Structural Study of the  12 Virus By:Elizabeth Brown

Electron microscopy in structural biology what is electron microscopy used for in structural biology? interactions of electrons with atoms: elastic & inelastic scattering, damage to biological samples what is the principle of a microscope? what is the maximum resolution of an electron microscope? what is contrast and how is it achieved? principles of structure determination by cryo-EM tomographic reconstruction We decided to use this virus because it attacks bacteria and can cause gastro enteritis

Interactions of electrons with atoms Elastic scattering Inelastic scattering No energy is deposited, wavelength electron unaffected Energy is deposited, inducing damage in the sample, wavelength electron increases

What is the scattering cross-section? p s = probability that an electron is scattered S = cross-section of atom Scattering cross-section = p s S

Electrons (200 keV) X-rays (1.5 Å) Inelastic / elastic scattering events 310 Energy deposited per inelastic event 20 eV8 keV Energy deposited relative to electrons per elastic event Scattered photons per scattered electron Current resolution6-8 Å< 1 Å From Henderson (1995) Quart. Rev. Biophys. 28, 171 Comparing scattering of electrons & X-rays

Principle of cryo-EM Use ‘holey’ carbon EM grids Apply sample on grid Blot away excess fluid Very quickly freeze sample, making sure that the water does not crystallize, but forms amorphous, vitreous ice Transfer sample to the microscope Ensure contrast by defocusing Take short exposures, otherwise damage the sample

Schematic of  12, (  6) P6 P9, P10, P13 phospholipids P5 P8 P1, P2 P4, P7 P12 * P3a P3c * non-structural protein PX NC Lipid Envelope

+30 nm AB C D A. Regular cryo- electron micrograph of the entire  12 virus particles in vitreous ice. The particles contain a nucleocapsid beneath the phopholipid envelope (black arrow). Two of the particles (white arrows) appear to be cores released from the virion particle. B, C, D. Tomographic reconstruction of  12 virions in vitreous ice. The tilt series consisted of 100 images taken at tilt angles between -70 and +70o with a total electron dose in the range of electrons/Å2. After tomographic reconstruction, the contrast was inverted and a low pass filter was applied to the 3D volume. B, C, and, D represent 7Å thick sections through this volume at levels indicated in the lower left corner. B. Ring like structures (black arrow) are seen in the +30 nm slice. In several instances they appear to be clustered (white arrow). Reconstruction analysis associates all of them with the virus particle surface. C. The white arrow in this 0 nm slice indicates a possible stem connection to the envelope. Two density areas (black arrow) are seen above the stem. D. In the -15 nm slice the two densities are seen to meld into one (black arrow) suggesting a ring-like structure. These structures are most likely the P3 receptor attachment complex. B-C different rotational slices. 3 of about 70

Acknowledgements Professor Paul Gottlieb Hui Wei Sasha Dr. Sat Harlem Children Society