1 Structure of the Lassa virus nucleoprotein revealed by X-ray crystallography, small-angle X-ray scattering, and electron microscopy. Brunotte et al. 2011

2 Lassa virus (LASV) The virus causes hemorrhagic Lassa fever (West Africa) Two negative-strand RNA segments (L and S) containing four genes: The L segment encodes small matrix Z protein and a 220 kDa L protein. L protein: harbors the viral RNA-dependent RNA polymerase (required for viral mRNA synthesis) The S segment encodes the glycoprotein precurser and the nucleoprotein (NP): NP: major component of the viral ribonucleoprotein complex (Associates with itself) and acts as a interferon antagonist. Full-lenght NP and its C-terminal domain: Nucleotide binding site in the N terminus 3’-5’-exonuclease in the C terminus NP and L protein: minimum components for replication and transcription. Results: crystal structure of full-length NP of LASV (expressed in Sf9 cells) complemented by SAXS and EM studies

3 X-ray crystallizationSitting-drop technique with reservior (7% PEG300, 10% glycerol, and 0.5 M KCl). Conditions for x-ray crystallization: resolution of 2.45 Å (-173 °C). Molecular replacement to solve the phase problem

4 Small angle X-ray scattering (SAXS)Method: small angle scattering techniques and you are able to determine size, shape and orientation of structures in a sample. Conditions: wavelength of 1.54 Å (20 °C)

5 Electron Microscopy (EM)Method: use a beam of electrons to create an image of the structure. reveal to see smaller objects and more details. Conditions: unknown temperature.

6 Results X-ray crystallization: Determined the structures of the NP monomers and arranged in asymmetric and symmetric trimers. Note: Residues 1-7, , , and could not be identified in the electron density. Table 1: resolution of 2.45 Å, completeness of 99.9%, I/sigma(I) of 23.8 (2.7), and Rmerge of 14.8%. EM: symmetric trimer identical to the data found in x-ray crystallization. SAXS: indication of trimer where the symmetric trimer is most likely in aqueous solution. Note: found mass of 160 ± 10 kDa (inconsistent with the estimated mass of 250 kDa)

7 Discussion Why use SAXS and EM when they got the same results?Why use different temperaturs?