Periodic Reporting for period 4 - ProtonPump (Structural mechanism coupling the reduction of oxygen to proton pumping in living cells)
Periodo di rendicontazione: 2023-07-01 al 2024-12-31
Sintesi del contesto e degli obiettivi generali del progetto
Every breath you take delivers oxygen to mitochondria within the cells of your body. Mitochondria are energy transducing organelles that accept electrons liberated from the food that you eat in order to generate a transmembrane proton concentration gradient. Cytochrome c oxidase is an integral membrane protein complex in the mitochondria that accepts four electrons and reduces molecular oxygen to two water molecules while simultaneously pumping protons against a transmembrane potential. Cytochrome c oxidase homologues are found in almost all living organisms. Because oxygen is the final destination of the transferred electrons, this enzyme family is referred to as the terminal oxidases. Crystal structures of terminal oxidases have been known for more than two decades and these enzymes have been studied with virtually all available biophysical and biochemical methods. Despite this scrutiny, it is unknown how the reduction of oxygen to water at the enzyme’s active-site are coupled to proton pumping across the biological membrane.
This ERC project, Proton Pump, aimed to record a three-dimensional movie that reveals how proton exchange steps between key amino acid residues are controlled by structural changes in the protein cytochrome c oxidase. In this work we utilized state-of-the-art methods of time-resolved serial crystallography at X-ray free electron lasers (XFELs) and synchrotron radiation facilities to observe how structural changes in terminal oxidases develop over time. For technical reasons, we utilized a bacterial enzyme that is highly stable from which we could grow well diffracting crystals. Our experiments required a new approach that would rapidly deliver oxygen into the protein’s active site and thereby initiate the oxygen reduction reaction in microcrystals of cytochrome c oxidase. This project therefore involved multiple challenges including microcrystal quality and density, chemical stability, reaction initiation within the microcrystals, the delivery of microcrystals into an X-ray beam, and data-processing and analysis. Structural results from this project have given new insight into one of the most important chemical reactions in biology. We have also developed new tools and methods that will help to grow the field of time-resolved structural biology and allow structural studies that go beyond a handful of naturally occurring light-driven systems. Although this project pursued blue-sky research aiming to understand a critical reaction in nature, our approaches have the potential to expand the field and allow other reactions of high medical interest to be studied by time-resolved crystallography.
This ERC project, Proton Pump, aimed to record a three-dimensional movie that reveals how proton exchange steps between key amino acid residues are controlled by structural changes in the protein cytochrome c oxidase. In this work we utilized state-of-the-art methods of time-resolved serial crystallography at X-ray free electron lasers (XFELs) and synchrotron radiation facilities to observe how structural changes in terminal oxidases develop over time. For technical reasons, we utilized a bacterial enzyme that is highly stable from which we could grow well diffracting crystals. Our experiments required a new approach that would rapidly deliver oxygen into the protein’s active site and thereby initiate the oxygen reduction reaction in microcrystals of cytochrome c oxidase. This project therefore involved multiple challenges including microcrystal quality and density, chemical stability, reaction initiation within the microcrystals, the delivery of microcrystals into an X-ray beam, and data-processing and analysis. Structural results from this project have given new insight into one of the most important chemical reactions in biology. We have also developed new tools and methods that will help to grow the field of time-resolved structural biology and allow structural studies that go beyond a handful of naturally occurring light-driven systems. Although this project pursued blue-sky research aiming to understand a critical reaction in nature, our approaches have the potential to expand the field and allow other reactions of high medical interest to be studied by time-resolved crystallography.
Lavoro eseguito dall’inizio del progetto fino alla fine del periodo coperto dalla relazione e principali risultati finora ottenuti
Proton Pump began January 1st 2019 and ended on December 31 2024. A twelve month no-cost extension was allowed to address lost time and opportunities due to the Covid-19 pandemic. Implementation of the project initially focused on building a team of biologists, chemists and other scientists with analytical skills in computing so as to address this challenging project. There was also initially a transitional phase as students who recovered preliminary results that contributed to the grant application, trained the next generation of PhD students before graduating. As such two PhD students defended their theses in 2020. Five additional PhD students joined the team and completed their education within this project. One of these graduated in 2023, two in 2024, and the remaining two in the first months of 2025.
