Structure Club
Structure Club invites scientists to present one of their papers to a broad audience. Listeners will hear about cutting edge science from the scientists themselves.
Structure Club
Venigalla Rao
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Venigalla Rao discusses his recent paper, “In situ structures of the portal-neck-tail complex of bacteriophage T4 inform a viral genome positioning mechanism”
Welcome to Structure Club. I'm Scott Stagg. I'm a professor at Florida State University where I use CryoEM to determine the molecular mechanisms, meas mechanisms of brain remodeling.
SPEAKER_03And I am Mashwund Francis, an assistant professor at Florida State University. My lab focuses on the structural biology of viruses and how they interact with host cells. Together, Scott and I created Structure Club as a journal club podcast and YouTube channel where the papers are given by the authors themselves. Today's speaker is Dr. Menigala Rao. Welcome, Rao.
SPEAKER_00Thank you.
SPEAKER_03And he has been there ever since. He will be talking with us about his recent paper, In-C2 structures of the Portal Necktail Complex of Bacteriophages T4 in form, a vital genome positioning mechanism. The manuscript came out in the journal Nature Communications in February 2026. Verigala, take it away.
SPEAKER_00All right. First, I'd like to thank you both for inviting me to share our work with you today. The title of my presentation is structural mechanisms of forced packaging genome flow in bacteria FHT4. Papers actually published as two back-to-back papers in Nature Communications recently, as you mentioned. I'm going to focus on the mechanistic features informed by our cryoum structures. Most of these are asymmetric reconstructions. If you have any detailed questions on the reconstruction itself, please feel to free feel free to follow up with any mail after my presentation. Myself or my collaborator Andre Fauquin will be happy to respond to your questions. This research started at the beginning of the COVID pandemic about six years ago, as a collaboration between four laboratories. My lab at Catholic, Andre Foucaine at Purdue, Layson at Fudon, and Shanglin Fang and Sun at Sean University. I should also mention that my lab is part of the Bacteria Phage Medical Research Center I founded about five years ago. More information on the center's mission can be obtained from the website listed here, T4phageCures.com. Research in my lab is funded by NIAD, NIDA, and NSF. The overarching interest of my laboratory has been to elucidate the mechanisms of genome flow and their potential translation to biomedical applications. Genome flow is the movement of genetic material from one generation to the next. It's a fundamental process in biology. Everything else is built around it. In viruses, it involves movement of a nucleic acid genome into and out of a proteceous capsule. Viruses must recover their newly replicated genome into a protective capsid shell, referred to as packaging, and then safely reintroduce it into a new host to initiate infection. Bacteria of HT4 is our model. T4 is a virus that infects E. coli bacteria. It has a large 120 by 86 nanometer prolate capsid or head into which around 171 Kb double standard DNA genome is packaged. Shown here is a video clip of the virus based on the near atomic cryo EM structure of the entire T4 virion. It's a complex structure consisting of at least 31 different proteins and 2560 subunits. A large part of our research over more than 45 years has been to tease out the basic mechanisms of virus assembly and genome packaging while harnessing the basic knowledge through translational research to develop novel biomedical applications, including vaccines, gene therapy, cancer therapy, and stem cell therapies. This is an overview of T4 infection. It starts with a single virus infecting the bacterial cell. In about 25 minutes, the virus uses the bacterial cell as a manufacturing facility to assemble progeny viruses. Numerous processes, including genome replication, capsid assembly, genome packaging, tail attachment, and so on, must occur within this time frame. Around 100 progeny viruses burst open the cell and released into the environment that are now ready to infect healthy bacterial cells. The T4 virus is indeed an incredibly fast and efficient replication machine. This is an overview of T4 virus assembly. Starting with the assembly of a dodacameric portal on the inner membrane of E. coli, an icosahedral shell is assembled with a scaffolding inner core. The core is then removed or digested by a viral-coded protease that is also part of the core. The empty capsid is then released into the cytosol. A pantameric packaging motor then assembles at the unique portal vortex and initiates DNA packaging. During the packaging process, the dimensions of the head are increased by about 15% to accommodate all the viral genome by a process known as head expansion. Two non-essential decoration proteins, namely hawk, highly antigenic outer capsule protein, and SOC, small outer capsule protein, assemble on the surface of the capsule. After headful packaging, which encapsulates about 102% of the viral genome, packaging is terminated with the ejection of the motor. A pre-assembled neck assembles followed by tail and tail fiber assembly to generate an infectious virion. While a lot of attention has been paid over the years to study the mechanisms of head assembly and genome packaging, very little mechanistic information is available on post-packaging processes. Today I will share with you our studies on the post-packaging mechanisms. These include, these studies led to the discovery of two new mechanisms, genome retention mechanism and genome positioning mechanisms.
