- Email: [email protected]
Tidy up cryo-EM sample grids with 3D printed tools
Journal Pre-proofs Tidy up cryo-EM sample grids with 3D printed tools Tasuku Hamaguchi, Koji Yonekura PII: DOI: Reference:
S1047-8477(19)30235-7 https://doi.org/10.1016/j.jsb.2019.107414 YJSBI 107414
To appear in:
Journal of Structural Biology
Received Date: Revised Date: Accepted Date:
11 September 2019 31 October 2019 2 November 2019
Please cite this article as: Hamaguchi, T., Yonekura, K., Tidy up cryo-EM sample grids with 3D printed tools, Journal of Structural Biology (2019), doi: https://doi.org/10.1016/j.jsb.2019.107414
This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
© 2019 Published by Elsevier Inc.
Tidy up cryo-EM sample grids with 3D printed tools
Tasuku Hamaguchi 1, *, Koji Yonekura 1, 2, * 1
Biostructural Mechanism Laboratory, RIKEN SPring-8 Center, 1-1-1 Kouto, Sayo,
Hyogo 679-5148, Japan 2
Advanced Electron Microscope Development Unit, RIKEN-JEOL Collaboration
Center, RIKEN Baton Zone Program, 1-1-1 Kouto, Sayo, Hyogo 679-5148, Japan
KEYWORDS: 3D printer; cryo-EM; grid case; surface polishing; single-particle analysis; CRYO ARM
* To whom correspondence should be addressed. E-mail: [email protected]
or
[email protected]
Phone: +81-791-58-2837
1
Abstract Cryo-EM technology has developed to the point of high-throughput structure determination of biological macromolecules embedded in vitreous ice. Nonetheless, challenging targets need extensive sample screening, often of many cryo-EM sample grids prepared under various conditions. We have designed and made tools for manipulating sample grids in storage cases.
These tools are made of a plastic fiber
using a wide-use 3D printer, a fused deposition modeling type, and polished under acetone gas.
A grid case stacker organizes many frozen-hydrated cryo-EM grids and
the stackers can be piled up inside a standard 50mL centrifuge tube. We have also introduced tools that facilitate handling of grid cases under liquid nitrogen and a stocker of the grid retainers contained in a CRYO ARM electron microscope. the tools named CryoGridTools are available from a GitHub site.
2
Blueprints of
1. Introduction Cryo-EM technology has developed rapidly in the last few years, such that several thousand movie stacks can now be collected in a single session in under a day using a direct detection detector with high speed readout and data acquisition schemes combining image and stage shifts (e.g. Zivanov et al., 2018; Schorb et al., 2019; (Danev et al., 2019; Mastronarde, 2005)). This greatly accelerates structure determination of protein molecules embedded in thin vitreous ice by single particle cryo-EM. Of course, data processing on high-performance CPUs and GPUs and sophisticated algorithms remain very important for increasing throughput (Punjani et al., 2017; Zivanov et al., 2018). However, preparation of suitable cryo-samples is still a limiting step for efficient application of this technique. In particular, challenging targets often require time-consuming screening of many samples, typically frozen under many different conditions.
In cryo-EM, metal grids (3 mmφ) coated with holey carbon film are generally used as a support structure onto which sample solution is directly applied. The grids are plunge-frozen in a cryogen such as liquid ethane and stored in small grid cases. In many laboratories, these grid cases are dropped into a centrifuge tube for storage in a liquid
3
nitrogen Dewar till use. Thus, before screening and data collection by cryo-EM large numbers of grid cases need to be processed and well-organized.
The situation is quite similar to macromolecular X-ray crystallography, which needs many crystals to be kept frozen and organized before transporting to synchrotron radiation facilities. Crystal mounters, typically, cryo-loops attached on a metal pin and a magnet base, are shelved in canes or metal containers. The latter, which typically holds 16 cryo-loops per container, can be linked with automated crystal mounting robots in many X-ray exposure facilities to dramatically increase the efficiency of screening for well-diffracting crystals. Several companies have now introduced similar-type containers with a diameter of ~70 mm to the cryo-EM field for storage of EM grid cases. One such container accommodates ~ 12 grid cases, and the containers slide into a tall shelf. Some products allow barcode-based registration of samples, which is useful when huge numbers of EM grids are processed. They are, however, heavy, sizable and costly, partly because the concept came from handling crystal mounters in X-ray crystallography. Similar designs have also been proposed in academic papers and are available from the authors (Scapin et al., 2017; Ultee et al., 2018), but production of these appears to be not easy for most users. Simplification of containers for cryo-EM
4
use should be possible, especially as automated mounting of grids into the sample storage unit is not yet feasible.
We designed and made several plastic tools to help process and organize cryo-EM sample grids using a fused deposition modeling (FDM) 3D printer. The grid case containers are light and stackable in a standard conical tube, and suitable for transporting to shared cryo-EM facilities. The 3D printing of many such stackers is easy and cheap. In addition, we designed tools that facilitate handling of grid cases in liquid nitrogen and one that stocks the grid retainer that is used to introduce a sample grid into the CRYO ARM electron microscope (Hamaguchi et al., 2019).
We name the tools
presented here CryoGridTools.