EXPERIMENTS PERFORMED AT X-RAY USER FACILITIES
The core objective of this project was to perform time-resolved X-ray crystallography studies of structural changes in cytochrome c oxidase, an enzyme which reduces oxygen to water while transporting protons across an energy transducing biological membrane. Our planned time-resolved crystallography studies required access to X-ray free electron lasers (XFELs), which are large international X-ray infrastructures that accept user experiments in open competition. The work was therefore based around a cycle of proposing an experiment; it being evaluated by the peer review panel in competition with other proposals; being allocated experimental time at an X-ray facility (called “beamtime”); making preparations for the experiment; travelling to and XFEL and collecting time-resolved X-ray diffraction data; analysing these data back in Sweden; and drawing scientific conclusions for publication or planning changes for new experiments. We also had beamtime at synchrotron radiation facilities which complemented these XFEL studies. After the COVID-19 pandemic began in the spring of 2019, access to XFEL and synchrotron radiation facilities became extremely restricted because these facilities did not allow external users on-site. Several experiments at European XFEL facilities were postponed or cancelled. After August 2021, access to European X-ray infrastructures begun to open up. As such, most of the main results from this project emerge from XFEL experiments performed between the autumn 2021 and late summer 2024.
EXPERIMENTS PERFORMED USING XFEL AND SYNCHROTRON RADIATION FACILITIES.
In June 2019 we travelled to the Japanese XFEL (SACLA) and performed time-resolved serial femtosecond X-ray crystallography studies of the photodissociation of bound carbon monoxide from the active-site of cytochrome c oxidase. Data from this experiment and an earlier experiment were combined into an important publication from this project (Safari et al., Science Advances 2023).
In September 2021 we travelled to the European-XFEL (Hamburg) and performed our first time resolved serial femtosecond X-ray crystallography studies of the reduction of molecular dioxygen to water by cytochrome c oxidase. Unfortunately, this experiment was plagued with technical problems due to the sample delivery system and issues with the X-ray detector.
In November, 2021 we travelled to SwissFEL (Villigen, Switzerland) and performed time resolved serial femtosecond X-ray crystallography studies of the reduction of molecular dioxygen to water by cytochrome c oxidase. This was a very successful experiment, and data from this study form a major component of Zoric et al. (2024).
In May 2022 we travelled to the LCLS (Stanford, USA) and performed a repeat study of time resolved serial femtosecond X-ray crystallography studies of the reduction of molecular dioxygen to water by cytochrome c oxidase. Although this experiment faced some technical difficulties, we recorded data complementary to the above experiment and is a major component of Zoric et al. (2024).
In October 2023 we travelled again to SwissFEL (Villigen) and performed a final control study for Zoric et al. (2024). We also recovered data from a new crystal form of cytochrome c oxidase grown at higher pH than the new form.
Our final XFEL study of the project was in February 2024 at the LCLS (Stanford, USA). In that study we built upon the high-pH crystals and attempted also a time-resolved study. Unfortunately, too many technical disruptions meant that the time-resolved study was incomplete, but data from this experiment contribute to another manuscript (Johannesson et al., manuscript, 2025).
Throughout the project we also performed several other studies using synchrotron radiation. These included multiple experiments at MAX IV laboratory to develop new tools for serial X-ray crystallography data-collection using synchrotron radiation. One of these works, the development of a flow-cell, is published (Ghosh et al., 2023) and a second work on the development of a simple fixed-target system is in manuscript form (Ghosh et al., 2023).
We also performed photo-reduction studies of cytochrome c oxidase using both single-crystals and microcrystals, at the ESRF (Grenoble, France) and PETRA III (Hamburg, Germany). These were very successful and nicely complement our time-resolved serial femtosecond X-ray crystallography data recorded using XFEL radiation. A manuscript describing this work will be presented in an upcoming PhD thesis (Kabbinale et al., 2025). Another approach was to modify the flow-cell developed in this project and perform mixing studies using synchrotron radiation. Data collected at the ESRF using this approach were successful and, along with other data collected at SwissFEL, form the basis of another manuscript (Kabbinale et al., 2025).
Finally, a series of X-ray spectroscopy experiments were performed at MAX IV Laboratory on samples of cytochrome c oxidase. This work was written up in a manuscript (Zoric et al., 2023) but may require additional studies to bring forward to publication. Likewise, we performed very successful studies of time-resolved X-ray solution studies of cytochrome c oxidase in solution using both photocaged oxygen and carbon monoxide at the ESRF (Grenoble, France). However, due to the need to model these data using molecular dynamics simulations, we have not yet brought that work to the point of a written manuscript.
