John Rubinstein
@johnrubinstein.bsky.social
CryoEM, Bioenergetics, V-ATPases (especially in synaptic vesicles and lysosomes), OxPhos (especially in mycobacteria as a TB drug target)
Scientist, The Hospital for Sick Children
Professor, U of Toronto
👨👩👧🔬🚲
rubinsteinlab.org
COI: structura.bio
Scientist, The Hospital for Sick Children
Professor, U of Toronto
👨👩👧🔬🚲
rubinsteinlab.org
COI: structura.bio
Oops, the picture of mycolic acids (from en.wikipedia.org/wiki/Mycolic...) in the previous skeet isn't displaying properly. Here it is again.
October 30, 2025 at 11:51 AM
Oops, the picture of mycolic acids (from en.wikipedia.org/wiki/Mycolic...) in the previous skeet isn't displaying properly. Here it is again.
Thanks! Yes, lots of great work has been done on related complexes!
The mycobacterial LCC is pretty weird compared to the others, though: it has one type of subunit (AccD5) for acetyl/propionyl-CoA and another (AccD4) for very long chain acyl-CoA, used to form the alpha branch of mycolic acid:
The mycobacterial LCC is pretty weird compared to the others, though: it has one type of subunit (AccD5) for acetyl/propionyl-CoA and another (AccD4) for very long chain acyl-CoA, used to form the alpha branch of mycolic acid:
October 30, 2025 at 11:41 AM
Thanks! Yes, lots of great work has been done on related complexes!
The mycobacterial LCC is pretty weird compared to the others, though: it has one type of subunit (AccD5) for acetyl/propionyl-CoA and another (AccD4) for very long chain acyl-CoA, used to form the alpha branch of mycolic acid:
The mycobacterial LCC is pretty weird compared to the others, though: it has one type of subunit (AccD5) for acetyl/propionyl-CoA and another (AccD4) for very long chain acyl-CoA, used to form the alpha branch of mycolic acid:
Interestingly, Yingke found some complexes bound to uncharacterized proteins MSMEG_0435 & 0436 (Rv0263c & 0264c in M. tuberculosis). A recent preprint from Carl Nathan shows that Rv0263c is needed for Mtb survival during transmission, suggesting it modulates mycolic acid synthesis in TB transmission
October 30, 2025 at 3:21 AM
Interestingly, Yingke found some complexes bound to uncharacterized proteins MSMEG_0435 & 0436 (Rv0263c & 0264c in M. tuberculosis). A recent preprint from Carl Nathan shows that Rv0263c is needed for Mtb survival during transmission, suggesting it modulates mycolic acid synthesis in TB transmission
A structure with short- and long-chain acyl-CoAs shows how the two types of carboxyltransferase (CT) subunit distinguish acyl-CoAs: the short-chain CT subunit binds CoA but can’t accommodate a long acyl chain; the long-chain CT subunit binds long acyl chains but doesn’t interact strongly with CoA.
October 30, 2025 at 3:21 AM
A structure with short- and long-chain acyl-CoAs shows how the two types of carboxyltransferase (CT) subunit distinguish acyl-CoAs: the short-chain CT subunit binds CoA but can’t accommodate a long acyl chain; the long-chain CT subunit binds long acyl chains but doesn’t interact strongly with CoA.
Adding short-chain acyl-CoA substrate (plus ATP & bicarbonate) causes biotin carboxyl carrier protein (BCCP) domains to travel between biotin carboxylase (BC) domains, which load a carboxyl group onto the BCCP biotin, and the carboxyltransferase (CT) domain, which transfers it to the acyl-CoA.
October 30, 2025 at 3:21 AM
Adding short-chain acyl-CoA substrate (plus ATP & bicarbonate) causes biotin carboxyl carrier protein (BCCP) domains to travel between biotin carboxylase (BC) domains, which load a carboxyl group onto the BCCP biotin, and the carboxyltransferase (CT) domain, which transfers it to the acyl-CoA.
The MCC complex is well studied, but the long-chain carboxylase (LCC) complex isn’t, and is essential for making mycolic acid for the mycobacterial cell wall. The LCC complex carboxylates both long-chain and short-chain acyl-CoA. A structure with propionyl-CoA shows how it binds short-chain acyl-CoA
October 30, 2025 at 3:21 AM
The MCC complex is well studied, but the long-chain carboxylase (LCC) complex isn’t, and is essential for making mycolic acid for the mycobacterial cell wall. The LCC complex carboxylates both long-chain and short-chain acyl-CoA. A structure with propionyl-CoA shows how it binds short-chain acyl-CoA
New lab preprint!
