Harry M. Williams
@harrywilliams.bsky.social
Structural biologist currently working on microsecond time-resolved cryo-EM | Currently based in Lausanne at EPFL 🇨🇭
Pinned
Microsecond Time-Resolved Cryo-EM Based on Jet Vitrification https://www.biorxiv.org/content/10.1101/2025.11.21.689681v1
Happy to see our latest work online. Here we present a new method for microsecond time-resolved cryo-EM based on jet vitrification! Details in the 🧵 below... we hope you enjoy reading! (Hint, there's some fun videos...) #cryoEM #structuralbiology
Reposted by Harry M. Williams
Organelles do NOT have a single uniform pH.
And if you think they must, because “protons diffuse fast,” this paper is for you.
A thread on why that assumption is wrong; and what we found instead. 🧵 1/n
And if you think they must, because “protons diffuse fast,” this paper is for you.
A thread on why that assumption is wrong; and what we found instead. 🧵 1/n
Organelles harbour pH gradients https://www.biorxiv.org/content/10.64898/2025.12.12.694065v1
December 17, 2025 at 12:46 AM
Organelles do NOT have a single uniform pH.
And if you think they must, because “protons diffuse fast,” this paper is for you.
A thread on why that assumption is wrong; and what we found instead. 🧵 1/n
And if you think they must, because “protons diffuse fast,” this paper is for you.
A thread on why that assumption is wrong; and what we found instead. 🧵 1/n
Reposted by Harry M. Williams
Experimental structures provide valuable hypotheses and accelerate but do not replace evolutionary and statistical analyses of protein sequences
AlphaFold predictions are valuable hypotheses and accelerate but do not replace experimental structure determination
www.nature.com/articles/s41...
www.nature.com/articles/s41...
AlphaFold predictions are valuable hypotheses and accelerate but do not replace experimental structure determination - Nature Methods
An analysis of AlphaFold protein structure predictions shows that while in many cases the predictions are highly accurate, there are also many instances where the predicted structures or parts of pred...
www.nature.com
December 15, 2025 at 9:38 AM
Experimental structures provide valuable hypotheses and accelerate but do not replace evolutionary and statistical analyses of protein sequences
Reposted by Harry M. Williams
An integrated workflow for structural virology with a 100 keV electron microscope www.biorxiv.org/content/10.64898/2025.12.08.693081v1 #cryoem
December 11, 2025 at 9:16 AM
An integrated workflow for structural virology with a 100 keV electron microscope www.biorxiv.org/content/10.64898/2025.12.08.693081v1 #cryoem
Congrats Dan!
December 2, 2025 at 4:32 PM
Congrats Dan!
As usual, there's a lot more cool stuff in this paper so naturally I encourage you to have a look! Thanks of course go to the entire team at EPFL, including the wonderful people at the DCI-Lausanne imaging centre who helped us collect something a lot of data.
November 22, 2025 at 11:26 AM
As usual, there's a lot more cool stuff in this paper so naturally I encourage you to have a look! Thanks of course go to the entire team at EPFL, including the wonderful people at the DCI-Lausanne imaging centre who helped us collect something a lot of data.
At 13.3 ms, we observe ErNaR in the O2 state. The chromophore has switched back to the all-trans configuration. We find the sidechain environment in the active center is starting to re-form. There also appears to be conformational dynamics taking place downstream along the proposed ion path.
November 22, 2025 at 11:26 AM
At 13.3 ms, we observe ErNaR in the O2 state. The chromophore has switched back to the all-trans configuration. We find the sidechain environment in the active center is starting to re-form. There also appears to be conformational dynamics taking place downstream along the proposed ion path.
At 107 µs after photoactivation, we observe the K2 state. As we show, the chromophore has switched from the all-trans configuration present in the dark state to the 13-cis configuration. A switch that is accompanied by sidechain movements around the chromophore in the ErNaR active center.
November 22, 2025 at 11:26 AM
At 107 µs after photoactivation, we observe the K2 state. As we show, the chromophore has switched from the all-trans configuration present in the dark state to the 13-cis configuration. A switch that is accompanied by sidechain movements around the chromophore in the ErNaR active center.