SPEAKER_03Dr. Ra, uh may I ask you a question, like uh biological just to understand. So there's this 170 kilobase pairs of DNA is packaged inside this head, right? Correct. And uh are there any additional spaces? Uh inside, can it package even more or less?
SPEAKER_00Yeah, it can't. Um, in fact, um uh T4 is uh known as strictly headful size. Uh it packages until the head is completely full. Okay. Um and there is very little room to incorporate anything else other than what's already in there.
SPEAKER_03Okay, thank you.
SPEAKER_00Um so in fact, to follow up that here is the headful packaging, it involves encapsulation of around 56 micrometer-long double standard DNA to near crystalline density, which is about 500 to 550 milligrams per ml, uh, and by an ATP-driven DNA packaging motor. This creates an internal pressure of 25 to 35 atmospheric pressure, which is five to seven times the pressure in a champagne bottle. This pressurizer capsid must retain the genome in its entirety during the next steps involving neck and tail assembly to generate an infectious urium. Even leakage of a few base pairs of DNA would be detrimental and could lead to abortive assembly. The question then is: how does the phage manage this highly vulnerable process with precision? In our studies, we generated several long sought-after high-resolution structures of post-packaging assemblies. These led to a series of surprising insights. The first surprise was when we generated a focused in-si-to reconstruction of the phage portal and compared it with our previously published structure of the portal in the empty head or the recombinantly produced portal. We found that the portal in the Virion undergoes a global conformational change. Probably the phage has evolved a sensing mechanism linked to headful packaging when a threshold internal pressure is reached. During this conformational change, the Dodacer Beric portal changes its shape from a flying saucer to a mushroom-like form. Portal is pushed down by about 10 angstroms outside the shell. Importantly, the portal clip domain, which is outside the shell, remodels, causing the ejection of the packaging motor while new docking sites appear for binding the neck protein GP13. The pressurized head is then sealed and the next structure containing with the neck structure containing a closed genome gut. We discovered this when we co-expressed the two known neck proteins in E. coli, the adapter protein GP13 and the stopper protein GP14. The second surprise was when we generated cryoeymore construction of the purified neck complexes. These structures consisted of three stacked discs. In addition to the expected dodacameric GP13 and the hexameric GP14, the third hexameric disc of unknown origin is seen covering the bottom of the neck without or with the portal. Here is a structure with the portal and the same HF, the unknown protein is stuck at the bottom. While the C terminal arms of GP13 are inserted between the grooves of the portal domain, clip domains of the portal. The density of the unknown protein here was well resolved, and we could generate the structure to near atomic resolution. When we searched the database, the structure matched with the structure of the E. coli host protein HFQ. HFQ is an abundant E. coli protein originally discovered as a host factor for phage Q beta replication. It was later characterized as a pleiotropic regulator commonly found in most bacterial genomes. Although HFQ primarily binds to RNA, it can also interact with a wide range of nucleic acids, including DNA, and regulates transcription, genome stability, and mRNA decay in the host. Apparently, T4 hijacks this host protein to form a reinforced double genome gate that plugs the pressurized package head as shown here. The GP14 hexamer shown here forms what we call as primary gate with it with six stopper loops shown here projected into the lumen of the next channel. It forms a closed primary genome gate. This is the primary gate. Then there are six additional loops projected into the channel provided by the HFQ, narrowing the channel further to about 10 angstrom, forming a second uh genome gate. Yeah.
SPEAKER_03So all these domains they have uh amino acids that can bind, interact with the DNA?
