Deciphering a hexameric protein complex with Angstrom optical resolution
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Evaluation Summary:
This paper will be of interest to the structural biology community and people working on cryogenic fluorescence microscopy. This paper is a clear step forward in the use of single-molecule localization microscopy at angstrom resolution, thanks to low-temperature polarized super-resolution imaging and advanced data processing algorithms.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 agreed to share their name with the authors.)
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Abstract
Cryogenic optical localization in three dimensions (COLD) was recently shown to resolve up to four binding sites on a single protein. However, because COLD relies on intensity fluctuations that result from the blinking behavior of fluorophores, it is limited to cases where individual emitters show different brightness. This significantly lowers the measurement yield. To extend the number of resolved sites as well as the measurement yield, we employ partial labeling and combine it with polarization encoding in order to identify single fluorophores during their stochastic blinking. We then use a particle classification scheme to identify and resolve heterogenous subsets and combine them to reconstruct the three-dimensional arrangement of large molecular complexes. We showcase this method (polarCOLD) by resolving the trimer arrangement of proliferating cell nuclear antigen (PCNA) and six different sites of the hexamer protein Caseinolytic Peptidase B (ClpB) of Thermus thermophilus in its quaternary structure, both with Angstrom resolution. The combination of polarCOLD and single-particle cryogenic electron microscopy (cryoEM) promises to provide crucial insight into intrinsic heterogeneities of biomolecular structures. Furthermore, our approach is fully compatible with fluorescent protein labeling and can, thus, be used in a wide range of studies in cell and membrane biology.
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Evaluation Summary:
This paper will be of interest to the structural biology community and people working on cryogenic fluorescence microscopy. This paper is a clear step forward in the use of single-molecule localization microscopy at angstrom resolution, thanks to low-temperature polarized super-resolution imaging and advanced data processing algorithms.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 agreed to share their name with the authors.)
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Reviewer #1 (Public Review):
The work by Mazal et al. demonstrates the possibility to reconstruct information from proteins structure at an angstrom resolution, using key elements in super-resolution microscopy: working at low temperature to accumulate an important number of photons per molecule (thus increasing localization precision), and discriminating different labels, positioned at precise locations of the proteins oriented in 3D, with the use of their polarization emission behavior. This approach is elaborated based on previous works by the authors, and is applied here for the first time to protein complexes in 3D. This is achieved thanks to additional steps of data processing and more advanced analysis algorithms, to (1) extract, out of step-wise time dependencies, the presence of molecule's identified by their different …
Reviewer #1 (Public Review):
The work by Mazal et al. demonstrates the possibility to reconstruct information from proteins structure at an angstrom resolution, using key elements in super-resolution microscopy: working at low temperature to accumulate an important number of photons per molecule (thus increasing localization precision), and discriminating different labels, positioned at precise locations of the proteins oriented in 3D, with the use of their polarization emission behavior. This approach is elaborated based on previous works by the authors, and is applied here for the first time to protein complexes in 3D. This is achieved thanks to additional steps of data processing and more advanced analysis algorithms, to (1) extract, out of step-wise time dependencies, the presence of molecule's identified by their different orientations and (2) use supervised particle classification for the reconstruction of the final structure information.
This work is a step forward in the use of optical super-resolution for ultra-high resolution analyses that come in close complementarity to electron-microscopy, and can be applied to proteins structures that are delicate to crystallize. While it shows that it can reproduce information from a structure as complex as a hexameric geometry, it is however not clear how this approach will be applied to unknown protein structures. The approach seems robust but is also still limited to a very specific framework of low-level labeling, immobilized and low-concentration proteins, therefore not yet likely to apply to dynamic studies as mentioned in the manuscript.Some points need to be addressed to clarify the findings, the conclusions, and the broader claims.
It is known that point spread functions from 3D-oriented dipoles, as found in the present study, are not symmetric Gaussians but rather deformed shapes that might lead to significant localization inaccuracies as compared to the localization precision reached here. The effect of this bias needs to be discussed. Related to this point, the authors should address and consider the possibility to directly measure the 3D orientation of fluorophores, which might be very relevant for the discrimination method developed in this work.
The method assumes that there is no possible rotational flexibility of the dipoles attached to the studied protein. Even though the measurements are performed at low temperature, it does not prevent different probable orientations to be explored for each fluorophore.
