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ABSTRACT Cyanobacteria are photosynthetic organisms responsible for ∼25% of organic carbon fixation on the Earth. These bacteria began to convert solar energy and carbon dioxide into

bioenergy and oxygen more than two billion years ago. Cyanophages, which infect these bacteria, have an important role in regulating the marine ecosystem by controlling cyanobacteria

community organization and mediating lateral gene transfer. Here we visualize the maturation process of cyanophage Syn5 inside its host cell, _Synechococcus_, using Zernike phase contrast

electron cryo-tomography (cryoET)1,2. This imaging modality yields dramatic enhancement of image contrast over conventional cryoET and thus facilitates the direct identification of

subcellular components, including thylakoid membranes, carboxysomes and polyribosomes, as well as phages, inside the congested cytosol of the infected cell. By correlating the structural

features and relative abundance of viral progeny within cells at different stages of infection, we identify distinct Syn5 assembly intermediates. Our results indicate that the procapsid

releases scaffolding proteins and expands its volume at an early stage of genome packaging. Later in the assembly process, we detected full particles with a tail either with or without an

additional horn. The morphogenetic pathway we describe here is highly conserved and was probably established long before that of double-stranded DNA viruses infecting more complex organisms.

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support SIMILAR CONTENT BEING VIEWED BY OTHERS CRYO-EM STRUCTURE OF CYANOPHAGE P-SCSP1U OFFERS INSIGHTS INTO DNA GATING AND EVOLUTION OF T7-LIKE VIRUSES Article Open access 13 October 2023

FREQUENCY OF MISPACKAGING OF _PROCHLOROCOCCUS_ DNA BY CYANOPHAGE Article Open access 14 September 2020 STRUCTURE OF A THYLAKOID-ANCHORED CONTRACTILE INJECTION SYSTEM IN MULTICELLULAR

CYANOBACTERIA Article Open access 14 February 2022 ACCESSION CODES ACCESSIONS ELECTRON MICROSCOPY DATA BANK * EMD-5742 * EMD-5743 * EMD-5744 * EMD-5745 * EMD-5746 DATA DEPOSITS The averaged

density maps of the procapsid, expanded capsid and the DNA-containing capsid have been deposited in the EBI under accession codes EMD-5742, EMD-5743, EMD-5744, EMD-5745 and EMD-5746,

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Chem._ 25, 1605–1612 (2004) Article  CAS  Google Scholar  Download references ACKNOWLEDGEMENTS This research was supported by grants from the Robert Welch Foundation (Q1242) and National

Institutes of Health (P41GM123832 to W.C.; AI0175208 and PN2EY016525 to W.C. and J.A.K.; GM080139 to S.J.L.; T15LM007093 through the Gulf Coast Consortia to W.D. and R.H.R.; T32GM007330

through the MSTP to R.H.R.). We thank J. G. Galaz-Montoya and R. N. Irobalieva for editing of the manuscript. AUTHOR INFORMATION Author notes * John Flanagan Present address: Present

address: FEI, 5350 Dawson Creek Drive, Hillsboro, Oregon 97124, USA., AUTHORS AND AFFILIATIONS * Verna and Marrs Mclean Department of Biochemistry and Molecular Biology, National Center for

Macromolecular Imaging, Baylor College of Medicine, Houston, 77030, Texas, USA Wei Dai, Caroline Fu, John Flanagan, Htet A. Khant, Xiangan Liu, Ryan H. Rochat, Steve J. Ludtke, Michael F.

Schmid & Wah Chiu * Department of Biology, Massachusetts Institute of Technology, Cambridge, 02139, Massachusetts, USA Desislava Raytcheva, Cameron Haase-Pettingell & Jonathan A.

King * Department of Biology, Northeastern University, Boston, 02115, Massachusetts, USA Desislava Raytcheva & Jacqueline Piret * Program in Structural and Computational Biology and

Molecular Biophysics, Baylor College of Medicine, Houston, 77030, Texas, USA Ryan H. Rochat, Steve J. Ludtke, Michael F. Schmid & Wah Chiu * National Institute for Physiological

Sciences, National Institutes of Natural Sciences, 5-1 Higashiyama, Myodaiji, Okazaki 444-8787, Japan , Kuniaki Nagayama Authors * Wei Dai View author publications You can also search for

this author inPubMed Google Scholar * Caroline Fu View author publications You can also search for this author inPubMed Google Scholar * Desislava Raytcheva View author publications You can

also search for this author inPubMed Google Scholar * John Flanagan View author publications You can also search for this author inPubMed Google Scholar * Htet A. Khant View author

publications You can also search for this author inPubMed Google Scholar * Xiangan Liu View author publications You can also search for this author inPubMed Google Scholar * Ryan H. Rochat

View author publications You can also search for this author inPubMed Google Scholar * Cameron Haase-Pettingell View author publications You can also search for this author inPubMed Google

Scholar * Jacqueline Piret View author publications You can also search for this author inPubMed Google Scholar * Steve J. Ludtke View author publications You can also search for this author

inPubMed Google Scholar * Kuniaki Nagayama View author publications You can also search for this author inPubMed Google Scholar * Michael F. Schmid View author publications You can also

search for this author inPubMed Google Scholar * Jonathan A. King View author publications You can also search for this author inPubMed Google Scholar * Wah Chiu View author publications You

can also search for this author inPubMed Google Scholar CONTRIBUTIONS W.D., D.R. and C.F. prepared the samples and conducted the infection experiments under the advice of C.H.-P. and J.P.