2. Design 2.1 Grid case stacker We designed a grid case stacker for a standard disk-shaped grid case with a diameter of ~14 mm, which typically has four slits into which EM sample grids fit (Fig. 1A). The grid case stacker holds three such grid cases standing vertically on three distinct slots (Fig. 1B). Each grid case settles nicely onto one slot of the stacker. The stackers fit
5
comfortably inside a 50 mL centrifuge tube with an inner diameter of 25 ~ 27 mm (Fig.1C), such as the widely-used conical type tubes of Falcon / Corning (Corning Inc., USA) with blue and orange caps (Fig. 2A). One tube accommodates up to five stackers and an extra single grid case can be placed underneath at the bottom (Figs. 1C and 2A right). In total 4 x 3 x 5 + 4 = 64 grids can be kept and organized in one tube (Fig. 2A right). Without the grid case stackers, ~ 22 grid cases randomly oriented can be placed in one tube (Fig. 2A left). Thus, while use of the stackers organizes grid cases, it does mean the number of sample grids in one tube is reduced by ~ 27%. We also designed a special grid case with six slits (Figs. 2B and C), which allows for precision tweezers to approach each slit from the same direction as in the standard case with four slits. The stacker fits well into a metal dipper filled with liquid nitrogen for safe transport (Supplementary Movie S1). 3D printing of stackers and grid cases in various colors would help with further organization of sample grids (Fig. 1A).
A metal screw on the stacker (Fig. 1A) has two functions: it helps to sink the stacker in liquid nitrogen (Fig. 1E) and to lift the stacker from / to the tube with a magnetic wand (Figs. 1D and 3; Supplementary Movie S1). The steel flat-head screw with a diameter of ~ 3.2 mm and a length of ~ 12 mm on the stacker is fitted with a
6
CrystalWand Magnetic (Hampton Research, USA), which has a release button (Fig. 1D lower). Using the 3D printer, we also made a simple pickup wand and then glued a small neodymium magnet (~ 6 mmφ) onto the tip (Fig. 1D upper). The stacker is easily released under liquid nitrogen by a wrenching action of the wand.
The stackers also settle well inside the conical tube where the number of stacks is less than five. In practice, a spacer is placed on top of the stacker for transport to the shared EM facility. We made several spacers for this purpose (Supplementary Fig. S1).
2.2 Grid case orientator The grid case must be placed in the stacker in a vertical position. We designed a tool for facilitating this, and named it the grid case orientator (Figs. 1F lower and G). This grid case orientator is fitted inside the outer space around the ethane vessel of the ethane container, which is supplied with Vitrobot Mark IV (Thermo Fisher Scientific, USA). The user can drop a grid case with plunge-frozen grids onto the orientator in liquid nitrogen, and the orientator supports the grid case in an upright position (Figs. 1G and 3; Supplementary Movie S1), thus facilitating the picking up of the grid case with tweezers and placing it in the stacker. The standard grid case holder, made of metal and
7
placed in the outer space of the ethane container, accepts four grid cases onto the hollows of four diagonal arms, but is not compatible with placing the orientator in the outer space. Thus, we replaced it with a 3D printed holder that lacks one arm, thereby affording enough room for the orientator (Figs. 1F upper, G and 3).
2.3 Retainer stocker for the CRYO ARM microscope Use of the CRYO ARM microscope requires that a frozen-hydrated grid is mounted on a sample retainer and attached with a C-ring clip (Fig. 4C and Supplementary Fig. S1 of Hamaguchi et al., 2019). Multiple EM grids are readily transferred into the sample storage unit in the microscope with this system. For alignment and calibration of the microscope, carbon grating and/or metal-coated carbon grids are routinely used but are usually too costly for onetime disposable use. However, detaching / attaching EM grids from / onto the retainer is cumbersome. We have already designed a special tool for detaching the C-ring clip (Hamaguchi et al., 2019), but found that even this tool may damage the grids.
It is more logical and convenient to keep the grids on the retainer.
Thus, we made a retainer stocker that accommodates four retainers (Supplementary Fig. S2). The stocker with retainers is usually kept inside a desiccator at ambient temperature till use.
8
3. 3D printing and surface polishing 3.1 3D printing All tools were designed with open source 3D creation software, Blender (https://www.blender.org/) and the data exported in STL format for 3D printing. We used a wide-use 3D printer, UP Box+ (Tiertime, China) equipped with acrylonitrile butadiene styrene (ABS) modeling material. This material tolerates liquid nitrogen, whereas typical photo curable resin used with a stereolithography 3D printer (Jasveer and Jianbin, 2018) breaks easily in liquid nitrogen. Models were built by stacking ABS filaments at 230˚C. Layer thickness and surface layers were 0.2 mm and three, respectively. Filling factor for printing was close-packing to increase strength and weight of the models. A slower head speed, a correct temperature (~ 100˚C) of the base plate and a controled cooling rate improved printing quality because of the linear expansion coefficient. With this setting, the production rarely fails even for large complex structures such as the grid case orientator as well as fine ones such as screw holes. Small defects can be retouched with surface polishing (see below). We used ABS filaments in five different colors for the stacker. 3D printing of a single stacker takes ~ 20 min and costs less than a half US dollar.
