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Brian Kelch @briankelch.bsky.social · 07/07/2025

I’d also like to thank all coauthors for their work on this manuscript, including lab alumni Drs. Jacob Landeck and Xingchen Liu, grad student Krishna Anand, and high school intern Sasha Litvak; UMass Chan cryo-EM facility staff Drs. Song, Chang & Ouch; and you for reading! (16/16)

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Brian Kelch @briankelch.bsky.social · 07/07/2025

I’d also like to point out that Josh never picked up a pipette before joining my lab! His PhD is in Mech Eng and purely computational! Yet in a short time he mastered biochem such that he was developing badass assays and his grid prep skillz are copied by others around UMass. (15/16)

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Brian Kelch @briankelch.bsky.social · 07/07/2025

At a time when science funding is so precious, we want to acknowledge that our work is NIH NIGMS funded, @joshua.pajak.bsky.social is funded by an American Cancer Society postdoc fellowship, and our MD sims used resources provided by NSF @accessforci.bsky.social and UMass Chan SCI. (14/16)

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Brian Kelch @briankelch.bsky.social · 07/07/2025

RFC is conceptually analogous to a GTPase, which has dedicated Nucleotide Exchange Factors and NTPase Activating Partners. What makes RFC unique is that its own substrates are the NEF (PCNA) and NAP (DNA). (13/16)

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Brian Kelch @briankelch.bsky.social · 07/07/2025

Based on our data, we propose a model whereby fast ATP binding to some subunits readies RFC to bind PCNA, and then PCNA completes RFC’s nucleotide exchange en route to clamp loading. We propose this prevents futile ATP hydrolysis and helps ensure RFC binds PCNA before DNA. (12/16)

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Brian Kelch @briankelch.bsky.social · 07/07/2025

Josh performed more MD sims to try to understand how PCNA could promote nucleotide exchange in RFC. His sims predict that the D/E interface weakly bridged by ADP can be pried open as D binds PCNA, accelerating release. (11/16)

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Brian Kelch @briankelch.bsky.social · 07/07/2025

Thus, “PCNA is a Nucleotide Exchange Factor for Clamp Loader ATPase Complex”! It only took us 10 posts to get to the title of the paper😉. But how does PCNA do this? (10/16)

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Brian Kelch @briankelch.bsky.social · 07/07/2025

So, what catalyzes nucleotide exchange? Josh developed a FRET assay using a labeled ATP analog to monitor ATP binding to RFC. RFC alone binds some ATPs fast but then binds more ATP slowly, consistent with A&B (and maybe C) exchanging faster than D. PCNA speeds up these slow binding steps! (9/16)

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Brian Kelch @briankelch.bsky.social · 07/07/2025

To test how interface opening plays a role in ADP release, Josh performed tRAMD(doi.org/10.1021/acs....), which is a technique that predicts relative release rates. He found that the half-off rate for C or D is slower than A or B, consistent with his cryo-EM. (8/16)

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Brian Kelch @briankelch.bsky.social · 07/07/2025

Next, we wondered how the dynamics of RFC influence ADP release? So, Josh performed MD sims. His sims predict that ATP-interfaces are tighter than ADP ones, and that the B/C interface can open farthest, offering an explanation why this interface is apo in our cryo-EM reconstructions. (7/16)

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Brian Kelch @briankelch.bsky.social · 07/07/2025

First, we looked at how RFC subunits respond to nucleotide binding by comparing Josh’s apo subunits to our lab’s previous ATPgS-bound subunits and found that the B&C subunits’ lids rotate in opposite directions! We had never seen this in a AAA+ before (if you know of any, please let us know!) (6/16)

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Brian Kelch @briankelch.bsky.social · 07/07/2025

We wanted to understand the functional relevance of why they hold ADP tightly, so we started looking for clues, perhaps the most fun part of science. (5/16)

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Brian Kelch @briankelch.bsky.social · 07/07/2025

Imagine our surprise when we saw that the C and D subunits were ADP-bound! Because he never added nucleotide, these ADPs must have co-purified with RFC over a 3 day prep, meaning that RFC releases ADP very slowly from these subunits. (4/16)

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Brian Kelch @briankelch.bsky.social · 07/07/2025