@zestytoast.bsky.social tagged a scarce mycobacterial protein in M. smegmatis with TwinStep but got… something? @kjamali.bsky.social's ModelAngelo built models & @martinsteinegger.bsky.social's FoldSeek IDed them as the biotin-containing MCC & LCC complexes
🧵
tinyurl.com/ukny4ptz
@zestytoast.bsky.social tagged a scarce mycobacterial protein in M. smegmatis with TwinStep but got… something? @kjamali.bsky.social's ModelAngelo built models & @martinsteinegger.bsky.social's FoldSeek IDed them as the biotin-containing MCC & LCC complexes
🧵
tinyurl.com/ukny4ptz
October 30, 2025 at 3:21 AM
New lab preprint!
@zestytoast.bsky.social tagged a scarce mycobacterial protein in M. smegmatis with TwinStep but got… something? @kjamali.bsky.social's ModelAngelo built models & @martinsteinegger.bsky.social's FoldSeek IDed them as the biotin-containing MCC & LCC complexes
🧵
tinyurl.com/ukny4ptz
@zestytoast.bsky.social tagged a scarce mycobacterial protein in M. smegmatis with TwinStep but got… something? @kjamali.bsky.social's ModelAngelo built models & @martinsteinegger.bsky.social's FoldSeek IDed them as the biotin-containing MCC & LCC complexes
🧵
tinyurl.com/ukny4ptz
Whoever designed the new landscaping around King's College Circle at @utoronto.ca has done a brilliant job!
All summer I've been getting off the subway early to walk down Philosopher's Walk and the new walkways on the St. George campus - a great half hour start to each day.
All summer I've been getting off the subway early to walk down Philosopher's Walk and the new walkways on the St. George campus - a great half hour start to each day.
August 15, 2025 at 5:36 PM
Whoever designed the new landscaping around King's College Circle at @utoronto.ca has done a brilliant job!
All summer I've been getting off the subway early to walk down Philosopher's Walk and the new walkways on the St. George campus - a great half hour start to each day.
All summer I've been getting off the subway early to walk down Philosopher's Walk and the new walkways on the St. George campus - a great half hour start to each day.
Returned from a wonderful symposium in honour of the 50th anniversary of Richard Henderson's and Nigel Unwin's 1975 paper on the structure of bacteriorhodopsin - the paper that started the field of membrane protein structural biology.
Also fulfilled my dream of ringing the LMB seminar bell.
Also fulfilled my dream of ringing the LMB seminar bell.
July 12, 2025 at 12:10 PM
Returned from a wonderful symposium in honour of the 50th anniversary of Richard Henderson's and Nigel Unwin's 1975 paper on the structure of bacteriorhodopsin - the paper that started the field of membrane protein structural biology.
Also fulfilled my dream of ringing the LMB seminar bell.
Also fulfilled my dream of ringing the LMB seminar bell.
Very proud of my amazing wife, Dr. Voula Kanelis, awarded a King Charles III coronation medal yesterday for her contributions to Heart & Stroke Canada (❤️ / ).
June 24, 2025 at 2:53 PM
Very proud of my amazing wife, Dr. Voula Kanelis, awarded a King Charles III coronation medal yesterday for her contributions to Heart & Stroke Canada (❤️ / ).
My brother, Dan Rubinstein's, latest book is out. Can't wait to dive in!
June 22, 2025 at 9:17 PM
My brother, Dan Rubinstein's, latest book is out. Can't wait to dive in!
Great to hear about this event. Derek was an awesome guy and one-of-a-kind. He made the world a better place.
Derek was a pioneer in cryo-EM. I first met him at the EMBO Workshop in 2000 when he was teaching people how to freeze grids.
Derek was a pioneer in cryo-EM. I first met him at the EMBO Workshop in 2000 when he was teaching people how to freeze grids.
June 7, 2025 at 5:43 PM
Great to hear about this event. Derek was an awesome guy and one-of-a-kind. He made the world a better place.
Derek was a pioneer in cryo-EM. I first met him at the EMBO Workshop in 2000 when he was teaching people how to freeze grids.
Derek was a pioneer in cryo-EM. I first met him at the EMBO Workshop in 2000 when he was teaching people how to freeze grids.
Wow. Amazing retirement symposium for the legendary Sergio Grinstein.
Staggering expanses of biology revealed.
But the heartfelt stories of generosity, support, and careers launched and sustained really got to me.