9
3.2 Surface polishing Printed models of filament layers were fused with acetone vapor. A small amount (10 20 mL) of acetone in a glass beaker was placed inside a well ventilated draft chamber, heated to 100˚C on a heat controller, and plastic wrap was used to cover the beaker to contain the vapor, as acetone gas is heavier than air. The surface of printed models exposed to the acetone vapor several times each for several seconds, melted quickly and became smooth. This process strengthened the structure, and also reduced bubbling in liquid nitrogen.
4. Working flow Our standard protocol for manipulating cases with cryo-EM grids is described below, where numbers correspond to Fig. 3. 1. Place a stacker and orientator in the outer space around the central ethane vessel of the ethane container. 2. Insert a grid case holder and anti-contamination ring inside the outer space and place an ethane vessel. 3. Place a metal cold finger inside the outer space. Fill the outer space with liquid
10
nitrogen, and then fill the ethane vessel with liquid ethane. Remove the cold finger when the vessel is full of liquid ethane. 4. Place labeled grid cases on holder arms and freeze the sample solution on the EM grid and insert the frozen grids into the slits of the case as usual. 5. Remove the anti-contamination ring. 6. Drop the grid cases onto the orientator. Place the anti-contamination ring, and repeat from freezing of Step 4 until the three positions of the orientator are full. Or proceed to the next step. 7. Remove the grid case holder and transfer the grid cases into the stacker with tweezers. 8. Catch the stacker with a magnetic wand. 9. Transfer a stacker in a metal dipper filled with liquid nitrogen to a centrifuge tube in liquid nitrogen, and store. Sample details, such as composition, locality and freezing conditions, are recorded in a log book. The procedure is demonstrated in Supplementary Movie S1.
5. Discussion and conclusion We have made several cryo-EM tools with a wide-use FDM 3D printer for use in
11
organizing cryo-EM sample grids, namely, a grid case stacker, a grid case orientator, a grid case holder with three arms, grid cases with six-, four- and two-slits, a pick-up wand, spacers, and a grid retainer stocker.
They have proved useful in our laboratory.
With the stacker (Figs. 1A and B), a total of 3 x 5 + 1 = 16 grid cases are stored in one 50 mL tube, and one 30 L liquid nitrogen Dewar can hold six tubes (Fig. 1E). In our four to five Dewars several thousand EM grids can be stored, and some are now well organized in stackers.
Users often carry frozen-hydrated samples on EM grids to shared cryo-EM facilities for data collection. 50 mL tubes suffices.
A typical number of grids is less than 50, and one or two
The stacker helps organize sample grids, although the number
of grid cases kept in the tube is reduced ~27 % because of the stacker volume. To obviate this, we made a grid case with six slits (Fig. 2C).
Commercial grid case containers do have benefits such as barcode-based registration of samples, easy access to all the containers from the side of the shelf, and so on. But the advantages, in our opinion, are not quite worth the expense.
12
Our stacker is light, simple and easy to use with the help of the other tools presented here. One – 3 grid cases and 1 - 5 stackers fit well on each stacker and tube, respectively (Figs. 1C and 2A right). A spacer can be placed on top of the stacker for added stabilization during transport to other facilities (Supplementary Fig. S1). Moving stackers around to access buried grids is not problematic, thanks to the magnetic wand (Figs. 1D, 3 and Supplementary Movie S1).
The wide-use FDM 3D printer can use various materials such as polylactic acid (PLA), ABS, polyethylene terephthalate (PET), aliphatic polyamides (Nylon), and so on. ABS is superior to other materials in its processibility, impact resistance, ease of printing and cost (Samykano et al., 2019). ABS is durable even in liquid nitrogen. A stereolithography 3D printer can produce models with a higher machining accuracy and a smoother surface from general photo curable resin, but this material is easily broken in liquid nitrogen. We finish the models by surface polishing with acetone vapor, which increases strength by fusing each layer tightly, prevents contamination of small ABS pieces, and produces a smooth surface and decreases bubbling in liquid nitrogen.
Our designs for the several cryo-EM tools, particularly for the grid case stacker,
13
are, of-course, not necessarily ideal for all users and instruments, but can be easily customized. The blueprints of all the tools in CryoGridTools are available from a GitHub site (https://github.com/YonekuraLab/CryoGridTools). We believe that 3D printing with surface polishing should be useful in not only the cryo-EM field but also in more general areas, and hope that this report will help accelerate its use.
Acknowledgments We thank Saori Maki-Yonekura and Radostin Danev for useful inputs on design of the grid case stacker. This work was partly supported by Japan Society for the Promotion of Science Grant-in-Aid for Scientific Research Grant 16H04757 (to K.Y.), Japan Society for the Promotion of Science Grant-in-Aid for Challenging Exploratory Research Grant 24657111 (to K.Y.), the RIKEN Pioneering Project, Dynamic Structural Biology (to T.H. and K.Y.), and the Cyclic Innovation for Clinical Empowerment (CiCLE) from the Japan Agency for Medical Research and Development, AMED (to K.Y.).
