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This issue features seven papers covering mass photometry and single-molecule measurements, cryo-EM model building and conformational ensembles, and membrane protein transport and lipid signaling.
Mass Photometry and Single-Molecule Measurements (3 papers)
01 · Quantitative mass imaging of single biological macromolecules
Science · Publication/issue date: 2018-04-27
Authors: Gavin Young, Nikolas Hundt, Daniel Cole, Adam Fineberg, Joanna Andrecka, Andrew Tyler, Anna Olerinyova, Ayla Ansari, Erik G. Marklund, Miranda P. Collier, Shane A. Chandler, Olga Tkachenko, Joel Allen, Max Crispin, Neil Billington, Yasuharu Takagi, James R. Sellers, Cédric Eichmann, Philipp Selenko, Lukas Frey, Roland Riek, Martin R. Galpin, Weston B. Struwe, Justin L. P. Benesch, Philipp Kukura
Proteins and their interactions exhibit structural and dynamic heterogeneity, making it difficult for ensemble-averaged measurements to distinguish individual components directly. This study uses interferometric scattering microscopy to quantify the mass of individual biological macromolecules in solution, reporting approximately 2% accuracy relative to sequence-derived mass, mass resolution as fine as 19 kDa, and precision of 1 kDa. The method resolves oligomer distributions across a broad dynamic range, detects small-molecule binding, and measures the mass of proteins containing lipid or carbohydrate components. The authors also apply it to glycoprotein crosslinking, amyloid aggregation, and actin polymerization, demonstrating how changes in mass can reveal molecular interactions. By converting optical scattering signals into single-molecule mass readouts, the work offers a complementary tool for studying the composition and dynamics of heterogeneous systems.
Highlight · Interferometric scattering imaging uses single-molecule mass readouts to distinguish oligomeric species and monitor a range of biomolecular interactions.
02 · Mass photometry enables label-free tracking and mass measurement of single proteins on lipid bilayers
Nature Methods · Publication/issue date: 2021-10
Authors: Eric D. B. Foley, Manish S. Kushwah, Gavin Young, Philipp Kukura
The stoichiometry and dynamics of molecular interactions at membrane surfaces are central to understanding cellular processes. However, conventional single-molecule methods often rely on fluorescent labels, with limitations arising from labeling efficiency, blinking, and photobleaching. This paper introduces dynamic mass photometry for label-free imaging, tracking, and mass measurement of individual membrane-associated proteins diffusing on supported lipid bilayers. Using the membrane-remodeling GTPase dynamin-1, the study identifies a heterogeneous mixture of oligomers built from dimers and reveals differences in mobility and membrane affinity across oligomeric states, as well as association and dissociation events in individual complexes. Measuring mass and trajectories together links the composition of protein complexes to their behavior on membranes. The authors also discuss how complementary measurement approaches could extend the method to integral membrane proteins, providing a methodological foundation for probing molecular mechanisms within and on lipid bilayers.
Highlight · Dynamic mass photometry links the mass of individual proteins on membranes to their diffusion and complex assembly behavior.
Authors: Dan Loewenthal, Roi Asor, Stephen Thorpe, Jack S. Peters, Konstantin C. Zouboulis, Merve Kaplan, Jan Christoph Thiele, Jack Bardzil, Seham Helmi, Sean A. Burnap, Tiong Kit Tan, Weston B. Struwe, Justin L. P. Benesch, Philipp Kukura
Biomolecular interactions often involve successive binding, dissociation, and state transitions, making it difficult to capture the full process in short observations. Continuous mass photometry uses single-molecule trapping to confine molecules on supported lipid bilayers within a region where they can be monitored continuously, while recording their motion and changes in mass. The authors demonstrate single-molecule observations lasting up to hours and report mass precision reaching the sub-kilodalton range and millisecond temporal resolution. Following the same molecule for longer allows interaction-induced mass states to be resolved sequentially over time, enabling analysis of binding events and dwell times. The paper also notes that greater temporal averaging improves mass precision at the expense of temporal resolution, so these performance levels are not achieved simultaneously across all timescales. The method provides continuous, quantitative traces for directly observing complex molecular processes.