As part of the project, a lot of work was performed on the organic chemistry of oxygen photocages. This work is now presented in two publications (Sandelin et al., 2024; Sandelin et al., 2024). We also made theoretical and practical developments to the study of structural changes in macromolecules using time-resolved serial crystallography (Vallejos et al, 2024) and time-resolved X-ray solution scattering (Sarabi et al., 2022).
EXPERIMENTS PERFORMED AT X-RAY USER FACILITIES
The core objective of this project was to perform time-resolved X-ray crystallography studies of structural changes in cytochrome c oxidase, an enzyme which reduces oxygen to water while transporting protons across an energy transducing biological membrane. Our planned time-resolved crystallography studies required access to X-ray free electron lasers (XFELs), which are large international X-ray infrastructures that accept user experiments in open competition. The work was therefore based around a cycle of proposing an experiment; it being evaluated by the peer review panel in competition with other proposals; being allocated experimental time at an X-ray facility (called “beamtime”); making preparations for the experiment; travelling to and XFEL and collecting time-resolved X-ray diffraction data; analysing these data back in Sweden; and drawing scientific conclusions for publication or planning changes for new experiments. We also had beamtime at synchrotron radiation facilities which complemented these XFEL studies. After the COVID-19 pandemic began in the spring of 2019, access to XFEL and synchrotron radiation facilities became extremely restricted because these facilities did not allow external users on-site. Several experiments at European XFEL facilities were postponed or cancelled. After August 2021, access to European X-ray infrastructures begun to open up. As such, most of the main results from this project emerge from XFEL experiments performed between the autumn 2021 and late summer 2024.
EXPERIMENTS PERFORMED USING XFEL AND SYNCHROTRON RADIATION FACILITIES.
In June 2019 we travelled to the Japanese XFEL (SACLA) and performed time-resolved serial femtosecond X-ray crystallography studies of the photodissociation of bound carbon monoxide from the active-site of cytochrome c oxidase. Data from this experiment and an earlier experiment were combined into an important publication from this project (Safari et al., Science Advances 2023).
In September 2021 we travelled to the European-XFEL (Hamburg) and performed our first time resolved serial femtosecond X-ray crystallography studies of the reduction of molecular dioxygen to water by cytochrome c oxidase. Unfortunately, this experiment was plagued with technical problems due to the sample delivery system and issues with the X-ray detector.
In November, 2021 we travelled to SwissFEL (Villigen, Switzerland) and performed time resolved serial femtosecond X-ray crystallography studies of the reduction of molecular dioxygen to water by cytochrome c oxidase. This was a very successful experiment, and data from this study form a major component of Zoric et al. (2024).
In May 2022 we travelled to the LCLS (Stanford, USA) and performed a repeat study of time resolved serial femtosecond X-ray crystallography studies of the reduction of molecular dioxygen to water by cytochrome c oxidase. Although this experiment faced some technical difficulties, we recorded data complementary to the above experiment and is a major component of Zoric et al. (2024).
In October 2023 we travelled again to SwissFEL (Villigen) and performed a final control study for Zoric et al. (2024). We also recovered data from a new crystal form of cytochrome c oxidase grown at higher pH than the new form.
Our final XFEL study of the project was in February 2024 at the LCLS (Stanford, USA). In that study we built upon the high-pH crystals and attempted also a time-resolved study. Unfortunately, too many technical disruptions meant that the time-resolved study was incomplete, but data from this experiment contribute to another manuscript (Johannesson et al., manuscript, 2025).
Throughout the project we also performed several other studies using synchrotron radiation. These included multiple experiments at MAX IV laboratory to develop new tools for serial X-ray crystallography data-collection using synchrotron radiation. One of these works, the development of a flow-cell, is published (Ghosh et al., 2023) and a second work on the development of a simple fixed-target system is in manuscript form (Ghosh et al., 2023).
We also performed photo-reduction studies of cytochrome c oxidase using both single-crystals and microcrystals, at the ESRF (Grenoble, France) and PETRA III (Hamburg, Germany). These were very successful and nicely complement our time-resolved serial femtosecond X-ray crystallography data recorded using XFEL radiation. A manuscript describing this work will be presented in an upcoming PhD thesis (Kabbinale et al., 2025). Another approach was to modify the flow-cell developed in this project and perform mixing studies using synchrotron radiation. Data collected at the ESRF using this approach were successful and, along with other data collected at SwissFEL, form the basis of another manuscript (Kabbinale et al., 2025).