References Danev, R., Yanagisawa, H., Kikkawa, M., 2019. Cryo-Electron Microscopy Methodology: Current Aspects and Future Directions. https://doi.org/10.1016/j.tibs.2019.04.008 Hamaguchi, T., Maki-Yonekura, S., Naitow, H., Matsuura, Y., Ishikawa, T., Yonekura, 14
K., 2019. A new cryo-EM system for single particle analysis. J. Struct. Biol. 207, 40–48. https://doi.org/10.1016/J.JSB.2019.04.011 Jasveer, S., Jianbin, X., 2018. Comparison of Different Types of 3D Printing Technologies. Int. J. Sci. Res. Publ. 8. https://doi.org/10.29322/IJSRP.8.4.2018.p7602 Mastronarde, D.N., 2005. Automated electron microscope tomography using robust prediction of specimen movements. J. Struct. Biol. 152, 36–51. https://doi.org/10.1016/J.JSB.2005.07.007 Punjani, A., Rubinstein, J.L., Fleet, D.J., Brubaker, M.A., 2017. cryoSPARC: algorithms for rapid unsupervised cryo-EM structure determination. Nat. Methods 14, 290–296. https://doi.org/10.1038/nmeth.4169 Samykano, M., Selvamani, S.K., Kadirgama, K., Ngui, W.K., Kanagaraj, G., Sudhakar, K., 2019. Mechanical property of FDM printed ABS: influence of printing parameters. Int. J. Adv. Manuf. Technol. 102, 2779–2796. https://doi.org/10.1007/s00170-019-03313-0 Scapin, G., Prosise, W.W., Wismer, M.K., Strickland, C., 2017. A novel storage system for cryoEM samples. J. Struct. Biol. 199, 84–86. https://doi.org/10.1016/j.jsb.2017.04.005 Ultee, E., Schenkel, F., Yang, W., Brenzinger, S., Depelteau, J.S., Briegel, A., 2018. An Open-Source Storage Solution for Cryo-Electron Microscopy Samples 24, 60–63. https://doi.org/10.1017/S143192761701279X Zivanov, J., Nakane, T., Forsberg, B.O., Kimanius, D., Hagen, W.J., Lindahl, E., Scheres, S.H., 2018. New tools for automated high-resolution cryo-EM structure determination in RELION-3. Elife 7. https://doi.org/10.7554/eLife.42166
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Figure Legends Figure 1. 3D printed tools for organizing cryo-EM grids. A, grid case stackers. 3D printed in skin color, grey, blue, black and yellow and polished under acetone vapor. One stacker has three holding slots for typical disk-shape grid cases shown in cyan here. B, design of the grid case stacker. A steel flat-head screw with a diameter of ~ 3.2 mm is inserted into the hole before use. C, cartoon of five grid case stackers piled up in a 50 mL centrifuge tube and one grid case at the bottom. Sixteen grid cases are stored in one tube here. D, magnetic wands for picking up the stacker from / to the 50 mL centrifuge tube. Upper: Designed and made in this report. Lower: a commercially available item, CrystalWand Magnetic (Hampton Research, USA). E, cartoon of one centrifuge tube with grid case stackers in a cross-sectional model of a Dewar filled with liquid nitrogen. F, grid case holder (upper) and orientator (lower). G, a schematic diagram for making a grid case upright on the grid case orientator in liquid nitrogen. This step can be performed inside the outer space of the ethane container.
Figure 2. Grid case stackers with grid cases. A, (left) typical way for storing grid cases in the conical tube. Twenty-two grid cases are disordered in one tube here. (right) sixteen grid cases were ordered with stackers printed in five-different colors. B, design
16
of a grid case with six slits. Sixteen grid cases with six holes can keep up to 96 grids, which is more than in 22 standard grid cases with four slits. C, 3D printed grid cases with six-, four-, and two-slits for various applications. The grid case with two slits is sometimes convenient for simpler operation and sorting.
Figure 3. Working flow to keep cryo-EM grids in grid cases organized by using the tools made here. Step 1: Place a grid case stacker and orientator in the outer space of the ethane container. Step 2: Insert a grid case holder, an ethane vessel, and an anti-contamination ring. Step 3: Place a metal cold finger. Fill the outer space with liquid nitrogen and then fill the central vessel with liquid ethane. Step 4: Place a grid case on the holder, and freeze sample solution on a grid and insert a frozen grid into a slit of the grid case. Step 5: Remove the anti-contamination ring. Step 6: Drop the grid case onto the orientator. Step 7: Remove the holder and transfer the grid case to the stacker. Step 8: Catch the stacker with a magnetic wand. Step 9: Transfer the stacker in a metal dipper to a centrifuge tube. Liquid nitrogen and liquid ethane are omitted here for clarity. See also Section 4 and Movie S1.
17
Tools for manipulating sample grids in storage cases are designed, made using a wide-use 3D printer and polished under acetone gas.
The grid case containers are light and stackable in a standard conical tube, and suitable for transporting to other facilities.
Supporting tools are also designed to facilitate handling of grid cases under liquid nitrogen.
These tools named CryoGridTools are useful for organizing many cryo-EM grids and blueprints of the tools are available for sharing in the community and easy customization.
