Batch Production of High-Quality Graphene Grids for Cryo-EM: Cryo-EM Structure of Methylococcus capsulatus Soluble Methane Monooxygenase Hydroxylase
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Curated by eLife
Evaluation Summary:
In this work, the authors describe a protocol for coating supports for transmission electron microscopy with graphene. The approach uses a commercially available PMMA/graphene pad and the authors provide a new process for PMMA removal after graphene transfer. This work has the potential to be an important methodological step forward - if a way were found to remove amorphous residue from the surface of graphene. The work has the potential to be of broad interest to the many structural biologists using cryo-EM techniques.
(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 and Reviewer #2 agreed to share their name with the authors.)
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Abstract
Cryogenic electron microscopy (cryo-EM) has become a widely used tool for determining protein structure. Despite recent technology advances, sample preparation remains a major bottleneck for several reasons, including protein denaturation at the air/water interface, the presence of preferred orientations, nonuniform ice layers, etc. Graphene, a two-dimensional allotrope of carbon consisting of a single atomic layer, has recently gained attention as a near-ideal support film for cryo-EM that can overcome these challenges because of its superior properties, including mechanical strength and electrical conductivity. Here, we introduce a reliable, easily implemented, and reproducible method to produce 36 graphene-coated grids within 1.5 days. To demonstrate their practical application, we determined the cryo-EM structure of Methylococcus capsulatus soluble methane monooxygenase hydroxylase (sMMOH) at resolutions of 2.9 and 2.4 Å using Quantifoil and graphene-coated grids, respectively. We found that the graphene-coated grid has several advantages, including less amount of protein required and avoiding protein denaturation at the air/water interface. By comparing the cryo-EM structure of sMMOH with its crystal structure, we identified subtle yet significant geometrical changes at the non-heme di-iron center, which may better indicate the active site configuration of sMMOH in the resting/oxidized state.
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Author Response
Reviewer #3 (Public Review):
The most interesting and novel part of the manuscript is the process for removing PMMA from the graphene after the transfer of the PMMA/graphene pad to electron microscopy grids. The authors use incubation in acetone followed by baking overnight at 200 Celsius. If this proves to be reproducible with easily obtained sources of commercial graphene, it will be a major aid in allowing more labs to generate electron microscopy grids with graphene. To further clarify the efficacy of this process and aid the reproducibility of the method, we ask the authors to improve the characterisation of the suspended graphene on the grids and add more detail in the description of the transfer and cleaning procedures.
In particular, in order to unambiguously demonstrate removal of the PMMA, Fig. 2 should …
Author Response
Reviewer #3 (Public Review):
The most interesting and novel part of the manuscript is the process for removing PMMA from the graphene after the transfer of the PMMA/graphene pad to electron microscopy grids. The authors use incubation in acetone followed by baking overnight at 200 Celsius. If this proves to be reproducible with easily obtained sources of commercial graphene, it will be a major aid in allowing more labs to generate electron microscopy grids with graphene. To further clarify the efficacy of this process and aid the reproducibility of the method, we ask the authors to improve the characterisation of the suspended graphene on the grids and add more detail in the description of the transfer and cleaning procedures.
In particular, in order to unambiguously demonstrate removal of the PMMA, Fig. 2 should include selected area electron diffraction (SAED) data where only the graphene layer suspended over the holes (no supporting foil) contributes to the diffraction pattern. This is easily achieved with a selected area aperture of the correct size. Patterns should be shown at each step of the process after the transfer. The authors should also clearly indicate in the TEM images the area of the sample that is illuminated to generate the SAED pattern. The SAED pattern as a function of tilt could also be examined to confirm a single graphene layer is present over the hole.
As reviewer#3 requested, we obtained the new SAED data and included in the revised Supplementary Materials (Figure S5, S6).
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Evaluation Summary:
In this work, the authors describe a protocol for coating supports for transmission electron microscopy with graphene. The approach uses a commercially available PMMA/graphene pad and the authors provide a new process for PMMA removal after graphene transfer. This work has the potential to be an important methodological step forward - if a way were found to remove amorphous residue from the surface of graphene. The work has the potential to be of broad interest to the many structural biologists using cryo-EM techniques.
(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 and Reviewer #2 agreed to share their name with the authors.)