Samples of ErNaR were activated with a nanosecond laser pulse and subsequently jet vitrified at 107 µs and 13.3 ms, to probe the K2 and O2 states of the photo-cycle, respectively. We also collected a dark state, which was not photo-activated, to serve as a control in our experiments.
November 22, 2025 at 11:26 AM
Samples of ErNaR were activated with a nanosecond laser pulse and subsequently jet vitrified at 107 µs and 13.3 ms, to probe the K2 and O2 states of the photo-cycle, respectively. We also collected a dark state, which was not photo-activated, to serve as a control in our experiments.
Next, to demonstrate that we could conduct time-resolved cryo-EM experiments, we turned to the bacterial rhodopsin ErNaR. Normally, ErNaR pumps sodium ions from the cytoplasm across the bacterial membrane to the ouside. Using our setup, we wanted to probe some of the earlier photocycle states.
November 22, 2025 at 11:26 AM
Next, to demonstrate that we could conduct time-resolved cryo-EM experiments, we turned to the bacterial rhodopsin ErNaR. Normally, ErNaR pumps sodium ions from the cytoplasm across the bacterial membrane to the ouside. Using our setup, we wanted to probe some of the earlier photocycle states.
To demonstrate this setup, we first prepared a sample of mouse heavy chain apoferritin. After the usual data collection and processing, we obtained a 1.3 Å volume demonstrating that we can produce high-quality cryo-samples. The volume for this is shown higher up in this thread (with sidechains).
November 22, 2025 at 11:26 AM
To demonstrate this setup, we first prepared a sample of mouse heavy chain apoferritin. After the usual data collection and processing, we obtained a 1.3 Å volume demonstrating that we can produce high-quality cryo-samples. The volume for this is shown higher up in this thread (with sidechains).
You can see these "timing lasers" and the "time of arrival laser" (or as I prefer to call it, the bouncy laser) working together here! In this video, the jet would normally come from the right hand side whereas the activation laser would be fired from the left hand side.
November 22, 2025 at 11:26 AM
You can see these "timing lasers" and the "time of arrival laser" (or as I prefer to call it, the bouncy laser) working together here! In this video, the jet would normally come from the right hand side whereas the activation laser would be fired from the left hand side.
We also have a "time of arrival laser". Informally, we call this the "bouncy laser" because it continuously bounces off the back-side of a grid during an experiment. When the jet reaches the grid, this laser is deflected and this gives us the actual time of arrival of the jet.
November 22, 2025 at 11:26 AM
We also have a "time of arrival laser". Informally, we call this the "bouncy laser" because it continuously bounces off the back-side of a grid during an experiment. When the jet reaches the grid, this laser is deflected and this gives us the actual time of arrival of the jet.
In other words, if we are interested in observing dynamics at 1 ms post-activation, we will fire the activation laser -1 ms before the predicted time of arrival of the cryogen jet. Simples!
November 22, 2025 at 11:26 AM
In other words, if we are interested in observing dynamics at 1 ms post-activation, we will fire the activation laser -1 ms before the predicted time of arrival of the cryogen jet. Simples!
Using this predicted time of arrival, we then fire the activation laser as the appropriate delay, which activates protein dynamics either by releasing a photoactive compound (e.g. caged-ATP) or exciting a photoactive component within a protein itself (as we demonstrate in this paper).
November 22, 2025 at 11:26 AM
Using this predicted time of arrival, we then fire the activation laser as the appropriate delay, which activates protein dynamics either by releasing a photoactive compound (e.g. caged-ATP) or exciting a photoactive component within a protein itself (as we demonstrate in this paper).
You can see these lasers in the first figure of this thread and can be divided into two. First, we have two "timing lasers", which detect the progress of the jet as it moves towards the grid. These allow us to calculate on-the-fly the predicted time of arrival of the jet at the grid.
November 22, 2025 at 11:26 AM
You can see these lasers in the first figure of this thread and can be divided into two. First, we have two "timing lasers", which detect the progress of the jet as it moves towards the grid. These allow us to calculate on-the-fly the predicted time of arrival of the jet at the grid.