18
Disordered
Organized
19
Figure 1 A
3.2
B
C 6
14
2
25
13 9
D E
F
4.5 and 6
G
Drop the grid case
The case is oriented for easy pickup
20
Figure 2 A
B
C
2.6 6
21
Figure 3
1
2
3
4
5
6
7
8
9
22
S1047-8477(19)30235-7 https://doi.org/10.1016/j.jsb.2019.107414 YJSBI 107414
To appear in:
Journal of Structural Biology
Received Date: Revised Date: Accepted Date:
11 September 2019 31 October 2019 2 November 2019
Please cite this article as: Hamaguchi, T., Yonekura, K., Tidy up cryo-EM sample grids with 3D printed tools, Journal of Structural Biology (2019), doi: https://doi.org/10.1016/j.jsb.2019.107414
This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
© 2019 Published by Elsevier Inc.
Tidy up cryo-EM sample grids with 3D printed tools
Tasuku Hamaguchi 1, *, Koji Yonekura 1, 2, * 1
Biostructural Mechanism Laboratory, RIKEN SPring-8 Center, 1-1-1 Kouto, Sayo,
Hyogo 679-5148, Japan 2
Advanced Electron Microscope Development Unit, RIKEN-JEOL Collaboration
Center, RIKEN Baton Zone Program, 1-1-1 Kouto, Sayo, Hyogo 679-5148, Japan
KEYWORDS: 3D printer; cryo-EM; grid case; surface polishing; single-particle analysis; CRYO ARM
* To whom correspondence should be addressed. E-mail: [email protected]
or
[email protected]
Phone: +81-791-58-2837
1
Abstract Cryo-EM technology has developed to the point of high-throughput structure determination of biological macromolecules embedded in vitreous ice. Nonetheless, challenging targets need extensive sample screening, often of many cryo-EM sample grids prepared under various conditions. We have designed and made tools for manipulating sample grids in storage cases.
These tools are made of a plastic fiber
using a wide-use 3D printer, a fused deposition modeling type, and polished under acetone gas.
A grid case stacker organizes many frozen-hydrated cryo-EM grids and
the stackers can be piled up inside a standard 50mL centrifuge tube. We have also introduced tools that facilitate handling of grid cases under liquid nitrogen and a stocker of the grid retainers contained in a CRYO ARM electron microscope. the tools named CryoGridTools are available from a GitHub site.
2
Blueprints of
1. Introduction Cryo-EM technology has developed rapidly in the last few years, such that several thousand movie stacks can now be collected in a single session in under a day using a direct detection detector with high speed readout and data acquisition schemes combining image and stage shifts (e.g. Zivanov et al., 2018; Schorb et al., 2019; (Danev et al., 2019; Mastronarde, 2005)). This greatly accelerates structure determination of protein molecules embedded in thin vitreous ice by single particle cryo-EM. Of course, data processing on high-performance CPUs and GPUs and sophisticated algorithms remain very important for increasing throughput (Punjani et al., 2017; Zivanov et al., 2018). However, preparation of suitable cryo-samples is still a limiting step for efficient application of this technique. In particular, challenging targets often require time-consuming screening of many samples, typically frozen under many different conditions.
In cryo-EM, metal grids (3 mmφ) coated with holey carbon film are generally used as a support structure onto which sample solution is directly applied. The grids are plunge-frozen in a cryogen such as liquid ethane and stored in small grid cases. In many laboratories, these grid cases are dropped into a centrifuge tube for storage in a liquid
3
nitrogen Dewar till use. Thus, before screening and data collection by cryo-EM large numbers of grid cases need to be processed and well-organized.
The situation is quite similar to macromolecular X-ray crystallography, which needs many crystals to be kept frozen and organized before transporting to synchrotron radiation facilities. Crystal mounters, typically, cryo-loops attached on a metal pin and a magnet base, are shelved in canes or metal containers. The latter, which typically holds 16 cryo-loops per container, can be linked with automated crystal mounting robots in many X-ray exposure facilities to dramatically increase the efficiency of screening for well-diffracting crystals. Several companies have now introduced similar-type containers with a diameter of ~70 mm to the cryo-EM field for storage of EM grid cases. One such container accommodates ~ 12 grid cases, and the containers slide into a tall shelf. Some products allow barcode-based registration of samples, which is useful when huge numbers of EM grids are processed. They are, however, heavy, sizable and costly, partly because the concept came from handling crystal mounters in X-ray crystallography. Similar designs have also been proposed in academic papers and are available from the authors (Scapin et al., 2017; Ultee et al., 2018), but production of these appears to be not easy for most users. Simplification of containers for cryo-EM
4
use should be possible, especially as automated mounting of grids into the sample storage unit is not yet feasible.
We designed and made several plastic tools to help process and organize cryo-EM sample grids using a fused deposition modeling (FDM) 3D printer. The grid case containers are light and stackable in a standard conical tube, and suitable for transporting to shared cryo-EM facilities. The 3D printing of many such stackers is easy and cheap. In addition, we designed tools that facilitate handling of grid cases in liquid nitrogen and one that stocks the grid retainer that is used to introduce a sample grid into the CRYO ARM electron microscope (Hamaguchi et al., 2019).