Highlight · Single-molecule trapping extends the observation window of mass photometry, allowing molecular interactions to be tracked continuously over time.
Authors: Baoquan Su, Kun Huang, Zhenling Peng, Alexey Amunts, Jianyi Yang
Building atomic models from cryo-EM density maps is a crucial step in translating experimental signals into explanations of molecular mechanisms. Building on ModelAngelo, CryoAtom incorporates AlphaFold2's modeling capabilities, replaces global attention with local attention, and uses three-dimensional rotary positional encoding to accommodate density-map information. The authors report that the method produces more complete models, works with lower-resolution maps, and speeds up model building. Applications to three large density maps demonstrate its ability to identify previously uncharacterized proteins, improve modeling of conformational changes, and separate out non-protein components. In one case, a complex containing 104 proteins was modeled within hours; comparing models from two maps also revealed subtle conformational differences in a single peripheral protein domain. The work highlights how experimental density constraints and deep learning can work together to make complex structures easier to interpret.
Highlight · CryoAtom combines local attention with density-map information to build more complete models of large cryo-EM structures more efficiently.
05 · Cooling fast and slow: Characterizing the effects of vitrification in cryo-EM and the subsequent recovery of equilibrium populations
Proceedings of the National Academy of Sciences · Publication/issue date: 2026-11-10
Authors: Robert Clark, Louis G. Smith, Matthew P. Leighton, Ryan J. Szukalo, Syma Khalid, Pablo G. Debenedetti, Pilar Cossio, Miro A. Astore, Sonya M. Hanson
Cryo-EM has the potential to reveal molecular conformational ensembles, but interpreting them depends on whether cooling during vitrification alters the original state populations. This study uses more than 50 milliseconds of aggregate molecular dynamics simulations of the Trp-cage miniprotein to compare seven cooling rates spanning three orders of magnitude, with the slowest approaching experimental rates. In the system studied, the protein did not alter the vitrification behavior of water. A Markov state model built from equilibrium sampling showed that states with characteristic timescales longer than the cooling process were more resistant to cooling-induced perturbations. Some states disappeared from low-temperature equilibrium ensembles yet persisted in nonequilibrium cooled ensembles. The authors also propose a thermodynamic inference framework for recovering equilibrium populations from vitrified ensembles, offering a testable approach to interpreting conformational distributions in cryo-EM.
Highlight · The effects of rapid cooling on conformational distributions depend on state-transition timescales, and equilibrium populations can be inferred using a thermodynamic framework.
Vesicular monoamine transporter 2 (VMAT2) loads neurotransmitters into presynaptic vesicles, and its modulation by drugs directly affects the intracellular distribution of monoamines. This study resolves cryo-EM structures of human VMAT2 bound to four amphetamine-type stimulants and to lobeline, revealing distinct recognition modes for the two classes of molecules. The amphetamines mimic endogenous monoamines and occupy the substrate-binding site; the structures support their role as substrates that contribute to redistributing monoamine pools between vesicles and the cytoplasm. Lobeline, by contrast, binds in a two-molecule arrangement that stabilizes the transporter in a vesicle-lumen-facing conformation, inhibiting uptake of monoamines and related stimulants. Dopamine-release experiments further suggest that its effects involve both VMAT2 and the dopamine transporter DAT. The study provides a mechanistic basis for understanding addiction-related pharmacology, though the structural and cellular findings remain basic research evidence.
Highlight · Distinct ligand-binding modes in VMAT2 reveal the structural basis for regulating monoamine storage and release.