The limitations of the method, in particular, the number of possibly discriminated orientations, required time dynamics of the photophysics of the fluorophores, required signal to noise level, required number of measurements given the very low yield of used molecules, should be clarified, in particular giving elements for possible improvements.
The applicability of the method to a larger framework is not entirely clear. Its application to unknown or dynamic proteins structures should be addressed, in particular with respect to the used supervised classification procedure.
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Reviewer #2 (Public Review):
The authors present polarCOLD, an extension of their recently presented technique of optical localization microscopy at cryogenic temperatures (COLD). They point out that the main limitation of COLD as being dependent on intensity fluctuations to separate individual emitters, which can then be localized. Such intensity fluctuations depend on the local environment and quantum efficiency of the fluorophore, which was previously shown to allow discrimination of up to four sites (Weissenburger, 2017). Recording the emission polarization as an additional parameter was recently presented by the authors (Böning, 2021) and shown to support fluorophore discrimination.
In the present manuscript, the authors now use polarCOLD in combination with partial labeling and particle classification to characterize more complex …
Reviewer #2 (Public Review):
The authors present polarCOLD, an extension of their recently presented technique of optical localization microscopy at cryogenic temperatures (COLD). They point out that the main limitation of COLD as being dependent on intensity fluctuations to separate individual emitters, which can then be localized. Such intensity fluctuations depend on the local environment and quantum efficiency of the fluorophore, which was previously shown to allow discrimination of up to four sites (Weissenburger, 2017). Recording the emission polarization as an additional parameter was recently presented by the authors (Böning, 2021) and shown to support fluorophore discrimination.
In the present manuscript, the authors now use polarCOLD in combination with partial labeling and particle classification to characterize more complex proteins with up to 6 binding sites. While DNA structures were used previously, the authors now demonstrate their technique by reconstructing binding sites in two multimeric proteins at Angstrom resolution. The new approach readily allows the discrimination of 5 fluorophores. By using a 3D particle reconstruction algorithm, they achieve a remarkable resolution of 4.9 Å for the simple trimeric protein PCNA. Towards more complex proteins, the authors exploit partial labeling together with particle classification to overcome the limitation of the number of fluorophores. They use a 50% labeled hexameric protein to accurately localize the remaining three fluorophores, which are then translated into a 3D structure using a combination of simulations, classification and template matching.
The authors extend their recently developed polarCOLD technique to the reconstruction of multimeric proteins, which was not shown previously. A major strength of cryogenic localization microscopy is that it can achieve Angstrom resolution. Although currently limited to purified proteins, which in principle makes the sample also compatible with cryoEM, COLD indeed allows discriminating binding sites within a complex which might not be readily possible with cryoEM. Still, both techniques operate in the same resolution regime and it will be interesting to explore the possibilities towards non-symmetric complexes or mixed biological samples.
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Reviewer #3 (Public Review):
The paper by Mazal, Wieser and Sandoghdar presents a method to image small molecular complexes with light microscopy, but obtain resolutions/localization uncertainties of fluorescent labels that have been limited to cryo-EM.
This submission by Mazal, Wieser and Sandoghdar builds upon earlier work from the same group (Weisenburger, Nature Methods, 2017 and Boning, ACS Photonics, 2021). The polarisation detection does not increase the sparsity compared to Weisenburger, but on the detection side they can identify different emitters based on their fixed dipole emission as shown in Boning. They further improve the sparsity, by explicit underlabeling, which later is compensated by particle registration and averaging. Compared to Boning the submission adds 3D reconstructions of two molecular complexes from 2D …
Reviewer #3 (Public Review):
The paper by Mazal, Wieser and Sandoghdar presents a method to image small molecular complexes with light microscopy, but obtain resolutions/localization uncertainties of fluorescent labels that have been limited to cryo-EM.
This submission by Mazal, Wieser and Sandoghdar builds upon earlier work from the same group (Weisenburger, Nature Methods, 2017 and Boning, ACS Photonics, 2021). The polarisation detection does not increase the sparsity compared to Weisenburger, but on the detection side they can identify different emitters based on their fixed dipole emission as shown in Boning. They further improve the sparsity, by explicit underlabeling, which later is compensated by particle registration and averaging. Compared to Boning the submission adds 3D reconstructions of two molecular complexes from 2D underlabeled structures. The polarisation and localization method is improved technically, but the concept was already there.
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