We name the tools
presented here CryoGridTools.
2. Design 2.1 Grid case stacker We designed a grid case stacker for a standard disk-shaped grid case with a diameter of ~14 mm, which typically has four slits into which EM sample grids fit (Fig. 1A). The grid case stacker holds three such grid cases standing vertically on three distinct slots (Fig. 1B). Each grid case settles nicely onto one slot of the stacker. The stackers fit
5
comfortably inside a 50 mL centrifuge tube with an inner diameter of 25 ~ 27 mm (Fig.1C), such as the widely-used conical type tubes of Falcon / Corning (Corning Inc., USA) with blue and orange caps (Fig. 2A). One tube accommodates up to five stackers and an extra single grid case can be placed underneath at the bottom (Figs. 1C and 2A right). In total 4 x 3 x 5 + 4 = 64 grids can be kept and organized in one tube (Fig. 2A right). Without the grid case stackers, ~ 22 grid cases randomly oriented can be placed in one tube (Fig. 2A left). Thus, while use of the stackers organizes grid cases, it does mean the number of sample grids in one tube is reduced by ~ 27%. We also designed a special grid case with six slits (Figs. 2B and C), which allows for precision tweezers to approach each slit from the same direction as in the standard case with four slits. The stacker fits well into a metal dipper filled with liquid nitrogen for safe transport (Supplementary Movie S1). 3D printing of stackers and grid cases in various colors would help with further organization of sample grids (Fig. 1A).
A metal screw on the stacker (Fig. 1A) has two functions: it helps to sink the stacker in liquid nitrogen (Fig. 1E) and to lift the stacker from / to the tube with a magnetic wand (Figs. 1D and 3; Supplementary Movie S1). The steel flat-head screw with a diameter of ~ 3.2 mm and a length of ~ 12 mm on the stacker is fitted with a
6
CrystalWand Magnetic (Hampton Research, USA), which has a release button (Fig. 1D lower). Using the 3D printer, we also made a simple pickup wand and then glued a small neodymium magnet (~ 6 mmφ) onto the tip (Fig. 1D upper). The stacker is easily released under liquid nitrogen by a wrenching action of the wand.
The stackers also settle well inside the conical tube where the number of stacks is less than five. In practice, a spacer is placed on top of the stacker for transport to the shared EM facility. We made several spacers for this purpose (Supplementary Fig. S1).
2.2 Grid case orientator The grid case must be placed in the stacker in a vertical position. We designed a tool for facilitating this, and named it the grid case orientator (Figs. 1F lower and G). This grid case orientator is fitted inside the outer space around the ethane vessel of the ethane container, which is supplied with Vitrobot Mark IV (Thermo Fisher Scientific, USA). The user can drop a grid case with plunge-frozen grids onto the orientator in liquid nitrogen, and the orientator supports the grid case in an upright position (Figs. 1G and 3; Supplementary Movie S1), thus facilitating the picking up of the grid case with tweezers and placing it in the stacker. The standard grid case holder, made of metal and
7
placed in the outer space of the ethane container, accepts four grid cases onto the hollows of four diagonal arms, but is not compatible with placing the orientator in the outer space. Thus, we replaced it with a 3D printed holder that lacks one arm, thereby affording enough room for the orientator (Figs. 1F upper, G and 3).
2.3 Retainer stocker for the CRYO ARM microscope Use of the CRYO ARM microscope requires that a frozen-hydrated grid is mounted on a sample retainer and attached with a C-ring clip (Fig. 4C and Supplementary Fig. S1 of Hamaguchi et al., 2019). Multiple EM grids are readily transferred into the sample storage unit in the microscope with this system. For alignment and calibration of the microscope, carbon grating and/or metal-coated carbon grids are routinely used but are usually too costly for onetime disposable use. However, detaching / attaching EM grids from / onto the retainer is cumbersome. We have already designed a special tool for detaching the C-ring clip (Hamaguchi et al., 2019), but found that even this tool may damage the grids.
It is more logical and convenient to keep the grids on the retainer.
Thus, we made a retainer stocker that accommodates four retainers (Supplementary Fig. S2). The stocker with retainers is usually kept inside a desiccator at ambient temperature till use.
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3. 3D printing and surface polishing 3.1 3D printing All tools were designed with open source 3D creation software, Blender (https://www.blender.org/) and the data exported in STL format for 3D printing. We used a wide-use 3D printer, UP Box+ (Tiertime, China) equipped with acrylonitrile butadiene styrene (ABS) modeling material. This material tolerates liquid nitrogen, whereas typical photo curable resin used with a stereolithography 3D printer (Jasveer and Jianbin, 2018) breaks easily in liquid nitrogen. Models were built by stacking ABS filaments at 230˚C. Layer thickness and surface layers were 0.2 mm and three, respectively. Filling factor for printing was close-packing to increase strength and weight of the models. A slower head speed, a correct temperature (~ 100˚C) of the base plate and a controled cooling rate improved printing quality because of the linear expansion coefficient. With this setting, the production rarely fails even for large complex structures such as the grid case orientator as well as fine ones such as screw holes. Small defects can be retouched with surface polishing (see below). We used ABS filaments in five different colors for the stacker. 3D printing of a single stacker takes ~ 20 min and costs less than a half US dollar.