Authors: Yunhai Li, Qingning Yuan, Zhenyu Cao, Liuqing Zhu, Jiuyin Xu, Cheng-Cen Guo, James Jiqi Wang, Youwei Xu, Shuai Li, Tianyu Li, Yang Li, Yong Li, H. Eric Xu, Canrong Wu
Phospholipid phosphatase PLPP1 helps terminate lipid signals such as lysophosphatidic acid signaling, but how it assembles and catalyzes reactions at the membrane interface has remained unclear. This study presents cryo-EM structures of human PLPP1 in ligand-free and product-bound states at resolutions of 1.87 Å and 2.21 Å, respectively, showing that it forms a homotetramer with fourfold symmetry. Ordered lipid densities at the subunit interfaces are assigned to phosphatidylcholine, phosphatidic acid, and phosphatidylinositol bisphosphate. Structure-guided mutagenesis shows that residues surrounding these interfacial lipids affect enzymatic activity, but loss of activity does not simply correspond to tetramer disassembly. The product-bound structure also reveals the catalytic pocket and membrane-facing channels, suggesting possible substrate-entry routes; no associated ionic conductance was detected under the conditions tested. These findings provide a structural framework linking the lipid environment, oligomeric assembly, and catalytic function.
Highlight · PLPP1 structures link interfacial lipids, tetramer assembly, and the catalytic pocket, while showing that changes in enzymatic activity cannot be explained by oligomer disassembly alone.
This issue features seven papers on single-molecule measurements, cryo-electron microscopy, and membrane protein mechanisms. Mass photometry enables label-free mass measurements, tracking on membranes, and continuous observation. CryoAtom streamlines structural modeling, while research on cooling reveals shifts in conformational distributions and suggests an approach to inferring equilibrium populations. Structural studies of VMAT2 and PLPP1 elucidate ligand regulation and lipid-associated assembly and catalysis, respectively.
This issue features seven papers covering mass photometry and single-molecule measurements, cryo-EM model building and conformational ensembles, and membrane protein transport and lipid signaling.
Mass Photometry and Single-Molecule Measurements (3 papers)
01 · Quantitative mass imaging of single biological macromolecules
Science · Publication/issue date: 2018-04-27
Authors: Gavin Young, Nikolas Hundt, Daniel Cole, Adam Fineberg, Joanna Andrecka, Andrew Tyler, Anna Olerinyova, Ayla Ansari, Erik G. Marklund, Miranda P. Collier, Shane A. Chandler, Olga Tkachenko, Joel Allen, Max Crispin, Neil Billington, Yasuharu Takagi, James R. Sellers, Cédric Eichmann, Philipp Selenko, Lukas Frey, Roland Riek, Martin R. Galpin, Weston B. Struwe, Justin L. P. Benesch, Philipp Kukura
Proteins and their interactions exhibit structural and dynamic heterogeneity, making it difficult for ensemble-averaged measurements to distinguish individual components directly. This study uses interferometric scattering microscopy to quantify the mass of individual biological macromolecules in solution, reporting approximately 2% accuracy relative to sequence-derived mass, mass resolution as fine as 19 kDa, and precision of 1 kDa. The method resolves oligomer distributions across a broad dynamic range, detects small-molecule binding, and measures the mass of proteins containing lipid or carbohydrate components. The authors also apply it to glycoprotein crosslinking, amyloid aggregation, and actin polymerization, demonstrating how changes in mass can reveal molecular interactions. By converting optical scattering signals into single-molecule mass readouts, the work offers a complementary tool for studying the composition and dynamics of heterogeneous systems.
Highlight · Interferometric scattering imaging uses single-molecule mass readouts to distinguish oligomeric species and monitor a range of biomolecular interactions.
