. The second crystallographic dimer occurs between the magenta and black monomers. The third dimer is formed by the green and blue monomers.

D. Stereo view of the electron density map for the residues building the phosphate ion binding pocket. The phosphate ion is shown as sphere; the electron density is contoured at 1.5 sigma level.

Proposed configuration of BpN-PilO2 with respect to the IM

Citation

Lassaux P, Conchillo-Solé O, Manjasetty BA, Yero D, Perletti L, Belrhali H, Daura X, Gourlay LJ, Bolognesi M (2014)
Redefining the PF06864 Pfam Family Based on Burkholderia pseudomallei PilO2Bp S-SAD Crystal Structure.
PLoS ONE 9(4): e94981. doi:10.1371/journal.pone.0094981

molecule visualisation
Burkholderia pseudomallei N-PilO2 Crystal structure by S-SAD
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Crystallographic data collection statistics
molecule visualisation
N-PilO2Bp 3D structure and intermolecular association
molecule visualisation
N-PilO2Bp protein
Beamline BM14

BM14 was bending magnet 14 beamline at the ESRF, optimised for exploiting anomalous scattering methods MAD/SAD in macromolecular structure determination.