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Л 0 ы И Q " Q И Ќ Ќ И 0 Q ш ` е 0 " И ' C с е е е $ $ Ќ Ќ $ а Ђ Ќ Ќ а Purification of Epsilon15 phage: A 1 liter culture of exponentially growing Salmonella anatum was infected by 6 plaque plugs of Epsilon15. Cultures were incubated at 37АC until lysis. After clearing, 5 ml of CHCl3 were added to lyse remaining cells. Cellular debris was removed by centrifugation and the phages were precipitated by 10% PEG8000, 500 mM NaCl and stirring at 4АC for 2 hours. The phage particles were pelleted and resuspended in 10 ml of 50 mM Tris pH 7.5, 25 mM NaCl, 10 mM MgCl2, and 0.1% TritonX-100. The phages were further purified by gradient centrifugation at 100K RCF for 105 minutes using a step gradient of 20% sucrose, CsCl d=1.4, and CsCl d=1.6. The phages that sedimented between the 1.4 and 1.6 density layers were harvested. The phages were dialyzed first in 50 mM Tris 7.5, 300 mM NaCl, and 10 mM MgCl2 and then against two changes of 10 mM Tris 7.5, 25 mM NaCl and 5 mM MgCl2. The final phage titer was ~1013 pfu/ml.
CryoEM imaging: 3 - 5 Мl o f s a m p l e w a s a p p l i e d o n Q u a n t i f o i l g r i d ( Q u a n t i f o i l M i c r o T o o l s G m b H , J e n a , G e r m a n y ) a n d r a p i d l y f r o z e n i n e t h a n e s l u s h a t l i q u i d n i t r o g e n t e m p e r a t u r e . T h e i m a g e s ( F i g . 1 a ) f o r t h e r e c o n s t r u c t i o n s w i t h o u t s y m m e t r y i m p o s i t i o n w e r e t a k e n o n a J E M 2 0 1 0 F electron microscope operated at 200kV accelerating voltage with a Gatan liquid nitrogen cryoholder. The images were taken at 55,200x magnification and recorded on a Gatan 4kx4k CCD camera using the JAMES imaging system ADDIN EN.CITE Booth2004202017Booth, C. R.Jiang, W.Baker, M. L.Zhou, Z. H.Ludtke, S. J.Chiu, W.Program in Structural and Computational Biology and Molecular Biophysics, Baylor College of Medicine, Houston, TX 77030, USA.A 9 Х single particle reconstruction from CCD captured images on a 200 kV electron cryomicroscopeJ Struct BiolJ Struct Biol116-271472Cryoelectron Microscopy/*methodsImaging, Three-Dimensional/*methodsReoviridae/chemistry/ultrastructureResearch Support, Non-U.S. Gov'tResearch Support, U.S. Gov't, P.H.S.Virion/*chemistry/*ultrastructure2004Aug15193640http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=151936401. The images (Supplementary Fig. 6a) for the icosahedral reconstruction were taken on a JEM3000SFF electron microscope operated at 300kV accelerating voltage and a specimen temperature at 4.2K. These images were recorded at 60,000x magnification on KODAK SO163 films, which were developed using standard procedure and scanned using Nikon SuperCoolScan 9000ED film scanner.
3-D icosahedral reconstruction: In both data sets, the individual phage particle images were initially selected using ethan program ADDIN EN.CITE Kivioja2000222217Kivioja, T.Ravantti, J.Verkhovsky, A.Ukkonen, E.Bamford, D.Department of Computer Science, University of Helsinki, 00014, Finland.Local average intensity-based method for identifying spherical particles in electron micrographsJ Struct BiolJ Struct Biol126-341312DNA, Viral/analysisFalse Positive ReactionsHepatitis B Virus, Duck/ultrastructureHerpesvirus 1, Human/ultrastructureImage Processing, Computer-Assisted/*methodsMicroscopy, Electron/*methodsParticle SizeReproducibility of ResultsResearch Support, Non-U.S. Gov'tSoftwareTectiviridae/genetics/ultrastructureViruses/genetics/*ultrastructure2000Aug11042083http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=110420832 in batch mode and were then manually screened using the graphic program boxer in EMAN package ADDIN EN.CITE Ludtke19994417Ludtke, S. J.Baldwin, P. R.Chiu, W.Verna and Marrs McLean Department of Biochemistry, National Center for Macromolecular Imaging, Houston, Texas 77030, USA.EMAN: semiautomated software for high-resolution single-particle reconstructionsJ Struct BiolJ Struct Biol82-971281AlgorithmsBluetongue virusCapsid/ultrastructure*Cryoelectron MicroscopyImage Processing, Computer-AssistedInternetModels, MolecularProgramming LanguagesProtein Structure, SecondaryResearch Support, Non-U.S. Gov'tResearch Support, U.S. Gov't, P.H.S.*Software1999Dec 110600563http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=106005633. The contrast transfer function parameters of the images were fitted using a new automated fitting program fitctf.py (Yang, C. and Jiang, W. et al., in preparation). The SAVR software package ADDIN EN.CITE Jiang20011117Jiang, W.Li, Z.Zhang, Z.Booth, C. R.Baker, M. L.Chiu, W.Program in Structural and Computational Biology and Molecular Biophysics, Baylor College of Medicine, Houston, Texas 77030, USA.Semi-automated icosahedral particle reconstruction at sub-nanometer resolutionJ Struct BiolJ Struct Biol214-251363AutomationCryoelectron Microscopy/methodsFourier AnalysisImage Processing, Computer-AssistedResearch Support, Non-U.S. Gov'tResearch Support, U.S. Gov't, Non-P.H.S.Research Support, U.S. Gov't, P.H.S.Sensitivity and SpecificitySoftwareViruses/*ultrastructure2001Dec12051901http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=120519014 was then used to process the particle images and reconstruct the 3-D maps assuming icosahedral symmetry. The 9.5 Х map with icosahedral symmetry imposition was generated using ~6,000 particle images from ~200 micrographs at a defocus range ~0.5-2 m№m .