To get insights into RFC recycling, postdoc @joshuapajak.bsky.social determined the structure of RFC in the apo state. He got great overall resolution of the core RFC complex, but the AAA+ domain of the A subunit is missing, suggesting it is flexibly tethered when apo. (3/16)

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Brian Kelch @briankelch.bsky.social · 07/07/2025

Recently, our lab showed how an ATP-bound RFC loads PCNA onto DNA during replication and repair (doi.org/10.7554/eLif...). But RFC loads PCNA extremely fast, so that when RFC has done its job once, how does it prep for the next round? (2/16)

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Brian Kelch @briankelch.bsky.social · 07/07/2025

Every day we make enough DNA to go to the moon and back. So, all our DNA replication enzymes work fast, right? In our latest preprint, we show that a key enzyme, the clamp loader ATPase RFC, releases ADP much slower than it loads PCNA. What gives? (1/16) doi.org/10.1101/2025...

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Krista Freeman @kfreeman.bsky.social · 16/04/2025

Hurray, it is finally out! Meet bacteriophage Bxb1 - the subject of my first full-phage cryo-EM study. My structures are beautifully complemented by the Park Lab’s cryo-ET analysis, shedding light on mycobacteriophage structural changes during infection. www.sciencedirect.com/science/arti...

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Structure and infection dynamics of mycobacteriophage Bxb1

Mycobacteriophage Bxb1 is a well-characterized virus of Mycobacterium smegmatis with double-stranded DNA and a long, flexible tail. Mycobacteriophages…

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Brian Kelch @briankelch.bsky.social · 18/04/2025

It’s been a long road, so massive props to everyone who worked on this project, including lab alum Emily Agnello, PhD, graduate students Julia Hobaugh and Rakeyah Ahsan, and Chen Xu, PhD and Kangkang Song, PhD at the fantastic UMass Chan CryoEM facility! /13

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Brian Kelch @briankelch.bsky.social · 18/04/2025

Putting that together with decades of research into phage genome packaging, we propose a speculative model for how the Portal switches from DNA-pumping mode to Tail attachment mode, using the pressure-induced piston motion of Portal to displace the motor. /12

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Brian Kelch @briankelch.bsky.social · 18/04/2025

So how does the Portal know it’s time to close the pore? We got a clue by comparing the position of the Portal in capsids before and after DNA has been pumped in. The pressure from DNA pushes the Portal lower in the capsid aperture like a piston! /11

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Brian Kelch @briankelch.bsky.social · 18/04/2025

On the left, Bayfield et al. showed the pore through Portal in an early, immature capsid. A specialized motor pumps the genome into the capsid through this open, hydrophilic pore. Then, it constricts in the mature phage, becoming narrow and hydrophobic. /10

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Brian Kelch @briankelch.bsky.social · 18/04/2025

In 2020, Ollie Bayfield and Fred Antson predicted that one component of the Neck complex, the Portal protein (dark blue), would be constricted to hold DNA in the capsid of this phage. And they were right! /9 Bayfield et al elifesciences.org/articles/55517

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Brian Kelch @briankelch.bsky.social · 18/04/2025

We know that the Neck of this phage is sufficient to hold its DNA inside the capsid because we also isolated phage that have broken tails, and therefore nothing left but the Neck to retain DNA. /8

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Brian Kelch @briankelch.bsky.social · 18/04/2025

But the phage has another problem—it needs a channel through the Neck and Tail to inject its genome into the host when it infects, but it can’t shoot its shot until it finds the right host. How does it keep that channel closed? /7

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Brian Kelch @briankelch.bsky.social · 18/04/2025

Instead of beefing up electrostatic or hydrophobic interactions between these proteins, the phage intertwines different subunits to create topological linkages. This is the same strategy used to make a wicker basket strong! /6

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Brian Kelch @briankelch.bsky.social · 18/04/2025

If this aperture were to dilate, it would allow the genome to explode out of the capsid and kill the phage! So how does the phage hold this aperture together at high temperatures, with tons of DNA pressing against it? /5

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Brian Kelch @briankelch.bsky.social · 18/04/2025

With 105 protein chains and 5 DNA segments, across 4 different symmetries, this is the largest structure the Kelch Lab has ever built (2.4 MDa)! The proteins in pinks/reds are part of the capsid shell, and they form an aperture that the rest of the Neck sits in. /4