SPEAKER_00Uh we we don't have the structure with the DNA at the moment. We have we in fact we tried very hard to get that structure, but all we got was a structure within without the DNA, and we have modeled the DNA into that structure. Okay. Um so this the this is by narrowing the neck channel with these genome gates, um, it stops the DNA from passing through the neck channel, thereby plugging the pressurized capsid and preventing any premature leakage of the genome from the packaged head. The third surprise was when we generated focused reconstructions of various sections of the infectious virion. Here we found a continuous stretch of cylindrical density that uh cylindrical density in the innermost core of the virion that extends from the portal crown right at the top all the way to the bottom until the base plate. The surprise was that the genome gate in the neck from the portal genome gate from the neck is no longer closed. Previously, it was believed that the seal formed by the neck assembly might be remained, retained intact in the virion and opens only at the time of infection. Contrary to this notion, we found that the gate is completely open in the virion and HFQ is no longer present as part of the virion structure. This means that the tail attachment caused dynamic conformational transitions in the neck structure, which is illustrated in the next slide. This is a cryoeum reconstruction of the portal neck connecting the head and the tail. At the top of the tail is the tail terminator shown here in blue, GP15. GP15 terminates the polymerization of the external gene uh sheath, tail sheath of GP18. Just below is another ring, GP13, shown in magenta, that caps the inner tube. It sets the length of the tube. This the below, yeah. So presumably the when the tail docks onto the neck, GP14 undergoes a dramatic conformational change. The stopper loops okay, uh please just a minute, please excuse me. Um yeah, so the stopper loops of the GP uh 14 that I talked to you before now rotate 90 degrees downward and interact with the GP15 ring, GP15 loops, where the C terminal region of GP14 forms a cluster of 30 salt bridges with GP15 locking in the neck with the tail. These movements eject the HFQ stopper, transforming the once closed neck into a fully open channel. That this is the uh closed one in vitro assembly intermediate, and it is in the virion structure shown here. These movements presumably eject the HFQ stopper, transforming the closed neck into a fully open channel that now lines up with the tails hollow tube. The DNA under pressure then can flow through this open neck channel down into the tail tube. The fourth and final surprise was when exam when we examined the innermost rod-like density in the Virion uh tunnel. And here the the this density is clearly resolved into two parts. At the top is the featureless DNA rod, and at the bottom is an extended tape measure protein or TMP coded by GP29, which we could model as a six-stranded helical tube. There are six copies of TMP, each containing long helices assembled as a coiled coiled tubular structure. It therefore forms a tube within the GP19 tail tube. This means that when uh TMP is actually a key component of the tail assembly, it acts as a ruler to set the tail length hence the name tape measure protein. So therefore, the tip of TMP is expected to be at the tip of the tail, as shown here by the arrow. However, um when I examined when we examine the density, the tip of the TMP moved to the bottom of the second disk of the tail tube. This means that when the DNA travels through the neck channel and reach the tip of the tail, its end is apparently captured by the tip of the TMP, and the DNA pressure probably pushed the DNA TMP complex further down into the tail tube, compressing the TMP's coil-coil segments like a spring. In fact, we collected three large data sets, each containing 30,000, 100,000, and 300,000 particles, and generated independent reconstructions. In each case, the DNA descended precisely to the same position into the tail tube, and the TMP is also precisely compressed to the same position. In total, the DNA has traveled around 17 nanometers from its previous position in the neck. Additionally, our structures show that the n-terminal end of TMP, which is at the top, interacts with the genomic DNA. Whereas the C-terminal tip is at the bottom of the tail bound to the base plate hub protein GP27. Sequence alignments show a cluster of conserved positively charged amino acids at the N-terminus of TMP. Shown here are six arginine and lysine residues in the N-terminus of TMP. Mutagenesis and genetic studies showed that these positive charges are important for function. Alanine scanning mutagenesis showed that although removal of one positive charge did not affect plaque formation, a double mutant, most of the triple mutants, and all of the quadruple mutants resulted in lethality. Taking together all of all our observations, these uh the structural uh observations, our studies on the post-packaging conformational transitions lead to an intricate and dynamic pressure-suspended genome positioning mechanism I have shown in this video. For clarity, I will pause the video at different steps in this in the sequence and again replay it at the end without pausing. First, after helpful packaging, a global conformational change in the portal ejects the packaging motor and remodels its clip domain to expose binding sites for the neck protein GP13. So here is the global conformational change remodeling