Finally, a series of X-ray spectroscopy experiments were performed at MAX IV Laboratory on samples of cytochrome c oxidase. This work was written up in a manuscript (Zoric et al., 2023) but may require additional studies to bring forward to publication. Likewise, we performed very successful studies of time-resolved X-ray solution studies of cytochrome c oxidase in solution using both photocaged oxygen and carbon monoxide at the ESRF (Grenoble, France). However, due to the need to model these data using molecular dynamics simulations, we have not yet brought that work to the point of a written manuscript.
As part of the project, a lot of work was performed on the organic chemistry of oxygen photocages. This work is now presented in two publications (Sandelin et al., 2024; Sandelin et al., 2024). We also made theoretical and practical developments to the study of structural changes in macromolecules using time-resolved serial crystallography (Vallejos et al, 2024) and time-resolved X-ray solution scattering (Sarabi et al., 2022).
Progressi oltre lo stato dell’arte e potenziale impatto previsto (incluso l’impatto socioeconomico e le implicazioni sociali più ampie del progetto fino ad ora)
We have made several important methodology developments concerning the analysis of time-resolved serial crystallography data and time-resolved X-ray solution scattering data. These include developing new tools to make synchrotron radiation studies easier for ourselves and others. We also developed new theoretical tools for the quantification and analysis of electron density changes in time-resolved crystallography studies, as well as combined molecular dynamics simulations of proteins within detergent micelles to analyse time-resolved X-ray solution scattering data. Our repeated experiments using photocaged oxygen to study structural changes in cytochrome c oxidase pushed the boundaries of the field of time-resolved X-ray crystallography, since this represents an important step beyond the study of naturally light-driven systems.
Since the project is now at an end, we report below all publications from the project, manuscripts presented in PhD theses, and PhD theses that were supported by the ERC grant.
PUBLICATIONS THAT ACKNOWLEDGE ERC SUPPORT:
R. Andersson, C. Safari, P. Båth, R. Bosman, A. Shilova, P. Dahl, S. Ghosh, A. Dunge, R. Kjeldsen-Jensen, J. Nan, R.L. Shoeman, M. Kloos, R.B. Doak, U. Mueller, R. Neutze, G. Brändén, Well-based crystallization of lipidic cubic phase microcrystals for serial X-ray crystallography experiments. Acta Crystallogr D Struct Biol 75, 937-946. (2019).
R. Dods, P. Båth, D. Morozov, V.A. Gagnér, D. Arnlund, H.L. Luk, J. Kübel, M. Maj, A. Vallejos, C. Wickstrand, R. Bosman, K.R. Beyerlein, G. Nelson, M. Liang, D. Milathianaki, J. Robinson, R. Harimoorthy, P. Berntsen, E. Malmerberg, L. Johansson, R. Andersson, S. Carbajo, E. Claesson, C.E. Conrad, P. Dahl, G. Hammarin, M.S. Hunter, C. Li, S. Lisova, A. Royant, C. Safari, A. Sharma, G.J. Williams, O. Yefanov, S. Westenhoff, J. Davidsson, D.P. DePonte, S. Boutet, A. Barty, G. Katona, G. Groenhof, G. Brändén, R. Neutze, Ultrafast structural changes within a photosynthetic reaction centre. Nature 589: 310-314. (2021).
Branden, G., R. Neutze, Advances and challenges in time-resolved macromolecular crystallography. Science 373, eaba0954 (2021).
P. Båth, A. Banacore, P. Börjesson, R. Bosman, C. Wickstrand, C. Safari, R. Dods, S. Ghosh, P. Dahl, G. Ortolani, T. Björg Ulfarsdottir, G. Hammarin, M.J. García-Bonete, A. Vallejos, L. Ostojic, P. Edlund, J.B. Linse, R. Andersson, E. Nango, S. Owada, R. Tanaka, K. Tono, Y. Joti, O. Nureki, F. Luo, D. James, K. Nass, P.J.M. Johnson, G. Knopp, D. Ozerov, C. Cirelli, C. Milne, S. Iwata, G. Brändén, R. Neutze, Lipidic cubic phaser serial femtosecond crystallography structure of a photosynthetic reaction centre, Acta Crystallographica D78, 698–708 (2022).
D. Sarabi, L. Ostojić, R. Bosman, A. Vallejos, J.-B. Linse, M. Wulff, M. Levantino, R. Neutze, Modelling difference X-ray scattering observations from an integral membrane protein within a detergent micelle, Structural Dynamics 9, 054102 (2022).
