Ribosome preparation from turquoise killifish skeletal muscle for cryo-EM

Summary Ribosomes are composed of a mix of ribosomal RNAs and proteins; this composition varies depending on time, condition, and organism. Here, we present an optimized protocol for preparation of intact ribosomes from the skeletal muscle of the turquoise killifish. We also detail the steps for ribosome quantification and cryo-EM grid preparation. This protocol can enable the identification of heterogeneous ribosome structures that vary by fish age or in response to specific conditions.


MATERIALS AND EQUIPMENT
Preparation of the turquoise killifish sacrifice buffer Dissolve 1.5 g of tricaine methanosulfonate  in 1 L of tank water (pH 7.0, 700 mS). Adjust pH to 7.0 with sodium hydrogen carbonate. Store the buffer at 4 C and use it within 3 months. Warm the solution to 28 C before placing fish in the solution.
Prepare sterilized ribosome isolation buffers by filtering them through 0.2 mm filters. Note: Buffer compositions were optimized for turquoise killifish muscle by adapting from a previous publication (Forsberg et al., 2017).

STEP-BY-STEP METHOD DETAILS
Preparation of skeletal muscle from the turquoise killifish Timing: 2-6 h 1. Transfer fish (5-6 weeks after hatching, after sexual maturation) to 1.5 g/L tricaine methanosulfonate solution until gill movement stops completely. 2. Remove skin after cutting yellow lines, as shown in Figure 1. 3. Collect the skeletal muscle area, indicated as a black line in Figure 1, while avoiding contamination with other tissues. 4. Freeze in liquid nitrogen and store tissue at À80 C before use. 5. Grind the frozen muscle finely (particle size < 100 mm) with a mortar and pestle in liquid nitrogen.
[Troubleshooting 2] Note: Due to the hardness of frozen muscle, we have also tried to grind tissue with a bead beater using different sizes of tungsten beads ranging in diameter from 3 mm to 1 cm. However, the desired fineness was not achieved.
CRITICAL: It is hard to measure particle size while grinding, but particle fineness to be optimal when the muscle powder sticks to the mortar wall tightly even in the presence of liquid nitrogen, or you might grind 600 mg of skeletal muscle for 15 min.
6. Add 200 mg aliquots of tissue powder to e-tubes. 7. Store the tubes at À80 C.

Ribosome purification
Timing: 2 days-Ribosome purification should be performed on ice.
8. Homogenize 600 mg of finely ground skeletal muscle in 30 mL of homogenization buffer. [Troubleshooting 1] Note: To purify enough ribosomes for a single Cryo-electron microscopy (EM) experiment, we generally use 600 mg of tissue.
Note: Because the ultra-clear centrifuge tubes have a narrow neck, it is much easier to use a Pasteur pipet as a funnel for filling sucrose from the bottom.
16. Load cleared solution from step 14 onto sucrose cushion. Make sure not to disrupt the sucrose cushion layer ( Figure 2D). 17. Heat-seal the top of the tube after balancing with a seal former and cordless tube topper. 18. Centrifuge at 150,0003g for 12 h at 4 C. 19. Remove the supernatant very carefully so as not to lose the ribosome pellet ( Figure 2E). 20. Very carefully wash the pellets three times with 1 mL of washing buffer.
Note: Make the washing buffer flow the surface of the pellet couple of times per wash. Try not to touch pellet while washing.
CRITICAL: It is easy to lose a ribosome pellet in this step, so make sure that the ribosome pellet stays on the tube wall every wash. [Troubleshooting 2] 21. Check to see if a transparent ribosome pellet is visible at the bottom of the tube ( Figure 2F). 22. Collect ribosome pellets with a tip and resuspend them in 50 mL of resuspension buffer ( Figures  2G and 2H).
Note: The pipette tip should be cut diagonally for easier collection of the ribosome pellet. The ribosome pellet is sticky, making it difficult to collect from the tube. Transferring a ribosome pellet can be facilitated by touching the e-tube wall. The remaining ribosome pellets should be collected by adding resuspension buffer directly to the centrifuge tube.

OPEN ACCESS
This step determines the quality and quantity of the isolated ribosomes prior to making a cryo-EM grid.
Note: OD 8-12 at 260 nm is a good quantity range. If OD is higher than 12, adjust ribosome into OD 8-12 with resuspension buffer.
Note: We used the Gatan Plasma System (US100, Gatan) using the instructions on the screen of the equipment. b. Load 4 mL of isolated ribosome onto the grid and incubate for 30 sec. c. Wash the grid twice by placing the grid briefly onto a drop of distilled water. d. Remove excess water from the grid by touching the grid edge with filter paper. e. Incubate the grid in uranyl acetate for 30 sec. f. Wash the grid twice by placing the grid briefly onto a drop of distilled water. g. Remove excess water from the grid by touching the grid edge with filter paper. h. Dry the grid for 5 min at 25 C.
Note: Ribosomes usually stained darker than background in our trials. It is critical to check ribosome particle distribution, aggregation and contamination.