the clip domain and that. Causes the ejection of the motor and opening the binding sites for the neck protein. Then a pre-assembled neck with its 12 C terminal arms extended on the surface docks on the portal clip domain. This is the pre-assembled neck containing GP13, GP14, and HFQ with this C terminal arms extended now, is docking on the portal, and the arms are inserted between the grooves of the clip domains of the of the portal. Then a major conformational change in the GP13 neck protein leads to extensive interactions of 13 with the portal and the carcassid, thus reinforcing head and neck connection. After insertion, there is a major conformational change that reinforces the neck neck portal connector structure. The packaged head is now sealed by the double genome gate formed by GP fourteen and HFQ hexamers. A dramatic informational change in GP fourteen here leads to opening of the primary genome gate while the HFQ gate is expelled. This is the pre-assembled tail and expelling of the HFQ because there are strong interactions between GP14 and GP15, and basically leading to internal conformational changes opening the genome gate. Genomic DNA then flows through the open neck channel and travels through the neck towards the tip of the tail where the N-terminal uh tip of TMP resides. The positively charged N terminus then captures the DNA. The DNA pressure compresses the TMP tube and the DNA TMP complex is pushed further down into the tail tube until it reaches the bottom of the second disk of the tail tube. The genomic DNA is thus positioned in a spring-loaded metastable state, poised for rapid and efficient delivery during infection. Then what appears to hold the twenty-five to thirty-five atmospheric DNA pressure in this state is the uh base plate hub at the very bottom, between the at the at the bottom of the tail, there is a hub structure and there is a puncturing device. This is where there is an interaction between P and the hub, and that seemed to hold the DNA pressure and ready to be um open up when uh the the host recognition occurs through the tail fibers and so on. So in conclusion, I would play the uh the one without pausing. Um we can actually uh go through the the process uh in a in in in sequence. This is the portal conformational change, ejection of the motor, and the pre-assemble uh neck very nicely, the arms extended on the top, docking between this uh the eclipse domains and that sealing the packaged head at this point, and the the pre-assembled tail structure is ready to now attach to the bottom of the neck through these strong interactions with GP14 that expels HFQ and the genome gate of GP14 opens. DNA now is free to go through, captured by TMP, and the pressure pushes down, and now it is actually spring-loaded in a metastable state in the in the central tunnel. So, in conclusion, our structural snapshots of post-packaging assemblies reveal that the phage genome is not merely stored in the virion particle, a series of dynamic conformational transitions are orchestrated to perfectly position the genome in the innermost compartment of the virion, such that its leading end is poised to flow smoothly and rapidly through the tunnel when a signal for genome delivery is received upon host recognition. Given such mechanistic precision, it is not surprising that bacteria FHT4 is known to be one of the most infectious viruses reported, with its infection efficiency reaching near 100%. And I think yeah.
SPEAKER_02So um in this model, uh does the TMP get pushed out as well? Does it go into the the bacteria?
SPEAKER_00Yeah. So um what the when the tail fibers recognize uh the receptor on the on the E. coli surface, which is the um uh this uh OMC receptor and so on, um the previous studies have shown the base plate actually have a major conformational change, opening the plug at the bottom. And the uh the the hub protein uh when it opens, the whole TMP actually travels. Uh the DNA is still attached to the anterminus of TMP. In fact, TMP now uh is used as a chaperone to uh basically pilot the genome through the tail channel uh towards the E. coli membrane, and then it is pushed out and remodels into a membrane pore, actually.
SPEAKER_02So it's like a needle with the with the DNA as a thread. So it's like the TMP is like really the thing that's penetrating through so that the more um flexible DNA can come along behind it.
SPEAKER_00Yeah, the TMP and the tip of the tail tube and the hub, these all form a complex pore that penetrates both the membranes, outer membrane. It actually forms a tube within the membrane so that DNA is already chaperoned to the very bottom of that pore and it actually flows through freely into the cytosol. DNA doesn't encounter any membrane as such because it forms a clear channel for the membrane to go through. And in fact, we have a paper, we actually submitted three papers, that was the third paper. Somehow the reviewers are not very happy, they wanted even more detailed structures because we didn't have a very high-resolution structure of the membrane pore, and the basically the the um reviewers and the editor rejected that paper, it's now on a review in another journal. So, actually, that's the idea to combine all these three papers together, but that paper didn't come through at the same time. But you're absolutely correct. Uh, these are amazing uh molecules, uh, these are structural proteins, measuring protein, and and stabilizing the DNA in this way, but then they all remodel into a membrane pore. I see.