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3.2 Surface polishing Printed models of filament layers were fused with acetone vapor. A small amount (10 20 mL) of acetone in a glass beaker was placed inside a well ventilated draft chamber, heated to 100˚C on a heat controller, and plastic wrap was used to cover the beaker to contain the vapor, as acetone gas is heavier than air. The surface of printed models exposed to the acetone vapor several times each for several seconds, melted quickly and became smooth. This process strengthened the structure, and also reduced bubbling in liquid nitrogen.
4. Working flow Our standard protocol for manipulating cases with cryo-EM grids is described below, where numbers correspond to Fig. 3. 1. Place a stacker and orientator in the outer space around the central ethane vessel of the ethane container. 2. Insert a grid case holder and anti-contamination ring inside the outer space and place an ethane vessel. 3. Place a metal cold finger inside the outer space. Fill the outer space with liquid
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nitrogen, and then fill the ethane vessel with liquid ethane. Remove the cold finger when the vessel is full of liquid ethane. 4. Place labeled grid cases on holder arms and freeze the sample solution on the EM grid and insert the frozen grids into the slits of the case as usual. 5. Remove the anti-contamination ring. 6. Drop the grid cases onto the orientator. Place the anti-contamination ring, and repeat from freezing of Step 4 until the three positions of the orientator are full. Or proceed to the next step. 7. Remove the grid case holder and transfer the grid cases into the stacker with tweezers. 8. Catch the stacker with a magnetic wand. 9. Transfer a stacker in a metal dipper filled with liquid nitrogen to a centrifuge tube in liquid nitrogen, and store. Sample details, such as composition, locality and freezing conditions, are recorded in a log book. The procedure is demonstrated in Supplementary Movie S1.
5. Discussion and conclusion We have made several cryo-EM tools with a wide-use FDM 3D printer for use in
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organizing cryo-EM sample grids, namely, a grid case stacker, a grid case orientator, a grid case holder with three arms, grid cases with six-, four- and two-slits, a pick-up wand, spacers, and a grid retainer stocker.
They have proved useful in our laboratory.
With the stacker (Figs. 1A and B), a total of 3 x 5 + 1 = 16 grid cases are stored in one 50 mL tube, and one 30 L liquid nitrogen Dewar can hold six tubes (Fig. 1E). In our four to five Dewars several thousand EM grids can be stored, and some are now well organized in stackers.
Users often carry frozen-hydrated samples on EM grids to shared cryo-EM facilities for data collection. 50 mL tubes suffices.
A typical number of grids is less than 50, and one or two
The stacker helps organize sample grids, although the number
of grid cases kept in the tube is reduced ~27 % because of the stacker volume. To obviate this, we made a grid case with six slits (Fig. 2C).
Commercial grid case containers do have benefits such as barcode-based registration of samples, easy access to all the containers from the side of the shelf, and so on. But the advantages, in our opinion, are not quite worth the expense.
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Our stacker is light, simple and easy to use with the help of the other tools presented here. One – 3 grid cases and 1 - 5 stackers fit well on each stacker and tube, respectively (Figs. 1C and 2A right). A spacer can be placed on top of the stacker for added stabilization during transport to other facilities (Supplementary Fig. S1). Moving stackers around to access buried grids is not problematic, thanks to the magnetic wand (Figs. 1D, 3 and Supplementary Movie S1).
The wide-use FDM 3D printer can use various materials such as polylactic acid (PLA), ABS, polyethylene terephthalate (PET), aliphatic polyamides (Nylon), and so on. ABS is superior to other materials in its processibility, impact resistance, ease of printing and cost (Samykano et al., 2019). ABS is durable even in liquid nitrogen. A stereolithography 3D printer can produce models with a higher machining accuracy and a smoother surface from general photo curable resin, but this material is easily broken in liquid nitrogen. We finish the models by surface polishing with acetone vapor, which increases strength by fusing each layer tightly, prevents contamination of small ABS pieces, and produces a smooth surface and decreases bubbling in liquid nitrogen.
Our designs for the several cryo-EM tools, particularly for the grid case stacker,
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are, of-course, not necessarily ideal for all users and instruments, but can be easily customized. The blueprints of all the tools in CryoGridTools are available from a GitHub site (https://github.com/YonekuraLab/CryoGridTools). We believe that 3D printing with surface polishing should be useful in not only the cryo-EM field but also in more general areas, and hope that this report will help accelerate its use.
Acknowledgments We thank Saori Maki-Yonekura and Radostin Danev for useful inputs on design of the grid case stacker. This work was partly supported by Japan Society for the Promotion of Science Grant-in-Aid for Scientific Research Grant 16H04757 (to K.Y.), Japan Society for the Promotion of Science Grant-in-Aid for Challenging Exploratory Research Grant 24657111 (to K.Y.), the RIKEN Pioneering Project, Dynamic Structural Biology (to T.H. and K.Y.), and the Cyclic Innovation for Clinical Empowerment (CiCLE) from the Japan Agency for Medical Research and Development, AMED (to K.Y.).