DOI: 10.1126/science.aar5839
02 · Mass photometry enables label-free tracking and mass measurement of single proteins on lipid bilayers
Nature Methods · Publication/issue date: 2021-10
Authors: Eric D. B. Foley, Manish S. Kushwah, Gavin Young, Philipp Kukura
The stoichiometry and dynamics of molecular interactions at membrane surfaces are central to understanding cellular processes. However, conventional single-molecule methods often rely on fluorescent labels, with limitations arising from labeling efficiency, blinking, and photobleaching. This paper introduces dynamic mass photometry for label-free imaging, tracking, and mass measurement of individual membrane-associated proteins diffusing on supported lipid bilayers. Using the membrane-remodeling GTPase dynamin-1, the study identifies a heterogeneous mixture of oligomers built from dimers and reveals differences in mobility and membrane affinity across oligomeric states, as well as association and dissociation events in individual complexes. Measuring mass and trajectories together links the composition of protein complexes to their behavior on membranes. The authors also discuss how complementary measurement approaches could extend the method to integral membrane proteins, providing a methodological foundation for probing molecular mechanisms within and on lipid bilayers.
Highlight · Dynamic mass photometry links the mass of individual proteins on membranes to their diffusion and complex assembly behavior.
DOI: 10.1038/s41592-021-01261-w
03 · Continuous mass photometry by single-molecule trapping
Nature Methods · Publication/issue date: 2026-10-26
Authors: Dan Loewenthal, Roi Asor, Stephen Thorpe, Jack S. Peters, Konstantin C. Zouboulis, Merve Kaplan, Jan Christoph Thiele, Jack Bardzil, Seham Helmi, Sean A. Burnap, Tiong Kit Tan, Weston B. Struwe, Justin L. P. Benesch, Philipp Kukura
Biomolecular interactions often involve successive binding, dissociation, and state transitions, making it difficult to capture the full process in short observations. Continuous mass photometry uses single-molecule trapping to confine molecules on supported lipid bilayers within a region where they can be monitored continuously, while recording their motion and changes in mass. The authors demonstrate single-molecule observations lasting up to hours and report mass precision reaching the sub-kilodalton range and millisecond temporal resolution. Following the same molecule for longer allows interaction-induced mass states to be resolved sequentially over time, enabling analysis of binding events and dwell times. The paper also notes that greater temporal averaging improves mass precision at the expense of temporal resolution, so these performance levels are not achieved simultaneously across all timescales. The method provides continuous, quantitative traces for directly observing complex molecular processes.
Highlight · Single-molecule trapping extends the observation window of mass photometry, allowing molecular interactions to be tracked continuously over time.
DOI: 10.1038/s41592-026-03252-1
Cryo-EM Model Building and Conformational Ensembles (2 papers)
04 · CryoAtom improves model building for cryo-EM
Nature Structural & Molecular Biology · Publication/issue date: 2026-02
Authors: Baoquan Su, Kun Huang, Zhenling Peng, Alexey Amunts, Jianyi Yang
Building atomic models from cryo-EM density maps is a crucial step in translating experimental signals into explanations of molecular mechanisms. Building on ModelAngelo, CryoAtom incorporates AlphaFold2's modeling capabilities, replaces global attention with local attention, and uses three-dimensional rotary positional encoding to accommodate density-map information. The authors report that the method produces more complete models, works with lower-resolution maps, and speeds up model building. Applications to three large density maps demonstrate its ability to identify previously uncharacterized proteins, improve modeling of conformational changes, and separate out non-protein components. In one case, a complex containing 104 proteins was modeled within hours; comparing models from two maps also revealed subtle conformational differences in a single peripheral protein domain. The work highlights how experimental density constraints and deep learning can work together to make complex structures easier to interpret.
Highlight · CryoAtom combines local attention with density-map information to build more complete models of large cryo-EM structures more efficiently.