SPEAKER_02That blows my mind, like so, like the whole thing blows my mind, but the uh but the the TMP forming a pore together with the other stuff, yeah. So it must remodel itself because probably now it's too narrow in the tail, so it has to expand in some way, right?
SPEAKER_00Yeah, uh everything is compressed into a remodeled pore, and the tail puncturing device you are seeing here, it has lysosome activity and all that. Yeah, it actually clears the somehow the membrane by creating a pore, by physically creating a hole in the membrane. These proteins just come out and remodel through interaction with the membrane proteins to form this you know nice uh long pore that that actually connects both the inner and outer membranes. Which makes so much sense because you have to, right?
SPEAKER_02Like some like you know, there's get getting through one is not enough. You've got to have some mechanism to shuttle the whole thing.
SPEAKER_00Oh wow. So there was actually in this context, there was old model, people have proposed that the DNA is released into the periplasmic space. But I think you recall that, yeah. That's not correct. In fact, it is it is really ejected straight into the cytosol, really beautiful way, uh forming a tube from all the way from the capsid into the into the cytosol. Wow.
SPEAKER_03Dr. Rav, so you know, I had this other question, you know, it's uh it's just so wonderful uh you know how much of insights we are getting into bacteria fat is where we learn so much. You know, so that you know, in the in the model in the video, it like it appears that you know the DNA is being pumped inside, and then it has to be cleaved at some point. Yes. What's the consequence of cleavage and where does that extra DNA go for the bacteriophage life cycle?
SPEAKER_00So so you are you referring to the after helpful genome is packaged, it has to be cut, right? Yeah, because it's a concatameric DNA. Uh concatameric DNA meaning it has no particular end, it's an endless DNA. It is produced as a very long polymer. So the motor itself actually cuts the DNA before it is ejected. Uh so the end is temporarily restrained by the portal while the neck is getting attached there.
SPEAKER_03I see. So, how does this extra piece of DNA be synthesized? Because now you're going to have a shorter genome packaged into the virion, right? In the second round of infection, right? So now this genome is going to go into a bacterial cell and it has to multiply and then it has to get back into another. And then where does this extra length come or is the genome shortened progressively as bacteria?
SPEAKER_00Yeah. No, what uh we don't have the complete story, but what we do know or what we believe uh is this two to three percent redundancy at the ends. Uh T4 is a strictly um uh what can I say, it it is known as circularly permitted uh terminally redundant phage, meaning it doesn't start at a specific sequence, it starts at a sequence and then packages 102%, 103% from that from that point, which is equal to roughly one headful. Uh the head structure presumably evolved along with the genome length, basically, to compact it. Uh now uh for to your question, it presumably circularizes using the terminal redundancy or use it to concatamarize it. So there is there will never be a loss or cutting off of the of the ends, basically. It's uh basically used for replication purposes. Very cool. Yeah.
SPEAKER_02So why don't we move on to the question um asking part of the of the of the um uh uh podcast? So um we have some questions, some interview questions that we prepared for you. Um and uh so we'll back we'll go back and forth, and Francis has the first question, then we'll take turns. So Francis, take it away.
SPEAKER_03So uh Karel, this was just fantastic, I should say. You know, hats off. It was beautiful structures, and you know, it's uh I'm still learning more, you know, from bacteria fagis. It's the first virus that people have been studying for a very, very long time, right? So I wanted to understand for the this particular work, uh these three papers what was the breakthrough moment that you thought that you know is going to be a story, like you know, so impactful?
