A cryo-electron microscopy support film formed by 2D crystals of hydrophobin HFBI

Cryo-electron microscopy (cryo-EM) has become a powerful tool to resolve high-resolution structures of biomacromolecules in solution. However, air-water interface induced preferred orientations, dissociation or denaturation of biomacromolecules during cryo-vitrification remains a limiting factor for many specimens. To solve this bottleneck, we developed a cryo-EM support film using 2D crystals of hydrophobin HFBI. The hydrophilic side of the HFBI film adsorbs protein particles via electrostatic interactions and sequesters them from the air-water interface, allowing the formation of sufficiently thin ice for high-quality data collection. The particle orientation distribution can be regulated by adjusting the buffer pH. Using this support, we determined the cryo-EM structures of catalase (2.29 Å) and influenza haemagglutinin trimer (2.56 Å), which exhibited strong preferred orientations using a conventional cryo-vitrification protocol. We further show that the HFBI film is suitable to obtain high-resolution structures of small proteins, including aldolase (150 kDa, 3.28 Å) and haemoglobin (64 kDa, 3.6 Å). Our work suggests that HFBI films may have broad future applications in increasing the success rate and efficiency of cryo-EM.

The additional experiments indeed show the protein adsorption's pH (electrostatic) dependency (supplemental Figure 11). But it does not explain the "worm-like" structures. In my view, this indicates that the interaction force between the particles is not obscured anymore by other forces, e.g., the interaction with the interface with the particles enforcing top views. Furthermore, unfortunately, manuscript Fig. 5a provides only relative information. It would be essential to know the absolute number of particles picked, discarded, and used for the reconstruction. And, finally, how many of the particles were integrated into the "worms." I believe the proposed methodology should be shared with the community asap and deserves publication in nature communications.
## Minor comments -Abstract: "air-water interface-induced": Missing coma, or should second hyphenation be removed? (I'm not a native English speaker) - Supplementary Fig 4: The contamination is also indicated by a cross, which is not described in the legend.

Letter to response reviewers
We would like to appreciate the original reviewers to check our revision again and highly recommend our work. According to the minor comments of Reviewer #3, we made a final revision of our manuscript with the following editions.
1. Providing the absolute numbers of particles picked and used for the reconstruction in Fig. 5a.
2. Adding more discussions about the formation of "worm-like" structure of GDH.

Corrections of typos indicated by the reviewer.
The point-by-point responses to the reviewers' suggestions and comments are given below.

[To Reviewer #2]
Remarks to the Author: The authors have taken a great effort to address all the reviewers' comments and the manuscript is greatly improved. This work is now ready to be published in a timely manner.
Response #1: We appreciate this reviewer for his/her high evaluation of our work. We believe our work will make a great contribution to the cryoEM community.

[To Reviewer #3]
Remarks to the Author: The manuscript was extensively revised and improved significantly. Notably, the figures are enhanced considerably. In addition, the additional experiments mostly clarified my concerns about the electrostatic interaction of the particles with the new interface.
Response #1: We are very happy to know our revision has satisfied this reviewer.

2
The additional experiments indeed show the protein adsorption's pH (electrostatic) dependency (supplemental Figure 11). But it does not explain the "worm-like" structures. In my view, this indicates that the interaction force between the particles is not obscured anymore by other forces, e.g., the interaction with the interface with the particles enforcing top views. Furthermore, unfortunately, manuscript Fig. 5a provides only relative information. It would be essential to know the absolute number of particles picked, discarded, and used for the reconstruction. And, finally, how many of the particles were integrated into the "worms." Response #2: Thanks a lot for this suggestion.
For the "worm-like" structure, we agree with this reviewer that it indicates a significant top-to-top interaction between GDH particles, which is the intrinsic nature of GDH protein. At the condition without HFBI film, the interaction between air-water interface (AWI) and the top side of GDH is stronger than the top-to-top interaction itself, which enforces the majority of GDH top views. At the condition with HFBI film, the interaction between the new interface and the top side of GDH is regulated efficiently by pH and becomes weaker than the top-to-top interaction of GDH when pH is larger than 7.0, which allows significant population of side views. Indeed, the "worm-like" structures were also observed from the recent report when the property of AWI is changed by adding amount of cationic detergent (see Figure S7 in PMID 34454014).
In our observations, there are a large portion of side view particles integrated into the "worms", which is also consistent with this report (PMID 34454014). These discussions have been added into the revise text as follows.
"The "worm-like" structure of GDH ( Supplementary Fig. 11a-g) suggests an intrinsic top-to-top interaction between GDH particles. At the condition without HFBI film, the interaction between air-water interface and the top side of GDH is stronger than the top-to-top interaction itself, which enforces the majority of GDH top views. At the condition with HFBI film, the interaction between the new interface and the top side 3 of GDH is regulated efficiently by pH and becomes weaker than the top-to-top interaction of GDH when pH is larger than 7.0, which allows significant population of side views. Indeed, the "worm-like" structures were also observed from the recent report when the property of air-water interface is changed by adding amount of cationic detergent." The absolute numbers of particles picked and used for the reconstruction in Fig.   5a have been provided in the revised text as follows.
## Minor comments -Abstract: "air-water interface-induced": Missing coma, or should second hyphenation be removed? (I'm not a native English speaker) Response #4: We have removed the second hyphenation.
- Supplementary Fig 4: The contamination is also indicated by a cross, which is not described in the legend.
Response #5: The yellow cross does not indicate the contamination but the position of ice at AWI. It was generated in IMOD and left in the snapshot. This information was added in the revised legend. We also added a description of green lines in the legend.