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Brian Kelch @briankelch.bsky.social · 18/04/2025

Postdoc @emma.sedivy.bsky.social determined the structure of the Neck complex that joins the capsid head to the tail. We only knew the identity of 4 out of 8 of the proteins in the Neck, but we were able to identify the remaining 4 using ModelAngelo by @kjamali.bsky.social. /3

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Brian Kelch @briankelch.bsky.social · 18/04/2025

This phage not only lives in hot springs but also has the longest tail of any known phage. Here you see 3 viruses infecting their host, T. thermophilus. One has released its genome into the host, but two still have DNA-filled heads. /2

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Brian Kelch @briankelch.bsky.social · 18/04/2025

Did you know that there is so much DNA packed inside a phage capsid that the pressure is 10X higher than in a bottle of champagne? In our latest preprint, we wondered how that is contained in a phage that lives at extremely high temperatures. /1 www.biorxiv.org/content/10.1...

Meme describing types of headaches, with the most all encompassing headache being the stress of holding copious amounts of DNA in a phage head.

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Brian Kelch @briankelch.bsky.social · 18/04/2025

I'm a little late to the game, but I'm happy to announce that our paper with my @umasschan.bsky.social colleagues in the Travis Thomson lab has now been published in @plosbiology.org! Thanks to all involved on this fun collaboration!

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Josh Dubnau @joshdubnau.bsky.social · 20/02/2025

Fun primer with @ForeverYHChang highlighting a lovely Travis Thomson lab paper! Did animals co-opt viral gag for intercellular (trans synaptic) communication? Or did viruses co-opt retrotransposon gags that animals had previously co-opted for intercellular use?

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Elinor Karlsson @elinork.bsky.social · 02/11/2023

Looking for TT faculty job? At #ASHG23? We’re recruiting! We = Genomics and Comp Bio dept at UMass Chan Medical School in Worcester, MA (not Boston but near Boston) academicjobsonline.org/ajo/jobs/25641 pop genomics, imaging, stat. genetics, machine learning aka cool science w big data.

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University of Massachusetts Chan Medical School, Department of Genomics and Computational Biology

Full service online faculty recruitment and application management system for academic institutions worldwide. We offer unique solutions tailored for academic communities.

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Jonathan Watts @jwattsgroup.bsky.social · 19/09/2024

Hey, senior PhD students working on RNA and moving toward leading independent labs: Apply for the Maraganore Early Independence Fellow program at the UMass Chan RNA Therapeutics Institute and come work with us! www.linkedin.com/posts/angela...

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Angela Messmer-Blust on LinkedIn: #diversityinstem #underrepresentedinscience #rnabiology…

I am unbelievably excited and proud to launch the RNA Therapeutic Institute's new Maraganore Early Independence Fellows Program at UMass Chan Medical School…

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Amir Mitchell @amitchell.bsky.social · 20/09/2023

Systems and synthetic biologists, we are recruiting at the department of Systems Biology at the @UMassChan. Position is open to faculty at all levels. academicjobsonline.org/ajo/jobs/254…

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University of Massachusetts Chan Medical School, Departme...

Full service online faculty recruitment and application m...

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Sven Klumpe @svenklumpe.bsky.social · 24/02/2024

Over 4 years in the making, I am extremely happy to share our pre-print on the “In-cell structure and snapshots of copia retrotransposons in intact tissue”. We used cryo-ET to resolve the copia capsid structure to subnanometer resolution inside cells: www.biorxiv.org/content/10.1...

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Brian Kelch @briankelch.bsky.social · 23/02/2024

Check out this new preprint from first author @svenklumpe.bsky.social and coworkers (primarily Plitsko and Beck labs at Max Planck) describing Copia retrotransposon capsids inside cells. Fig 3 is so dope. Look at that capsid array! Beautiful work! 😍🤩 www.biorxiv.org/content/10.1...