SPEAKER_00Yeah, yeah, thank you for for for uh for your interest and comments. I really appreciate it. Um as I mentioned, there are these were surprises that we did not um previously. People thought there would be complexity there, didn't really uh we didn't really imagine this very dynamic, this intricate, precise movements uh that are quite fascinating to but uh to your question. So these these surprises really initially we you know we were you know struck by that. But one thing that really was a key surprise, or want to call it breakthrough moment, is when we found there is the innermost density at the very uh core of the vireon, all the way from the top to the bottom. And as we looked at it more closely, uh we we said, oh, the TMP seemed to be attached to the DNA, and it is no longer at the tip of the tail, it's actually moved down into the tube. That was an entirely unanticipated result, and we couldn't um believe that that is actually because it has never been reported that way. Um, that's the part of the reason we went and generated independent reconstructions, um, as well as the delivery intermediate, which is the third paper, where the that DNA TMP complex moved all the way to the bottom, and the TMP is actually expelled outside that kind of that uh interaction still retained. So that was a really a moment that we were really struck by that and and uh didn't know how to explain it at that moment, but nevertheless, that was quite uh unexpected and and uh um really got us very curious about what's going on here and kind of drove our thinking and analysis uh from that point and everything else kind of pieced together around it, actually, finding the HFQ genome gate opening and all of that stuff.
SPEAKER_03As is always the case.
SPEAKER_00As is always the case. Yeah.
SPEAKER_02Okay, so what I'm supposed to ask you is what is the future of this work? But what I'm gonna ask you is so I'm really interested in this uh like this T4 phage um therapeutics kind of thing that uh uh I see of sort of an interest of yours. And so here's what my question is is like, so does what you do your work that you did now, does that relate to these these bigger ideas about like you know using phages as therapy and you know, maybe designing them and stuff like that?
SPEAKER_00Yeah, absolutely, absolutely. Um, in fact, um uh the idea of phage therapy, uh even vaccines, came from some of these analyses. Um as we progressed in the structure, biochemistry, genetics of T4, the information that we are generating, or what nature is basically teaching us as we progressed in this exploration, um, LEN itself for developing these biomedical applications. Uh now we have reduced the whole packaging to a simple in vitro packaging assay where we can take the empty capsides, basically using mutations and all that. We can just produce the accumulate the empty capsides in E. coli cell. In a simple purification procedure, we can isolate them and remove the decoration proteins which are not essential. So we have just shells, just a capsid shell with nothing inside, nothing outside. Now we can put whatever we want inside, DNA inside, whatever we want to put proteins outside. And T4 packaging system is very promiscuous, it doesn't require any sequence to start, to end. You can package oligonucleotides, you can package plasmid DNAs, you can package pretty much any DNA you want as long as it's a linear molecule. And it really, like a vacuum machine, it stops this nanoparticle until it is completely full. So we can do that taking the empty shell, package therapeutic DNAs, and put surface molecules like genome editing molecules, targeting molecules to human cells. We can deliver these nanoparticles into now human cells and carry out these editing recombination and other things. Because it has a large capacity, we can now think about correcting uh gene genetic functions or multiple genetic functions and so forth. We published a paper about, we have been publishing a series of papers. Uh the last one was in 23, and we have one on bioarchives now on stem cell gene therapy using T4. Uh so we are in a stepwise manner um uh developing, progressing on this nanoparticle uh delivery into human primary cells, and that is now um uh uh in a way integrated with our basic research. Any new information. Now we have the NAC proteins, we can actually prepare the heads full of therapeutic DNA, we can store them potentially for years for therapeutic proteins. Yeah, very stable.
SPEAKER_02And the the structure, uh the structural work really informs like how you would engineer these things, right? Like yes, that's really the yeah, yeah, that's really correct.
SPEAKER_00And we can functionalize the capsid with there are two loops on the capsid, and and those are relatively flexible loops. Now we are functionalizing the capsid so that we can actually prepare shells with already some of these belts and whistles incorporated into the capsid itself, then we'll have even more room to actually engineer the capsid on the surface. Very cool.
SPEAKER_03Yeah, absolutely.
SPEAKER_02Okay, so uh stepping back even broader now. Okay, so um uh you know, in our careers, in our scientific careers, every so often there'll be some piece of data that like gets burned in your head because it's like it, you know, it's like this sort of GWS kind of Eureka kind of moment. And those are so precious. So do you have um do you have examples of that that you would share with us?
