Bacteriophage lambda stabilization by auxiliary protein gpD: timing, location, and mechanism of attachment determined by cryo-EM.

Production and Purification of Lambda Virions Wild type bacteriophage lambda with genome length 48.5 kbp was produced by thermal induction of lysogenic Escherichia coli strain AE1. The AE1 strain was modified to grow without LamB protein expressed on its surface in order to increase the yield of phage induced in the cell. The culture was then lysed by temperature induction. Phage was purified by CsCl equilibrium centrifugation. The sample was dialyzed from CsCl against TM buffer (10 mM MgSO 4 and 50 mM Tris-HCl/pH 7.4). The final titer was 10 13 virions/ml, determined by plaque assay. Phage purification details are described previously ( Evilevitch et al., 2003 ). The prohead particles appeared in a preparation of the mutant phage lambda b221cl26, which packages a 37.7 kbp genome. Purification details are described previously ( Grayson et al., 2006 ).

Electron Microscopy

Lambda phages were prepared for CryoEM analysis by preservation in vitreous ice over a holey carbon substrate via rapid-freeze plunging. The holey carbon films used were developed at NRAMM and are currently commercially available from Protochips Inc. under the name Cflats. They consist of a 400 mesh copper grids onto which a layer of pure carbon fenestrated by 2 µm holes spaced 4 µm apart was applied. Grids were cleaned prior to freezing using a plasma cleaner (Fischione Instruments, Inc.) using 75% argon and 25% oxygen for 25 seconds. A 3 µl aliquot of sample was applied to the grids, which were loaded into an FEI Vitrobot (FEI company) with settings at 4° C, 100% humidity, and a blot offset of −2. The grids were double-blotted for 7 seconds at a time and immediately plunged into liquid ethane. Grids were stored in liquid nitrogen until being loaded into the microscope for data collection.

Data Collection Mature Virion

Data were acquired using a Tecnai F20 Twin transmission electron microscope operating at 200keV at liquid nitrogen temperatures, and equipped with a Tietz F415 4k × 4k pixel CCD camera (15µm pixel) and a Gatan side entry cryostage. Six datasets were collected from six different grids at a nominal magnification of 80,000x with a pixel size of 1.01 Å. A total of 2471, 788, 2090, 770, 686, and 1939 images were recorded during the six sessions using the Leginon automated electron microscopy package ( Suloway et al., 2005 ) with a randomized defocus range from −0.8 µm to −2.5µm at a dose of 19 e − /Å 2 . Concurrent with data collection, preliminary image analysis was performed with the use of a new software package, Appion, which is currently under development at NRAMM. Automated particle picking ( Roseman, 2003 ) and automated CTF estimation ( Mallick et al., 2005 ) were performed on each image as it was collected. Procapsids Data were acquired on the same model of microscope running under the same conditions as above, but outfitted with a Gatan 4k × 4k CCD (Gatan Inc), at a nominal magnification of 50,000x with a pixel size of 2.26Å. A total of 2683 images were collected, which were processed in the same manner as the above. Single Particle Reconstruction Mature Virion Initial particle selection by automated methods was inspected manually with an Appion module, yielding 5565, 1932, 7811, 7550, 4653, and 14344 particles from each of the six data collection sessions. Particles were extracted from the images whose CTF estimation had a confidence of 80% or higher using a box size of 768 pixels on an edge. The phases of the images were corrected according to the CTF estimation during creation of the stack, and the particles centered using functions included in the EMAN software package ( Ludtke et al., 1999 ). The resulting 31422-particle stack was binned by a factor of two for reconstruction by EMAN, using an icosahedrally-averaged reconstruction of bacteriophage P22 ( Lander et al., 2006 ) low-pass filtered to 40 Å as the starting model. Particles iterated through 20 rounds of refinement, beginning at an angular increment of 5° and decreased by 1° at four iteration intervals. An additional four rounds of refinement were then performed at an angular increment of 0.5°, the last round of which provided the density reported. The amplitudes of the resulting refined structure were adjusted with the SPIDER software package ( Frank et al., 1996 ) to fit the density Fourier amplitudes to an experimental 1D low-angle X-ray scattering curve. Procapsids Processing was carried out in the same manner as in the mature virion, using 4640 particle images with a box size of 384 pixels. The EMAN function “starticos” was used to create an initial model. Particles were not manually inspected as in the mature virion reconstruction, nor were the last four iterations of refinement at an angular increment of 0.5° performed. Rigid-body docking of crystal structures into the cryoEM density and graphical representations were produced with the Chimera visualization software package ( Goddard et al., 2007 ). The fourteen residue N-terminal polypeptide of gpD was modeled as a poly-alanine chain into the EM density with the Coot crystallography package ( Emsley and Cowtan, 2004 ).

Supplementary Material 01