DOI: 10.1038/s41594-025-01713-3
05 · Cooling fast and slow: Characterizing the effects of vitrification in cryo-EM and the subsequent recovery of equilibrium populations
Proceedings of the National Academy of Sciences · Publication/issue date: 2026-11-10
Authors: Robert Clark, Louis G. Smith, Matthew P. Leighton, Ryan J. Szukalo, Syma Khalid, Pablo G. Debenedetti, Pilar Cossio, Miro A. Astore, Sonya M. Hanson
Cryo-EM has the potential to reveal molecular conformational ensembles, but interpreting them depends on whether cooling during vitrification alters the original state populations. This study uses more than 50 milliseconds of aggregate molecular dynamics simulations of the Trp-cage miniprotein to compare seven cooling rates spanning three orders of magnitude, with the slowest approaching experimental rates. In the system studied, the protein did not alter the vitrification behavior of water. A Markov state model built from equilibrium sampling showed that states with characteristic timescales longer than the cooling process were more resistant to cooling-induced perturbations. Some states disappeared from low-temperature equilibrium ensembles yet persisted in nonequilibrium cooled ensembles. The authors also propose a thermodynamic inference framework for recovering equilibrium populations from vitrified ensembles, offering a testable approach to interpreting conformational distributions in cryo-EM.
Highlight · The effects of rapid cooling on conformational distributions depend on state-transition timescales, and equilibrium populations can be inferred using a thermodynamic framework.
DOI: 10.1073/pnas.2619560123
Other Papers (2 papers)
06 · Mechanisms of vesicular monoamine transporter 2 modulation by amphetamines and lobeline
Cell Reports · Publication/issue date: 2026-11-24
Authors: Di Wu, Chuanyu Liu, Hongshuang Wang, Cheng Chi, Yibo Wang, Wenhua Zhang, Xiaohui Wang, Daohua Jiang
Vesicular monoamine transporter 2 (VMAT2) loads neurotransmitters into presynaptic vesicles, and its modulation by drugs directly affects the intracellular distribution of monoamines. This study resolves cryo-EM structures of human VMAT2 bound to four amphetamine-type stimulants and to lobeline, revealing distinct recognition modes for the two classes of molecules. The amphetamines mimic endogenous monoamines and occupy the substrate-binding site; the structures support their role as substrates that contribute to redistributing monoamine pools between vesicles and the cytoplasm. Lobeline, by contrast, binds in a two-molecule arrangement that stabilizes the transporter in a vesicle-lumen-facing conformation, inhibiting uptake of monoamines and related stimulants. Dopamine-release experiments further suggest that its effects involve both VMAT2 and the dopamine transporter DAT. The study provides a mechanistic basis for understanding addiction-related pharmacology, though the structural and cellular findings remain basic research evidence.
Highlight · Distinct ligand-binding modes in VMAT2 reveal the structural basis for regulating monoamine storage and release.
DOI: 10.1016/j.celrep.2026.118205
07 · Lipid-organized tetramerization of PLPP1 defines the molecular basis of spatial lipid phosphate signaling
Nature Communications · Publication/issue date: 2026-10-09
Authors: Yunhai Li, Qingning Yuan, Zhenyu Cao, Liuqing Zhu, Jiuyin Xu, Cheng-Cen Guo, James Jiqi Wang, Youwei Xu, Shuai Li, Tianyu Li, Yang Li, Yong Li, H. Eric Xu, Canrong Wu
Phospholipid phosphatase PLPP1 helps terminate lipid signals such as lysophosphatidic acid signaling, but how it assembles and catalyzes reactions at the membrane interface has remained unclear. This study presents cryo-EM structures of human PLPP1 in ligand-free and product-bound states at resolutions of 1.87 Å and 2.21 Å, respectively, showing that it forms a homotetramer with fourfold symmetry. Ordered lipid densities at the subunit interfaces are assigned to phosphatidylcholine, phosphatidic acid, and phosphatidylinositol bisphosphate. Structure-guided mutagenesis shows that residues surrounding these interfacial lipids affect enzymatic activity, but loss of activity does not simply correspond to tetramer disassembly. The product-bound structure also reveals the catalytic pocket and membrane-facing channels, suggesting possible substrate-entry routes; no associated ionic conductance was detected under the conditions tested. These findings provide a structural framework linking the lipid environment, oligomeric assembly, and catalytic function.
Highlight · PLPP1 structures link interfacial lipids, tetramer assembly, and the catalytic pocket, while showing that changes in enzymatic activity cannot be explained by oligomer disassembly alone.
DOI: 10.1038/s41467-026-78196-3