SPEAKER_00Yeah, yeah, but uh I'm so fortunate to have many such moments, so because I've been here for a long time. So you guys are very young and and and uh still a lot more. So that's my gray hair. But um but uh I I can share with you one particular instance and and kind of um like you said, the right phrase burnt into my uh etched into my memory. Um you probably know Michael Rossman uh from Purdue University, and we had I had wonderful collaboration with him for 20 years. Um after he passed away, now we are progressing in this way. All the labs that I mentioned to you, they were postdocs with Michael at the time. And I worked with them uh during that period, and now we have uh they have their independent. Labs and so on. So just going back in the initial stages, going back almost 25 years ago, Michael asked me whether I want to collaborate with him, and I was so elated when he asked me that. We never did structures at the time. We are basically molecular biology and all of that. We wanted to get the structure of the packaging motor, and nobody had it. And Michael has been trying for a while and didn't get it. And he wanted to try T4. And he got really excited because we constructed literally hundreds of mutants testing their biochemistry and all of that. He thought that would be really a gold mine to apply structural biology. And we have been, we sent Michael hundreds of milligrams, maybe grams of protein of different mutants and never crystallized. And we kept on changing this mutant this way or the changing the domain and all of those things. Nothing is working. And one thing Michael and I have in common is we never, never, ever give up. We just keep going until we succeed. So one fine more we changed one of the mutants, we changed, because it's a too dynamic molecule, and we didn't were not able to trap a confirmation. So we constructed a mutant by changing DE, which is aspartic and glutamic acid, Walker B and catalytic glutamate. We changed it to ED, DE to ED, aspartic, glutamic to glutamic, aspartic, uh, and studied the biochemistry, it doesn't work at all, but it binds ATP. So we sent that mutant, just the ATP as domain of it. And one fine morning, um Michael called and said, Ah, your protein crystallized. Uh this he has to immediately share that news with me. And uh not only that, we have an X-ray machine down in our in-house. We got some diffractions done. It has beautiful ATP binding site, Rossman Fold. Rossman was the first to uh get this nuclear fold structure in uh in in 71. So and and then a few days later we were supposed to meet in a conference in uh in Portugal for a virus assembly conference, and and we first he was the first, he shared with me the first picture uh of that of that structure, and I was I was okay, this is it. I can even if I don't get one more result, I'm happy with this because the ATP is right where we predicted from the genetic and biochemical studies, and every single amino acid we predicted, interacting with ATP, exactly mirrored in this structure. And we were sitting on the sofa, both were kind of jumping on the sofa, looking at this first structure of the packaging ATPS domain, and we were thrilled to see that all the years, five, six years of failures suddenly now blew up in this beautiful structure, and we really both of us really enjoyed that moment, actually. So I never forget that. I always tell that to graduate students, they probably listen from me maybe dozens of times, but I never get tired of uh describing that that picture, especially Michael. He was like, Oh, this is coming back in like almost 50, 40 years after he discovered this Rossman 4, he could see that in that packaging motor, he was just um you know elated by by that uh by that uh structure. Oh, that's great.
SPEAKER_03So so here I'm going to take you back even further and ask you when did you realize and how did you realize science was your thing? And you wanted to pursue this and you know, just keep keeps you going even till today.
SPEAKER_00Yes, so uh all I can say is going back in my memory, or I was really um always curious about unknown. I want to know about things, and I was fascinated by physics, uh atoms, and I never imagined there's something like an atom, and within the atom there are these protons, neutrons, and uh, electrons and all of that. Um, and I wanted to be a physicist, learning more about it and go more deeply. Uh, it so happened my father, when I went to college, I used to go to the library, read the books on physics and all of that. And I went to college, applied for sent my application to college. He actually changed, I put MPC in India, it is math, physics, and chemistry that is the major. He basically scratched it out and said, B is at C, which is botany, zoology, and chemistry. He wanted me to become a doctor, and I never wanted to become a doctor. So we I said, okay, you in India you never say no to your parents, you know, parents know better, right? I mean, so I became a biology student, but then the closest I could come to physics was chemistry. So I put all my effort studying chemistry and then biochemistry, and even now I have collaborators doing biophysics, single molecule biophysics, now the structural biology. Uh, in a way, my I'm glad that my father, at the time I was not happy, but I'm I'm glad that he changed it. This way I can actually learn biology and physics and really close the loop and and really understand more more deeply. And um yeah, that's that's the story behind it. That's a fun story. Thanks.