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Reviewer #1 (Public Review):
This paper describes a straightforward method to produce graphene-coated cryo-EM grids, which does not require complicated machines or techniques. Thereby it has the potential to make these powerful types of grids more widely accessible to researchers. Besides a description of the method, the authors also present a structure soluble methane monooxydase hydroxylase (sMMOH) from Methylococcus capsulatus, as determined using both conventional Au Quantifoil grids and their graphene-coated grids. I am not entirely convinced of the comparison between the Au Quantifoil grids and the graphene-coated ones, nor of the differences between the EM and Xray structures (also see below), but these parts of the paper seem to be less important than the description of the new method.
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Reviewer #2 (Public Review):
This manuscript describes an easily made grid holder (3D printed transfer tool), shown in Figure S1, which is used to transfer commercially available graphene from pads, as provided, onto EM grids. In addition, a baking protocol (24 hr at 200 {degree sign}C in air) is used to remove most of the PMMA that remains on the transferred graphene, after washing with acetone. The use of these graphene-coated grids is demonstrated by preparing cryo-EM samples of soluble Methane Monoxoginase (sMMOH), a ~250 kDa particle, resulting in a high-resolution (2.4 Å} structure.
While using a transfer tool is a good idea, it represents a rather minor, incremental advance.
I am far less enthusiastic about the use of high-temperature oxidation, in air, as a way to remove residues of PMMA that remain after washing with acetone. …
Reviewer #2 (Public Review):
This manuscript describes an easily made grid holder (3D printed transfer tool), shown in Figure S1, which is used to transfer commercially available graphene from pads, as provided, onto EM grids. In addition, a baking protocol (24 hr at 200 {degree sign}C in air) is used to remove most of the PMMA that remains on the transferred graphene, after washing with acetone. The use of these graphene-coated grids is demonstrated by preparing cryo-EM samples of soluble Methane Monoxoginase (sMMOH), a ~250 kDa particle, resulting in a high-resolution (2.4 Å} structure.
While using a transfer tool is a good idea, it represents a rather minor, incremental advance.
I am far less enthusiastic about the use of high-temperature oxidation, in air, as a way to remove residues of PMMA that remain after washing with acetone. There is already extensive literature that shows that oxidation of the graphene is difficult to prevent when conditions are pushed far enough to remove all of the contamination. Indeed, Figure S2 C confirms that the surface of graphene remains contaminated over much or possibly all of the area of a hole in the supporting Quantifoil grid. A higher magnification image would have made this even more apparent.
In short, nothing new is presented that makes me believe that these grids will be more successful than those used in previous publications, whether used "as is", after glow discharge treatment, or when functionalized in other ways. If, on the other hand, a way could be found to remove all of the polymerized, amorphous residue, it might be possible that existing methods that have been tried to passivate or functionalize the graphene might have a better chance of success. I caution, however, that many have tried to achieve this goal (making atomically clean graphene on EM grids), without success.
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Reviewer #3 (Public Review):
The most interesting and novel part of the manuscript is the process for removing PMMA from the graphene after the transfer of the PMMA/graphene pad to electron microscopy grids. The authors use incubation in acetone followed by baking overnight at 200 Celsius. If this proves to be reproducible with easily obtained sources of commercial graphene, it will be a major aid in allowing more labs to generate electron microscopy grids with graphene. To further clarify the efficacy of this process and aid the reproducibility of the method, we ask the authors to improve the characterisation of the suspended graphene on the grids and add more detail in the description of the transfer and cleaning procedures.
In particular, in order to unambiguously demonstrate removal of the PMMA, Fig. 2 should include selected area …
Reviewer #3 (Public Review):
The most interesting and novel part of the manuscript is the process for removing PMMA from the graphene after the transfer of the PMMA/graphene pad to electron microscopy grids. The authors use incubation in acetone followed by baking overnight at 200 Celsius. If this proves to be reproducible with easily obtained sources of commercial graphene, it will be a major aid in allowing more labs to generate electron microscopy grids with graphene. To further clarify the efficacy of this process and aid the reproducibility of the method, we ask the authors to improve the characterisation of the suspended graphene on the grids and add more detail in the description of the transfer and cleaning procedures.
In particular, in order to unambiguously demonstrate removal of the PMMA, Fig. 2 should include selected area electron diffraction (SAED) data where only the graphene layer suspended over the holes (no supporting foil) contributes to the diffraction pattern. This is easily achieved with a selected area aperture of the correct size. Patterns should be shown at each step of the process after the transfer. The authors should also clearly indicate in the TEM images the area of the sample that is illuminated to generate the SAED pattern. The SAED pattern as a function of tilt could also be examined to confirm a single graphene layer is present over the hole.
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