References Danev, R., Yanagisawa, H., Kikkawa, M., 2019. Cryo-Electron Microscopy Methodology: Current Aspects and Future Directions. https://doi.org/10.1016/j.tibs.2019.04.008 Hamaguchi, T., Maki-Yonekura, S., Naitow, H., Matsuura, Y., Ishikawa, T., Yonekura, 14
K., 2019. A new cryo-EM system for single particle analysis. J. Struct. Biol. 207, 40–48. https://doi.org/10.1016/J.JSB.2019.04.011 Jasveer, S., Jianbin, X., 2018. Comparison of Different Types of 3D Printing Technologies. Int. J. Sci. Res. Publ. 8. https://doi.org/10.29322/IJSRP.8.4.2018.p7602 Mastronarde, D.N., 2005. Automated electron microscope tomography using robust prediction of specimen movements. J. Struct. Biol. 152, 36–51. https://doi.org/10.1016/J.JSB.2005.07.007 Punjani, A., Rubinstein, J.L., Fleet, D.J., Brubaker, M.A., 2017. cryoSPARC: algorithms for rapid unsupervised cryo-EM structure determination. Nat. Methods 14, 290–296. https://doi.org/10.1038/nmeth.4169 Samykano, M., Selvamani, S.K., Kadirgama, K., Ngui, W.K., Kanagaraj, G., Sudhakar, K., 2019. Mechanical property of FDM printed ABS: influence of printing parameters. Int. J. Adv. Manuf. Technol. 102, 2779–2796. https://doi.org/10.1007/s00170-019-03313-0 Scapin, G., Prosise, W.W., Wismer, M.K., Strickland, C., 2017. A novel storage system for cryoEM samples. J. Struct. Biol. 199, 84–86. https://doi.org/10.1016/j.jsb.2017.04.005 Ultee, E., Schenkel, F., Yang, W., Brenzinger, S., Depelteau, J.S., Briegel, A., 2018. An Open-Source Storage Solution for Cryo-Electron Microscopy Samples 24, 60–63. https://doi.org/10.1017/S143192761701279X Zivanov, J., Nakane, T., Forsberg, B.O., Kimanius, D., Hagen, W.J., Lindahl, E., Scheres, S.H., 2018. New tools for automated high-resolution cryo-EM structure determination in RELION-3. Elife 7. https://doi.org/10.7554/eLife.42166
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Figure Legends Figure 1. 3D printed tools for organizing cryo-EM grids. A, grid case stackers. 3D printed in skin color, grey, blue, black and yellow and polished under acetone vapor. One stacker has three holding slots for typical disk-shape grid cases shown in cyan here. B, design of the grid case stacker. A steel flat-head screw with a diameter of ~ 3.2 mm is inserted into the hole before use. C, cartoon of five grid case stackers piled up in a 50 mL centrifuge tube and one grid case at the bottom. Sixteen grid cases are stored in one tube here. D, magnetic wands for picking up the stacker from / to the 50 mL centrifuge tube. Upper: Designed and made in this report. Lower: a commercially available item, CrystalWand Magnetic (Hampton Research, USA). E, cartoon of one centrifuge tube with grid case stackers in a cross-sectional model of a Dewar filled with liquid nitrogen. F, grid case holder (upper) and orientator (lower). G, a schematic diagram for making a grid case upright on the grid case orientator in liquid nitrogen. This step can be performed inside the outer space of the ethane container.
Figure 2. Grid case stackers with grid cases. A, (left) typical way for storing grid cases in the conical tube. Twenty-two grid cases are disordered in one tube here. (right) sixteen grid cases were ordered with stackers printed in five-different colors. B, design
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of a grid case with six slits. Sixteen grid cases with six holes can keep up to 96 grids, which is more than in 22 standard grid cases with four slits. C, 3D printed grid cases with six-, four-, and two-slits for various applications. The grid case with two slits is sometimes convenient for simpler operation and sorting.
Figure 3. Working flow to keep cryo-EM grids in grid cases organized by using the tools made here. Step 1: Place a grid case stacker and orientator in the outer space of the ethane container. Step 2: Insert a grid case holder, an ethane vessel, and an anti-contamination ring. Step 3: Place a metal cold finger. Fill the outer space with liquid nitrogen and then fill the central vessel with liquid ethane. Step 4: Place a grid case on the holder, and freeze sample solution on a grid and insert a frozen grid into a slit of the grid case. Step 5: Remove the anti-contamination ring. Step 6: Drop the grid case onto the orientator. Step 7: Remove the holder and transfer the grid case to the stacker. Step 8: Catch the stacker with a magnetic wand. Step 9: Transfer the stacker in a metal dipper to a centrifuge tube. Liquid nitrogen and liquid ethane are omitted here for clarity. See also Section 4 and Movie S1.
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Tools for manipulating sample grids in storage cases are designed, made using a wide-use 3D printer and polished under acetone gas.
The grid case containers are light and stackable in a standard conical tube, and suitable for transporting to other facilities.
Supporting tools are also designed to facilitate handling of grid cases under liquid nitrogen.
These tools named CryoGridTools are useful for organizing many cryo-EM grids and blueprints of the tools are available for sharing in the community and easy customization.
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