SPEAKER_02Okay, last question. So um uh I'm always interested to hear about uh people's inspiration. So, like, do you have a scientific hero? And if so, like who and why?
SPEAKER_00Uh yeah, so again, I have many people I can say scientific heroes, including uh Michael, Michael Rossman. He was just an amazing, not only a collaborator, friend, mentor, but he's a phenomenal scientist, um, and a legendary scientist, I should say. He, as you well know, he started along with Peritz and Kindrew the structural biology, X racography, and all of that. Uh, but uh I I should also say that uh Brown and Goldstein uh have been my uh quite a great inspiration for me. Um uh these are two doctors um at uh you know University of Texas Southwestern just had an observation while they were in NIH actually. Um they found they saw a girl really have sky-high cholesterol uh levels in her blood, and very perplexed, never have seen that kind of a uh cholesterol level in a young girl, seven-year girl like that. So they were really curious what happened here and studied that, took it to UC Sight University of Texas Southwestern, and together in a collaborative way, spent their entire lifetime, 50 plus years, really step-by-step, rigorous, focused, dedicated way to really chip away that problem, to determine the entire pathway of cholesterol metabolism, LDL receptor processing, biogenesis, ultimately leading to statin discoveries, which is helping hundreds of millions of people. And that really struck me as such an amazing collaborative story, scientific story, such a great rigor, biochemistry and all that. So when I went to Baltimore, um just quite by accident, I was an enzymology PhD from Institute of Science Bangalore, and quite an accident I got an offer from Lindsay Black to work on phage. Uh, he he just found an enzyme which he thought as a packaging enzyme. Since I have an enzymology background, he offered me the position. Although I didn't know anything about phages, I just took up the position because if it's an enzymology type of position uh research. And and uh soon uh after I realized that no, this is not a packaging enzyme, and there are these other proteins, motor proteins. At that time, we don't know they're motor proteins. Uh, genetical AGP617 is the key protein. So Ulindsey actually graciously allowed me to switch my project to that. We published a couple of papers on the enzyme, but then switched to this. Um, and um so um so that that's the point where you know I said, okay, I will work on the nothing is known about bacteriophage packaging at the time, uh no biochemistry, nothing. Uh we just have a packaging essay that Lindsay has optimized, and then I jumped into that. We both together optimized the essay to essay for packaging, that's all we have. And um, and that's where we started. And I really, over a period of time, I really fell in love with that uh problem because we are discovering things as we went along. And I said, as I said, Mike Brown and Goldstein story really inspired me. If you want to learn something deeply, you you really want to spend your lifetime uh on this problem working through it. And oftentimes people said, Why do you work on T4? Why do you work on phages? Um, when I came to Catholic, my grant proposals were rejected left and right from NIH, NSF. Many people outside the department and inside the department, they said, Why do you work on this? Nobody cares about phages and packaging. Work on maybe harpice virus or something, you know. And um but but that in that inspired me. I said, look, I just am interested in this. I already invested a lot of years, and I stuck to it, and I stuck to it, and the rest is basically as a result of that uh that that uh you know commitment to keep working on this. Well, however many failures there are, that's okay. That's part of what we do, but but just keep working on it, and that's inspiration I would say uniquely uh left uh uh in me, and that remains as the old motivation to keep working on T4.
SPEAKER_03Oh wow, Dr. Vidigala Rao, that was such an inspirational story, and I think a lot of young investigators who listen to this will be inspired to have that kind of dedication towards your research. With that, you know, I would like to thank you so much for joining us today. We really appreciate you taking your time to talk to us about your science and your journey uh to where you are today.
SPEAKER_00Thank you both, Ashwant and uh Scott. I greatly appreciate it. I'm grateful that you invited me to share our results, and I'm also grateful that you are able to um uh you know share that share this with other people as well, especially the ag researchers, because that's one of the main things in this phase. I would like to contribute to uh to that generation as well. Thank you.
SPEAKER_02Great. Well, thank you so much. And that does it for this episode. We hope you all join us again for the next episode of Structure Club.