W.D. collected the image data and reconstructed the tomograms; C.F. and H.A.K. established the Zernike phase plate imaging conditions in the microscope; K.N. provided the phase plates for

imaging; R.H.R. performed the statistical analysis. W.D. and M.F.S. developed the imaging processing methods and solved the structures of the phage assembly intermediates; W.D. and X.L.

refined the structures; J.F. and S.J.L. developed the symmetry-search algorithm for subvolume alignment; W.D., M.F.S., J.A.K. and W.C. interpreted the structures and wrote the manuscript.

CORRESPONDING AUTHOR Correspondence to Wah Chiu. ETHICS DECLARATIONS COMPETING INTERESTS The authors declare no competing financial interests. EXTENDED DATA FIGURES AND TABLES EXTENDED DATA

FIGURE 1 ZPC IMPROVES CONTRAST OF CRYOET IMAGES AND REVEALS DETAILED STRUCTURAL FEATURES OF SYN5-INFECTED CELLS. A, A conventional EM image of a Syn5-infected WH8109 cell. B, A ZPC image of

the same cell as shown in A under the same imaging conditions. EXTENDED DATA FIGURE 2 ZPC-CRYOEM SINGLE-PARTICLE IMAGES OF BIOCHEMICALLY PURIFIED MATURE SYN5 PHAGE. The particles are shown

with the tail pointing down and the wavy horn pointing up. The tail fibres appear to have variable conformations. EXTENDED DATA FIGURE 3 GENERAL LINEAR MODELLING OF CELLULAR CARBOXYSOME

NUMBER WITH PROGRESSION OF INFECTION. The number of carboxysomes remains roughly constant as infection progresses, indicating that their variation does not correlate with progression of

infection. The solid line indicates linear regression; the dashed lines indicate the 95% confidence limits. SUPPLEMENTARY INFORMATION ZERNIKE PHASE CONTRAST TILT SERIES IMAGES OF A

SYN5-INFECTED WH8109 CELL AT AN INTERMEDIATE STAGE OF INFECTION Tilt series of a frozen, hydrated Syn5-infected WH8109 cell was collected manually with an electron energy of 200 kV under low

dose conditions on a 4kx4k CCD camera at 25,000× microscope magnification. The tilt angles ranged from -60° to 60° at 3° step increments. The accumulated electron exposure for the specimen

in this tilt series was 40-50 electrons/Å2. The sampling of the data was calibrated to be 4.52Å/pixel. (MOV 11476 kb) VOLUME RENDERING AND ANNOTATION OF THE TOMOGRAM SHOWN IN VIDEO S1 WITH

COLOUR REPRESENTATIONS AS IN FIG. 2C The tomogram was reconstructed using IMOD software. It is displayed both section-by-section and by volume rendering. Features are annotated by colours to

designate molecular components attached and inside the cells, including cell envelope, thylakoid membrane, carboxysome, P-granules, ribosomes, infecting phages and phage assembly

intermediates. (MOV 27885 kb) ZERNIKE PHASE CONTRAST TILT SERIES IMAGES OF A SYN5-INFECTED WH8109 CELL AT A LATE STAGE OF INFECTION WITH A RUPTURED CELL MEMBRANE Tilt series of frozen,

hydrated Syn5-infected WH8109 cell was collected manually with an electron energy of 200 kV under low dose conditions on a 4kx4k CCD camera at 25,000× microscope magnification. The tilt

angles ranged from -60° to 60° at 3° step increments. The accumulated electron exposure for the specimen in this tilt series was 40-50 electrons/Å2. The sampling of the data was calibrated

to be 4.52Å/pixel. (MOV 13083 kb) VOLUME RENDERING AND ANNOTATION OF THE TOMOGRAM SHOWN IN VIDEO S3 WITH COLOUR REPRESENTATIONS AS IN FIG. 1 The tomogram was reconstructed using IMOD

software. It is displayed both section-by-section and by volume rendering. Features are annotated by colours to designate molecular components attached and inside the cells, including cell

envelope, thylakoid membrane, carboxysome, P-granules, ribosomes, infecting phages and phage assembly intermediates. An increased number of DNA containing phage particles are visible, and

overall contrast is enhanced over that in video S2, because the cell is beginning to rupture at this late stage of infection. (MOV 28142 kb) POWERPOINT SLIDES POWERPOINT SLIDE FOR FIG. 1

POWERPOINT SLIDE FOR FIG. 2 POWERPOINT SLIDE FOR FIG. 3 POWERPOINT SLIDE FOR FIG. 4 RIGHTS AND PERMISSIONS Reprints and permissions ABOUT THIS ARTICLE CITE THIS ARTICLE Dai, W., Fu, C.,

Raytcheva, D. _et al._ Visualizing virus assembly intermediates inside marine cyanobacteria. _Nature_ 502, 707–710 (2013). https://doi.org/10.1038/nature12604 Download citation * Received:

31 May 2013 * Accepted: 27 August 2013 * Published: 09 October 2013 * Issue Date: 31 October 2013 * DOI: https://doi.org/10.1038/nature12604 SHARE THIS ARTICLE Anyone you share the following

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