Structural Studies On A Freshwater Cyanophage Myoviridae A-1(L) And DNA Packaging Motor Of A Freshwater Siphocyanophage Mic1

(Ⅰ)Structure and assembly mechanism of a freshwater cyanophage Myoviridae A-1(L)Cyanophages are bacteriophages that specifically infect cyanobacteria,which are distributed widely in aquatic environments.According to the tail morphology,cyanophages are classified into three families:Podoviridae,Siphoviridae and Myoviridae.Cyanophages can infect and lysis cyanobacteria,and are involved in regulating the population density and distribution of host cyanobacteria,thus they are considered as potential and novel biological agents to control the algal bloom.To date,only the capsid structures of two marine podophages and one freshwater siphophage have been reported.However,the structural information of myophage still remains unknown.Here we reported the cryo-EM structures of the capsid,portal,and helical tail of myophage A-1(L),which infects model cyanobacterium Anabaena PCC.7120.Structural analysis showed that A-1(L)is the first near atom-resolution phage capsid with a triangulation number T of 9.The icosahedral capsid of A-1(L)is composed of 535 copies of the major capsid protein gp4,which are assembled into 60 hexamers and 11 pentamers.Due to the missing of the insertion domain in gp4 and the absence of the cement protein,A-1(L)adopts a novel strategy to stabilize the capsid different from other bacteriophages.The adjacent capsomers form a perfect tenon-and-mortise structure based on the precise shape and electrostatic complementarity.Moreover,a three-layered interface at the quais-3-fold symmetric axis,where three neighbouring capsomers meet further improve the stability of A-1(L)capsid.We solved the structures of portal protein of A-1(L)in the mature state(in situ)and immature state(in vitro).Structural comparison showed that the portal protein undergoes conformational changes during maturation:the overall structure becomes more compact,and the diameter of the central DNA channel becomes narrow,which prevents the leakage of packaged genomic DNA.Besides,we solved the structure of A-1(L)helical tail,which is the highest resolution structure of phage tail to date.It clearly displays the interaction patterns between the sheath proteins and tube proteins.Notably,the interface of the sheath proteins displays a β-sandwich structure owing to the a helix at the C-terminus,which further strengthens the stability of the helical tail.The high-resolution structures of A-1(L)enable us to better understand the assembly mechanism of myocyanophage,and provide structural basis for future modification and design of artificial cyanophages for the application in controlling cyanobacterial blooms.(Ⅱ)Structural study on the DNA packaging motor of a freshwater siphocyanophage Mic1Genome packaging is an indispensable process during the maturation of viruses.Tailed dsDNA bacteriophages usually use portal-terminase complex as the packaging machine to translocate DNA into the procapsid.Notably,terminase is composed of large subunit TerL and small subunit TerS.However,how these proteins(portal,TerL and TerS)assemble into an effective DNA packaging motor remains unknown.Mic1,isolated from the Lake Chaohu,is a freshwater siphocyanophage infecting Microcystis.It possesses an isometric capsid with a diameter of～90 nm and a flexible tail of-400 nm in length.Here we solve the cryo-EM structure of Micl portal,a major component of its DNA packaging motor.Besides,we have made a lot of attempts to obtain the portal-TerL complex.Altogether,these results could lay the foundation for further studies on the structure and assembly of DNA packaging machine.