S. Ghosh, D. Zorić, P. Dahl, M. Bjelčić, J. Johannesson, E. Sandelin, P. Borjesson, A. Björling, A. Banacore, P. Edlund, O. Aurelius, M. Milas, J. Nan, A. Shilova, A. Gonzalez, U. Mueller, G. Brändén, R. Neutze, A simple goniometer compatible flow-cell for serial synchrotron X-ray crystallography, J. J. Appl. Cryst. 56, 449–460 (2023).
C. Safari, S. Ghosh, R. Andersson, J. Johannesson, P. Båth, O. Uwangue, P. Dahl, D. Zoric, E. Sandelin, A. Vallejos, E. Nango, R. Tanaka, R. Bosman, P. Börjesson, E. Dunevall, G. Hammarin, G. Ortolani, M. Panman, T. Tanaka, A. Yamashita, T. Arima, M. Sugahara, M. Suzuki, T. Masuda, H. Takeda, R. Yamagiwa, K. Oda, M. Fukuda, T. Tosha, H. Naitow, S. Owada, K. Tono, O. Nureki, S. Iwata, R. Neutze, G. Brändén, Time-resolved serial crystallography to track the dynamics of carbon monoxide in the active site of cytochrome c oxidase, Science Advances, 9, eadh4179 (2023).
E. Sandelin, J. Johannesson, O. Wendt, G. Brändén, R. Neutze, C. J. Wallentin, Characterization and evaluation of photolabile (µ‑peroxo)(µ‑hydroxo)bis[bis(bipyridyl)cobalt caged oxygen compounds to facilitate time‑resolved crystallographic studies of cytochrome c oxidase, Photochem. Photobiol. Sci. 23, 839–851 (2024).
E. Sandelin, L. Schilling, E. Saha, A. Ruiu, R. Neutze, H. Sundén, C.J. Wallentin, Spatiotemporal Release of Singlet Oxygen in Low Molecular Weight Organo-Gels Upon Thermal or Photochemical External Stimuli, Small 20, 2400827 (2024).
M. Bjelčić, O. Aurelius, J. Nan, R. Neutze, T. Ursby, Room-temperature serial synchrotron crystallography structure of Spinacia oleracea RuBisCO, Acta crystallogr. F 80, 117-124 (2024).
A. Vallejos, G. Katona, R. Neutze, Appraising protein conformational changes by resampling time-resolved serial x-ray, crystallography data. Struct. Dyn. 11, 044302 (2024).
R. Bosman, G. Ortolani, S. Ghosh, D. James, P. Norder, G. Hammarin, T. Björg Úlfarsdóttir, L. Ostojić, T. Weinert, F. Dworkowski, T. Tomizaki, J. Standfuss, G. Brändén, R. Neutze, Structural basis for the prolonged photocycle of Sensory Rhodopsin II revealed by serial synchrotron crystallography, Nature communications, accepted (2025).
UNPUBLISHED MANUSCRIPTS THAT ACKNOWLEDGE ERC SUPPORT AND ARE PRESENTED IN PHD THESES
D. Zorić, J. Johannesson, A. Vallejos, E. Sandelin, A. Kabbinale, S. Ghosh, A.D. Finke, M. Bjelčić, J. Rönnholm, L. Ostojić, J. Glerup, P. Dahl, E.V. Beale, C. Cirelli, F. Dworkowski, C. Bacellar, P.J.M. Johnson, D. Ozerov, A. Batyuk, S. Boutet, G.M. Gate, C. Kupitz, A. N. Peck, F. Poitevin, R. Sierra, S. Lisova, C.J. Wallentin, G. Brändén, R. Neutze, Structural changes in cytochrome c oxidase following the reduction of dioxygen to water, submitted manuscript (2024).
J. Johannesson, A. Kabbinale, D. Zorić, E. Sandelin, A. Vallejos, J. Rönnholm, J. Glerup, L. Ostojic, A. Aquila, G. Gate, F. Poitevin, S. Lisova, S. Mous, M. Kashipathy, C.J. Wallentin, R. Neutze, G. Brändén, Drastically altered structure of the cytochrome c oxidase active site visualized by serial femtosecond crystallography, manuscript (2024).
A. Kabbinale, J. Johannesson, G. Hammarin, A. Vallejos, N. Caramello, S. Engilberge, S. Rose, A. Royant, D. von Stetten, A. Pearson, G. Brändén, R. Neutze, Photoreduction induced structural changes in cytochrome c oxidase, manuscript (2025).