I spent the entire day verklempt
Brilliantly organized by @spencerfreeman.bsky.social
Staggering expanses of biology revealed.
But the heartfelt stories of generosity, support, and careers launched and sustained really got to me.
I spent the entire day verklempt
Brilliantly organized by @spencerfreeman.bsky.social
May 31, 2025 at 2:39 PM
Wow. Amazing retirement symposium for the legendary Sergio Grinstein.
Staggering expanses of biology revealed.
But the heartfelt stories of generosity, support, and careers launched and sustained really got to me.
I spent the entire day verklempt
Brilliantly organized by @spencerfreeman.bsky.social
Staggering expanses of biology revealed.
But the heartfelt stories of generosity, support, and careers launched and sustained really got to me.
I spent the entire day verklempt
Brilliantly organized by @spencerfreeman.bsky.social
We developed an EtfD assay!
By mixing mycobacterial inverted membrane vesicles (containing the electron transport chain) with purified proteins for fatty acid oxidation and e- transfer, we can detect when EtfD drives the ETC.
This assay will allow for detection and development of EtfD inhibitors.
By mixing mycobacterial inverted membrane vesicles (containing the electron transport chain) with purified proteins for fatty acid oxidation and e- transfer, we can detect when EtfD drives the ETC.
This assay will allow for detection and development of EtfD inhibitors.
May 16, 2025 at 2:30 PM
We developed an EtfD assay!
By mixing mycobacterial inverted membrane vesicles (containing the electron transport chain) with purified proteins for fatty acid oxidation and e- transfer, we can detect when EtfD drives the ETC.
This assay will allow for detection and development of EtfD inhibitors.
By mixing mycobacterial inverted membrane vesicles (containing the electron transport chain) with purified proteins for fatty acid oxidation and e- transfer, we can detect when EtfD drives the ETC.
This assay will allow for detection and development of EtfD inhibitors.
One of the iron-sulfur clusters (D1) is a linear [3Fe-4S] cluster that has been synthesized chemically and detected spectroscopically but has not been seen before in the structure of a protein under physiological conditions
May 16, 2025 at 2:30 PM
One of the iron-sulfur clusters (D1) is a linear [3Fe-4S] cluster that has been synthesized chemically and detected spectroscopically but has not been seen before in the structure of a protein under physiological conditions
The structure of EtfD reveals a wire of iron-sulfur clusters and a b heme that link the electron transfer flavoprotein (ETF) in the cytosol to a menaquinone binding site in the membrane. Two of the iron-sulfur clusters are pretty weird...
![A) Cryo-EM map and atomic model for EtfD
B) Schematic of domain architecture of EtfD
C) Cartoon showing the wire of four iron-sulfur clusters (D1-D4) and heme b linking the input of electrons from ETF to the menaquinone-reducing site in the membrane
D) Experimental density for D1-D4, heme b, and the menaquinone site
D1 is linear [3Fe-4S] cluster that has been synthesized and detected spectroscopically not been seen before in the structure of a protein under physiological conditions (!!!).
D2 is a non-cubane [4Fe-4S] cluster
D3 and D4 are cubane [4Fe-4S] clusters](https://cdn.bsky.app/img/feed_thumbnail/plain/did:plc:2h7m2pmlm4foweae4sdxrkmo/bafkreic2ezq6zgjht3r3mjlg2ofxoybihnedh5tmeyp2xfkuyipii7ke4i@jpeg)
May 16, 2025 at 2:30 PM
The structure of EtfD reveals a wire of iron-sulfur clusters and a b heme that link the electron transfer flavoprotein (ETF) in the cytosol to a menaquinone binding site in the membrane. Two of the iron-sulfur clusters are pretty weird...
EtfD links beta oxidation to OxPhos in mycobacteria - recent work suggests that targeting EtfD could shorten #tubeculosis treatment.
But how does EtfD work? And how can you assay its activity?
In his final PhD manuscript @courbongautier.bsky.social provides answers!
www.biorxiv.org/content/10.1...
But how does EtfD work? And how can you assay its activity?
In his final PhD manuscript @courbongautier.bsky.social provides answers!
www.biorxiv.org/content/10.1...
May 16, 2025 at 2:30 PM
EtfD links beta oxidation to OxPhos in mycobacteria - recent work suggests that targeting EtfD could shorten #tubeculosis treatment.
But how does EtfD work? And how can you assay its activity?
In his final PhD manuscript @courbongautier.bsky.social provides answers!
www.biorxiv.org/content/10.1...
But how does EtfD work? And how can you assay its activity?