3 - D n o n - i c o s a h e d r a l r e c o n s t r u c t i o n : T h e r e c o n s t r u c t i o n o f t h e 2 0 0 k V i m a g e s w a s i n i t i a l l y c a r r i e d o u t w i t h t h e p r o c e d u r e a s d e s c r i b e d a b o v e w i t h i c o s a h e d r a l s y m m e t r y . T h e n , t h e f o l l o w i n g p r o c e d u r e w a s a p p l i e d . A n i m a g e o f a s i n g l e p a r t i c l e w i t h a p r o n o u nced tail density was chosen to generate a cylindrically averaged 3D model. The tail from this model was computationally extracted and grafted on one of the five-fold vertices of an icosahedral reconstruction. The combined capsid/tail was used as the initial non-icosahedral model. The orientation of each particle image, which was determined initially with the icosahedral symmetry assumption, was assigned to be one of the 60 possible orientations by the projection matching between the non-icosahedral model projection and the particle image. This was performed using a new Python script symrelax.py implemented using EMAN library and parallelized using the EMAN runpar utility. The best determined orientations for all particles were used to reconstruct a 3-D map using EMAN program make3d with symmetry option c1.
Iteration of above steps was carried out until convergence using a new Python script asymrefine.py. Ten or more iterations were required to gradually break the cylindrical symmetry of the initial tail model and converge to 6-fold tail. Other non-icosahedral components (portal, hub, core, dsDNA terminus and genome) were resolved together with the tail. The 20 Х map without any symmetry imposition was generated using ~15,000 particle images from ~950 CCD frames at a defocus range of 1-4 m№m .
I n o r d e r t o e l i m i n a t e a n y p o s s i b l e b i a s f r o m t h e i n i t i a l m o d e l , a n a l t e r n a t i v e i n i t i a l m o d e l w a s g e n e r a t e d u s i n g o n l y t h e c a p s i d s h e l l a n d t h e c y l i n d r i c a l l y a v e r a g e d g e n o m e i n s t e a d o f t h e t a i l . T h i s a l t e r n a t i v e m o d e l r e s u l t e d i n a n i d e n t i c a l l y c o n v e r g ed 3-D structure. This agreement validated the correctness of the structure of the entire Epsilon15 phage.
The faithfulness of this map is substantiated by the excellent structural match of the capsomeres in the capsid with those seen in the 9.5 Х resolution map independently reconstructed from a separate data set using the well-established icosahedral reconstruction procedure (Supplementary Movie 2 and Supplementary Fig. 6b).
Note that the scripts (symrelax.py and asymrefine.py) are available upon request.
Structural analysis: Amira (http://www.amiravis.com) visualization software was used for most of the segmentation and graphics displays. Alignment and similarity comparison of the individual subunits of the shell protein were performed using the foldhunter program ADDIN EN.CITE Jiang20012217Jiang, W.Baker, M. L.Ludtke, S. J.Chiu, W.Program in Structural and Computational Biology and Molecular Biophysics, Baylor College of Medicine, TX 77030, USA.Bridging the information gap: computational tools for intermediate resolution structure interpretationJ Mol BiolJ Mol Biol1033-443085AlgorithmsComputational Biology/instrumentation/*methods*Computer SimulationDatabasesInternet*Models, MolecularMolecular WeightProtein FoldingProtein Structure, SecondaryProtein Structure, TertiaryProteins/*chemistry/metabolismResearch Support, Non-U.S. Gov'tResearch Support, U.S. Gov't, Non-P.H.S.Research Support, U.S. Gov't, P.H.S.*Software2001May 1811352589http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=113525895 . I d e n t i f i c a t i o n o f Б- h e l i c e s a n d В- s h e e t s w a s p e r f o r m e d u s i n g t h e A I R S p r o g r a m s , w h i c h p r o v i d e s a g r a p h i c i n t e r f a c e t o t h e h e l i x h u n t e r p r o g r a m A D D I N E N . C I T E <