A. Kabbinale, J. Johannesson, E. Sandelin, A. Vallejos, D. Zorić, S. Ghosh, P. Dahl, A. Banacore, G. Hammarin, D de Sanctis, S. Basu, J. Orlans, S. Rose, E. V. Beale, C. Cirelli, F. Dworkowski, C. Bacellar, P.J. M. Johnson, D. Ozerov, C.J. Wallentin, G. Brändén, R. Neutze, Observation of the accumulation of peroxide at the active site of cytochrome c oxidase in the presence of sodium dithionite, manuscript (2025).
S. Ghosh, A. Banacore, P. Börjesson, M. Bjelcic, A. Kabbinale, P. Nileshwar, G. Wellhander, D. de Sanctis, S. Basu, J. Orlans, L.M.G. Chavas, R. Neutze, G. Brändén, A user-friendly goniometer-compatible fixed-target platform for macromolecular crystallography at synchrotrons, manuscript (2025).
J. Johannesson, R. Wahlström, J. Gräfenstein, R. Neutze, G. Brändén, CASSCF/NEVPT2 study of the structural identity of the proton induced Q-band blue shift of the P to F transition in cytochrome c oxidase, manuscript (2024).
J. Johannesson, J. Gräfenstein, R. Neutze, G. Brändén, Quantum chemical investigation of the active site proton donor of cytochrome c oxidase suggests novel identity, manuscript (2024).
D. Zorić, S. Ghosh, M. Bjelčić, J. Johannesson, A. Kabbinale, E. Sandelin, Y. Chen, S. Nehzati, R. Neutze, K. Sigfridsson Clauss and G. Brändén. X-ray absorption spectroscopy as a tool to investigate details of the cytochrome c oxidase metal co-factors. Manuscript (2023)
One other significant result from the project has not yet been written as a manuscript. This concerns time-resolved X-ray solution scattering studies of cytochrome c oxidase using photocaged oxygen. Other resources have been committed to bring that work to completion.
PHD THESES PARTIALLY SUPPORTED BY THE ERC GRANT
Cecilia Safari, Microcrystallization in lipidic cubic phase and serial crystallography studies of cytochrome c oxidase, November 11, 2019.
Rebecka Andersson, Lipidic cubic phase microcrystallization and its application in serial crystallography, November 23, 2020.
PHD THESES FULLY SUPPORTED BY THE ERC GRANT
Doris Zoric, Structural studies of ba3-type cytochrome c oxidase using serial crystallography and X-ray absorption spectroscopy, August 29, 3023.
Emil Sandelin, Breaking the cage – implementing photocages to address spatiotemporal challenges in chemical biology, April 16, 2024.
Jonatan Johannesson, Time-Resolved X-ray Crystallography and Quantum Chemical Calculations of the Proton Pumping Mechanism in Cytochrome c Oxidase, December 13, 2024.
Adams Vallejos, Computational tools for the analysis of time-resolved serial X-ray crystallography data, November 11, 2024.
Arpitha Kabbinale, Structural and functional insights into ba3-type cytochrome c oxidase using X-ray crystallography and in crystallo spectroscopy, March 17, 2025.
Since the project is now at an end, we report below all publications from the project, manuscripts presented in PhD theses, and PhD theses that were supported by the ERC grant.
PUBLICATIONS THAT ACKNOWLEDGE ERC SUPPORT:
R. Andersson, C. Safari, P. Båth, R. Bosman, A. Shilova, P. Dahl, S. Ghosh, A. Dunge, R. Kjeldsen-Jensen, J. Nan, R.L. Shoeman, M. Kloos, R.B. Doak, U. Mueller, R. Neutze, G. Brändén, Well-based crystallization of lipidic cubic phase microcrystals for serial X-ray crystallography experiments. Acta Crystallogr D Struct Biol 75, 937-946. (2019).
R. Dods, P. Båth, D. Morozov, V.A. Gagnér, D. Arnlund, H.L. Luk, J. Kübel, M. Maj, A. Vallejos, C. Wickstrand, R. Bosman, K.R. Beyerlein, G. Nelson, M. Liang, D. Milathianaki, J. Robinson, R. Harimoorthy, P. Berntsen, E. Malmerberg, L. Johansson, R. Andersson, S. Carbajo, E. Claesson, C.E. Conrad, P. Dahl, G. Hammarin, M.S. Hunter, C. Li, S. Lisova, A. Royant, C. Safari, A. Sharma, G.J. Williams, O. Yefanov, S. Westenhoff, J. Davidsson, D.P. DePonte, S. Boutet, A. Barty, G. Katona, G. Groenhof, G. Brändén, R. Neutze, Ultrafast structural changes within a photosynthetic reaction centre. Nature 589: 310-314. (2021).