We also took high-speed video frames of a similar jet and you can see these in the figure here. Now, a big part of our experiments is time. We want to accurately and precisely arrest protein dynamics at given timepoints to observe biological phenomena. To do this, we use a combination of lasers.
November 22, 2025 at 11:26 AM
We also took high-speed video frames of a similar jet and you can see these in the figure here. Now, a big part of our experiments is time. We want to accurately and precisely arrest protein dynamics at given timepoints to observe biological phenomena. To do this, we use a combination of lasers.
Here you can see the the cryogen jet traveling towards the grid, ultimately striking the grid and, in doing so, arresting protein dynamics.
November 22, 2025 at 11:26 AM
Here you can see the the cryogen jet traveling towards the grid, ultimately striking the grid and, in doing so, arresting protein dynamics.
To conduct a time-resolved experiment, a sample grid is placed in the activation subunit of the jetting assembly, and protein dynamics are initiated with a laser pulse. The sample is then vitrified with a liquid ethane (or ethane/propane) jet that is generated in the jetting subunit.
November 22, 2025 at 11:26 AM
To conduct a time-resolved experiment, a sample grid is placed in the activation subunit of the jetting assembly, and protein dynamics are initiated with a laser pulse. The sample is then vitrified with a liquid ethane (or ethane/propane) jet that is generated in the jetting subunit.
Experimental concept: protein dynamics are initiated with a short laser pulse. As they unfold, the sample is vitrified with a time-delayed jet of a liquid cryogen, trapping the proteins in their transient configurations.
November 22, 2025 at 11:26 AM
Experimental concept: protein dynamics are initiated with a short laser pulse. As they unfold, the sample is vitrified with a time-delayed jet of a liquid cryogen, trapping the proteins in their transient configurations.
Happy to see our latest work online. Here we present a new method for microsecond time-resolved cryo-EM based on jet vitrification! Details in the 🧵 below... we hope you enjoy reading! (Hint, there's some fun videos...) #cryoEM #structuralbiology
Microsecond Time-Resolved Cryo-EM Based on Jet Vitrification https://www.biorxiv.org/content/10.1101/2025.11.21.689681v1
November 22, 2025 at 11:26 AM
Happy to see our latest work online. Here we present a new method for microsecond time-resolved cryo-EM based on jet vitrification! Details in the 🧵 below... we hope you enjoy reading! (Hint, there's some fun videos...) #cryoEM #structuralbiology
Always a good feeling!
November 19, 2025 at 5:41 PM
Always a good feeling!
Reposted by Harry M. Williams
MISO: microfluidic protein isolation enables single-particle cryo-EM structure determination from a single cell colony.
Or from a single dish of HEK cell culture in the case of two membrane proteins.
Out in Nature Methods now! lnkd.in/gpyBSceg
Wonderful collaboration with the Efremov lab.
Or from a single dish of HEK cell culture in the case of two membrane proteins.
Out in Nature Methods now! lnkd.in/gpyBSceg
Wonderful collaboration with the Efremov lab.
November 14, 2025 at 6:38 PM
MISO: microfluidic protein isolation enables single-particle cryo-EM structure determination from a single cell colony.
Or from a single dish of HEK cell culture in the case of two membrane proteins.
Out in Nature Methods now! lnkd.in/gpyBSceg
Wonderful collaboration with the Efremov lab.
Or from a single dish of HEK cell culture in the case of two membrane proteins.
Out in Nature Methods now! lnkd.in/gpyBSceg
Wonderful collaboration with the Efremov lab.
Reposted by Harry M. Williams
We are pleased to announce Doppio version 1.4! Available here www.ccpem.ac.uk/software/dow...
Downloads – CCP-EM
www.ccpem.ac.uk
November 4, 2025 at 5:38 PM
We are pleased to announce Doppio version 1.4! Available here www.ccpem.ac.uk/software/dow...
Hopefully leading to one or two new microscopes in Utrecht?
November 3, 2025 at 5:01 PM
Hopefully leading to one or two new microscopes in Utrecht?