In his final PhD manuscript @courbongautier.bsky.social provides answers!
www.biorxiv.org/content/10.1...
Trillium season in Ontario!
May 10, 2025 at 5:17 PM
Trillium season in Ontario!
I love how scientific ideas and techniques spread from mind to mind and place to place. Spotted this week in Cleveland, Ohio:
May 10, 2025 at 12:56 PM
I love how scientific ideas and techniques spread from mind to mind and place to place. Spotted this week in Cleveland, Ohio:
Hey University of Saskatchewan colleagues: I'm looking forward to giving a lecture there on Thur Mar 27th.
Membrane protein structural biology, mycobacterial bioenergetics, and TB drug discovery.
Come say 'Hi'
Membrane protein structural biology, mycobacterial bioenergetics, and TB drug discovery.
Come say 'Hi'
March 13, 2025 at 1:19 PM
Hey University of Saskatchewan colleagues: I'm looking forward to giving a lecture there on Thur Mar 27th.
Membrane protein structural biology, mycobacterial bioenergetics, and TB drug discovery.
Come say 'Hi'
Membrane protein structural biology, mycobacterial bioenergetics, and TB drug discovery.
Come say 'Hi'
My own institution doesn't do too badly in the Specialized/Pediatric category (no surprise, knowing my clinical colleagues)...
February 27, 2025 at 5:35 PM
My own institution doesn't do too badly in the Specialized/Pediatric category (no surprise, knowing my clinical colleagues)...
Newsweek ranks Toronto's University Health Network (UHN) as 3rd best hospital in world and
#1 Public hospital in the world.
The two hospitals above it are private businesses.
Is this the poor healthcare Donald Trump wants to save Canadian's from?
www.newsweek.com/rankings/wor...
#1 Public hospital in the world.
The two hospitals above it are private businesses.
Is this the poor healthcare Donald Trump wants to save Canadian's from?
www.newsweek.com/rankings/wor...
February 27, 2025 at 5:17 PM
Newsweek ranks Toronto's University Health Network (UHN) as 3rd best hospital in world and
#1 Public hospital in the world.
The two hospitals above it are private businesses.
Is this the poor healthcare Donald Trump wants to save Canadian's from?
www.newsweek.com/rankings/wor...
#1 Public hospital in the world.
The two hospitals above it are private businesses.
Is this the poor healthcare Donald Trump wants to save Canadian's from?
www.newsweek.com/rankings/wor...
🚨 Amazing Opportunity 🚨
U Alberta is looking for a director for their new #cryoEM facility
Support excellent researchers (who are really nice people) using state-of-the-art infrastructure.
+ reasonable cost of living
+ near some of most beautiful places in world
+ 🇨🇦
apps.ualberta.ca/careers/post...
U Alberta is looking for a director for their new #cryoEM facility
Support excellent researchers (who are really nice people) using state-of-the-art infrastructure.
+ reasonable cost of living
+ near some of most beautiful places in world
+ 🇨🇦
apps.ualberta.ca/careers/post...
February 22, 2025 at 2:47 AM
🚨 Amazing Opportunity 🚨
U Alberta is looking for a director for their new #cryoEM facility
Support excellent researchers (who are really nice people) using state-of-the-art infrastructure.
+ reasonable cost of living
+ near some of most beautiful places in world
+ 🇨🇦
apps.ualberta.ca/careers/post...
U Alberta is looking for a director for their new #cryoEM facility
Support excellent researchers (who are really nice people) using state-of-the-art infrastructure.
+ reasonable cost of living
+ near some of most beautiful places in world
+ 🇨🇦
apps.ualberta.ca/careers/post...
After a long and productive career at The Hospital for Sick Children and the University of Toronto, Prof. Charlie Deber has retired. Saw this in his lab space 🥲
February 21, 2025 at 2:28 PM
After a long and productive career at The Hospital for Sick Children and the University of Toronto, Prof. Charlie Deber has retired. Saw this in his lab space 🥲
We propose a revised integrated model of the mycobacterial electron transport chain and central carbon metabolism (Krebs), with the Krebs cycle tethered to the membrane in multiple places, and preferentially directing electrons to the CIII2CIV2 terminal oxidase and not the cyt. bd terminal oxidase.
February 19, 2025 at 9:24 PM
We propose a revised integrated model of the mycobacterial electron transport chain and central carbon metabolism (Krebs), with the Krebs cycle tethered to the membrane in multiple places, and preferentially directing electrons to the CIII2CIV2 terminal oxidase and not the cyt. bd terminal oxidase.