Branden, G., R. Neutze, Advances and challenges in time-resolved macromolecular crystallography. Science 373, eaba0954 (2021).
P. Båth, A. Banacore, P. Börjesson, R. Bosman, C. Wickstrand, C. Safari, R. Dods, S. Ghosh, P. Dahl, G. Ortolani, T. Björg Ulfarsdottir, G. Hammarin, M.J. García-Bonete, A. Vallejos, L. Ostojic, P. Edlund, J.B. Linse, R. Andersson, E. Nango, S. Owada, R. Tanaka, K. Tono, Y. Joti, O. Nureki, F. Luo, D. James, K. Nass, P.J.M. Johnson, G. Knopp, D. Ozerov, C. Cirelli, C. Milne, S. Iwata, G. Brändén, R. Neutze, Lipidic cubic phaser serial femtosecond crystallography structure of a photosynthetic reaction centre, Acta Crystallographica D78, 698–708 (2022).
D. Sarabi, L. Ostojić, R. Bosman, A. Vallejos, J.-B. Linse, M. Wulff, M. Levantino, R. Neutze, Modelling difference X-ray scattering observations from an integral membrane protein within a detergent micelle, Structural Dynamics 9, 054102 (2022).
S. Ghosh, D. Zorić, P. Dahl, M. Bjelčić, J. Johannesson, E. Sandelin, P. Borjesson, A. Björling, A. Banacore, P. Edlund, O. Aurelius, M. Milas, J. Nan, A. Shilova, A. Gonzalez, U. Mueller, G. Brändén, R. Neutze, A simple goniometer compatible flow-cell for serial synchrotron X-ray crystallography, J. J. Appl. Cryst. 56, 449–460 (2023).
C. Safari, S. Ghosh, R. Andersson, J. Johannesson, P. Båth, O. Uwangue, P. Dahl, D. Zoric, E. Sandelin, A. Vallejos, E. Nango, R. Tanaka, R. Bosman, P. Börjesson, E. Dunevall, G. Hammarin, G. Ortolani, M. Panman, T. Tanaka, A. Yamashita, T. Arima, M. Sugahara, M. Suzuki, T. Masuda, H. Takeda, R. Yamagiwa, K. Oda, M. Fukuda, T. Tosha, H. Naitow, S. Owada, K. Tono, O. Nureki, S. Iwata, R. Neutze, G. Brändén, Time-resolved serial crystallography to track the dynamics of carbon monoxide in the active site of cytochrome c oxidase, Science Advances, 9, eadh4179 (2023).
E. Sandelin, J. Johannesson, O. Wendt, G. Brändén, R. Neutze, C. J. Wallentin, Characterization and evaluation of photolabile (µ‑peroxo)(µ‑hydroxo)bis[bis(bipyridyl)cobalt caged oxygen compounds to facilitate time‑resolved crystallographic studies of cytochrome c oxidase, Photochem. Photobiol. Sci. 23, 839–851 (2024).
E. Sandelin, L. Schilling, E. Saha, A. Ruiu, R. Neutze, H. Sundén, C.J. Wallentin, Spatiotemporal Release of Singlet Oxygen in Low Molecular Weight Organo-Gels Upon Thermal or Photochemical External Stimuli, Small 20, 2400827 (2024).
M. Bjelčić, O. Aurelius, J. Nan, R. Neutze, T. Ursby, Room-temperature serial synchrotron crystallography structure of Spinacia oleracea RuBisCO, Acta crystallogr. F 80, 117-124 (2024).
A. Vallejos, G. Katona, R. Neutze, Appraising protein conformational changes by resampling time-resolved serial x-ray, crystallography data. Struct. Dyn. 11, 044302 (2024).
R. Bosman, G. Ortolani, S. Ghosh, D. James, P. Norder, G. Hammarin, T. Björg Úlfarsdóttir, L. Ostojić, T. Weinert, F. Dworkowski, T. Tomizaki, J. Standfuss, G. Brändén, R. Neutze, Structural basis for the prolonged photocycle of Sensory Rhodopsin II revealed by serial synchrotron crystallography, Nature communications, accepted (2025).