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Brian Kelch @briankelch.bsky.social · 04/01/2024

For details, plz read the preprint. This is a huge team effort primarily from the Thomson lab, with biochemical and structural work from twitterless Yumeng Liu from my lab. /7

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Brian Kelch @briankelch.bsky.social · 04/01/2024

This leads to a mechanistic hypothesis that Copia and Arc antagonize each others’ capsid assembly by competing for RNA. The functional relevance of Copia in synaptic dev also suggests that we might be watching Copia in process of ‘domestication’. /6

A) Image showing mutual antagonism between Copia and Arc. Arc is pro-plasticity, while Copia is inhibitory. B) Proposed mechanism for mutual antagonism, in which Arc and Copia capsids compete for RNA.

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Brian Kelch @briankelch.bsky.social · 04/01/2024

Amazingly, knockdown of Copia results in increased bouton development and synaptic plasticity, the opposite of an Arc knockout. Copia and Arc are antagonizing each other. 5/

A) Images of Drosophila NMJ showing that Copia knockdown causes increased boutons. B&C) quantification of bouton increase

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Brian Kelch @briankelch.bsky.social · 04/01/2024

We find that Copia requires RNA for proper assembly of the capsid structure. The structure shows pores that presumably allow small molecules to enter the capsid, presumably for reverse transcriptase activity. Arc lacks these pores. /4

Image of the Copia hexamer, showing the pore in the center is ringed w postiviely charged residues.

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Brian Kelch @briankelch.bsky.social · 04/01/2024

Here, they report that the retrotransposon Copia forms capsids that are also packaged into EVs that transport across the NMJ. We determined the structure of these virus-like particles, which are larger than Arc capsids, presumably to bind bigger Copia RNA. /3

A) images of Drosophila NMJs, showing presence of Copia protein at the NMJ. B) Negative stain EM micrograph of Copia capids. C) Structure of the Copia capsid. D) Structure of the dArc1 capsid (from the Briggs group).

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Brian Kelch @briankelch.bsky.social · 04/01/2024

The Thomson lab previously showed that the retrovirus-derived Arc gene forms virus-like capsid particles in extracellular vesicles (EVs). Arc capsids package and carry RNA across a neuromuscular junction (NMJ) for regulating synaptic plasticity. /2 pubmed.ncbi.nlm.nih.gov/29328915/

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Brian Kelch @briankelch.bsky.social · 04/01/2024

I'm back to do an in-depth dive into our most recent work up on biorxiv. Led by the Thomson Lab in the Neurobiology dept, w Yumeng Liu of the Kelch Lab. We find that the retrotransposon Copia forms capsids that antagonize Arc for controlling synaptic plasticity. /1 www.biorxiv.org/content/10.1...

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Brian Kelch @briankelch.bsky.social · 24/12/2023

Just in time for Festivus, another paper up on biorxiv. Led by Thomson Lab in Neurobiology dept @ UMass Chan Med School, w Yumeng Liu of the Kelch Lab. We find that the retrotransposon Copia forms capsids that antagonize Arc for controlling synaptic plasticity. www.biorxiv.org/content/10.1...

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Brian Kelch @briankelch.bsky.social · 04/12/2023

This is primarily the work of soon-to-be PhD Jacob Landeck, with help from postdocs Josh Pajak and Emma Sedivy, and rotation student Emily Norman. We thank NIGMS for funding, and the UMass Chan cryoEM facility for being awesome as always. 9/

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Brian Kelch @briankelch.bsky.social · 04/12/2023

So why the different mechanisms between euks and bacs? We suspect it has to do w arrangement of binding sites on the clamp. Euk clamp has strong-weak-strong-weak-strong-weak arrangement. Bac clamp is strong-weak-weak-strong-weak-weak, leading to different mechanisms. 8/

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Brian Kelch @briankelch.bsky.social · 04/12/2023

We also find an alternate state in the loader in which a pore opens at the same position as seen in the euk loader, which we & others had shown is important for unwinding DNA. Similar unwinding activity in Ecoli? We don’t see evidence. Jury is still out on pore’s function. 7/

Figure showing that the Alternate_collar state adopts an unanticipated conformation where a pore opens between the A and B subunits of the clamp loader.

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Brian Kelch @briankelch.bsky.social · 04/12/2023

Then the primer/template junction binds, which forces the assembly to adopt a spiral that tightly hugs the p/t. This state is nearly identical with an RNA or a DNA primer. The clamp then closes and the loader undergoes a step-wise disengagement from the clamp. 6/

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