UNPUBLISHED MANUSCRIPTS THAT ACKNOWLEDGE ERC SUPPORT AND ARE PRESENTED IN PHD THESES
D. Zorić, J. Johannesson, A. Vallejos, E. Sandelin, A. Kabbinale, S. Ghosh, A.D. Finke, M. Bjelčić, J. Rönnholm, L. Ostojić, J. Glerup, P. Dahl, E.V. Beale, C. Cirelli, F. Dworkowski, C. Bacellar, P.J.M. Johnson, D. Ozerov, A. Batyuk, S. Boutet, G.M. Gate, C. Kupitz, A. N. Peck, F. Poitevin, R. Sierra, S. Lisova, C.J. Wallentin, G. Brändén, R. Neutze, Structural changes in cytochrome c oxidase following the reduction of dioxygen to water, submitted manuscript (2024).
J. Johannesson, A. Kabbinale, D. Zorić, E. Sandelin, A. Vallejos, J. Rönnholm, J. Glerup, L. Ostojic, A. Aquila, G. Gate, F. Poitevin, S. Lisova, S. Mous, M. Kashipathy, C.J. Wallentin, R. Neutze, G. Brändén, Drastically altered structure of the cytochrome c oxidase active site visualized by serial femtosecond crystallography, manuscript (2024).
A. Kabbinale, J. Johannesson, G. Hammarin, A. Vallejos, N. Caramello, S. Engilberge, S. Rose, A. Royant, D. von Stetten, A. Pearson, G. Brändén, R. Neutze, Photoreduction induced structural changes in cytochrome c oxidase, manuscript (2025).
A. Kabbinale, J. Johannesson, E. Sandelin, A. Vallejos, D. Zorić, S. Ghosh, P. Dahl, A. Banacore, G. Hammarin, D de Sanctis, S. Basu, J. Orlans, S. Rose, E. V. Beale, C. Cirelli, F. Dworkowski, C. Bacellar, P.J. M. Johnson, D. Ozerov, C.J. Wallentin, G. Brändén, R. Neutze, Observation of the accumulation of peroxide at the active site of cytochrome c oxidase in the presence of sodium dithionite, manuscript (2025).
S. Ghosh, A. Banacore, P. Börjesson, M. Bjelcic, A. Kabbinale, P. Nileshwar, G. Wellhander, D. de Sanctis, S. Basu, J. Orlans, L.M.G. Chavas, R. Neutze, G. Brändén, A user-friendly goniometer-compatible fixed-target platform for macromolecular crystallography at synchrotrons, manuscript (2025).
J. Johannesson, R. Wahlström, J. Gräfenstein, R. Neutze, G. Brändén, CASSCF/NEVPT2 study of the structural identity of the proton induced Q-band blue shift of the P to F transition in cytochrome c oxidase, manuscript (2024).
J. Johannesson, J. Gräfenstein, R. Neutze, G. Brändén, Quantum chemical investigation of the active site proton donor of cytochrome c oxidase suggests novel identity, manuscript (2024).
D. Zorić, S. Ghosh, M. Bjelčić, J. Johannesson, A. Kabbinale, E. Sandelin, Y. Chen, S. Nehzati, R. Neutze, K. Sigfridsson Clauss and G. Brändén. X-ray absorption spectroscopy as a tool to investigate details of the cytochrome c oxidase metal co-factors. Manuscript (2023)
One other significant result from the project has not yet been written as a manuscript. This concerns time-resolved X-ray solution scattering studies of cytochrome c oxidase using photocaged oxygen. Other resources have been committed to bring that work to completion.
PHD THESES PARTIALLY SUPPORTED BY THE ERC GRANT
Cecilia Safari, Microcrystallization in lipidic cubic phase and serial crystallography studies of cytochrome c oxidase, November 11, 2019.
Rebecka Andersson, Lipidic cubic phase microcrystallization and its application in serial crystallography, November 23, 2020.
PHD THESES FULLY SUPPORTED BY THE ERC GRANT
Doris Zoric, Structural studies of ba3-type cytochrome c oxidase using serial crystallography and X-ray absorption spectroscopy, August 29, 3023.
Emil Sandelin, Breaking the cage – implementing photocages to address spatiotemporal challenges in chemical biology, April 16, 2024.
Jonatan Johannesson, Time-Resolved X-ray Crystallography and Quantum Chemical Calculations of the Proton Pumping Mechanism in Cytochrome c Oxidase, December 13, 2024.
Adams Vallejos, Computational tools for the analysis of time-resolved serial X-ray crystallography data, November 11, 2024.
Arpitha Kabbinale, Structural and functional insights into ba3-type cytochrome c oxidase using X-ray crystallography and in crystallo spectroscopy, March 17, 2025.