Timing: 2-4 h
This step describes preparing grids for cryo-EM. This step can be adjusted according to sample condition.
28. Glow discharge both sides of a new grid with argon gas for 30 s which is the same condition above (step 27a). 29. Coat the grid with graphene oxide. [Troubleshooting 4] a. Load 3 mL of graphene oxide solution onto the carbon side and incubate for 2 min b. Remove excess graphene oxide from the grid by touching the grid edge with filter paper and wash three times by placing the grid on distilled water drop. c. Remove excess water on the grid touching grid edge to filter paper. d. Dry for 5 min and use within a month without additional glow-discharge.
Note: This step is performed according to a described protocol (Bokori-Brown et al., 2016).

Cryo-EM grid preparation
a. Set-up Vitrobot for Cryo-EM grid preparation.
Note: We used VitrobotTM (FEI, Thermo Fisher Scientific) using the process parameters on the screen of the equipment.
b. Load 3 mL of sample onto the graphene oxide coated grid. c. Wait 30 s and blot the ribosomes for 3 s with 10 of blotting force.
Note: When you are trying to optimize sample blotting conditions for a new sample, it is recommended that 6-12 grids be prepared per sample and that different sample concentrations and waiting and blotting times to be tested.
31. Store grids in liquid nitrogen before imaging and data collection. 32. Collect data ( Figure 3C).

EXPECTED OUTCOMES
This protocol describes the overall procedure from skeletal muscle sample to cryo-EM grid preparation. The quantity and quality of isolated ribosomes can be monitored in steps 26 and 27 ( Figures  3, 4, and 5). These steps are critical to proceed to the next steps for Cryo-EM data collection. If the ribosome quantity and quality do not meet the suggested criteria, further steps are not warranted. Furthermore, graphene oxide coating also greatly affects ribosome loading onto the grid. Graphene oxide coatings are highly variable, and depending on the situation, which can cause different degrees of grid quality. Thus, it is necessary to determine whether one or two grids are coated with graphene oxide prior to sample loading. In addition, the best grids and holes should be screened with a microscope after sample loading. Ribosomes on grids can be imaged and data can be collected as shown in Figure 3C. This protocol helps to reduce errors that can occur during ribosome sample preparation from skeletal muscle. Tissue properties differ, making it necessary to adjust experimental conditions for different tissues.

LIMITATIONS
Ribosome preparation from animal tissue is largely dependent on the amount and properties of the tissue of interest. The turquoise killifish is a small teleost fish ranging from 2.5-6 cm in length. Collecting skeletal muscle tissue from young fish (5-6 weeks after hatching, after sexual maturation) is difficult due to its softness and small quantity. By contrast, harvesting skeletal muscle from old fish (16-18 weeks after hatching, around the median lifespan) is easier. However, ribosome yield from the muscle of old fish is generally lower than its yield from the muscle of young fish for unknown reasons. These variables affect sample size for the experiments. EM may be a limiting step to check ribosome quality and grid preparation. Finding the closest EM facility is always best when preparing grids. Ribosomes obtained under certain conditions, such as those from old fish, are unstable based on our experiences; thus, grids should be prepared immediately after ribosome isolation.

Potential solution
Ribosome yield can be dependent on sample quantity ( Figure 4A). To increase yield, use larger tissue samples, while increasing the starting sample amount and volumes of the homogenization buffers accordingly. Importantly, do not mix ribosomes isolated on different days. Ribosome pellets can also be lost when washing and transferring them to new tubes ( Figure 4B). In this case, the positions of ribosome pellets should be marked on the centrifuge tubes, to ensure the transfer of sticky ribosome pellets.

Potential solution
Not fully-ground muscle tissue and myosin are major sources of contamination ( Figures 5A and 5B). Muscle tissue should be ground as finely as possible. Muscle fiber contamination often occurs when the volume of homogenization buffer is insufficient. An optimal amount is 600 mg tissue per 30 mL of homogenization buffer. Increasing the volume of the homogenization buffer reduces muscle fiber contamination. The volume of this buffer, however, can be adjusted as needed.

Potential solution
Ribosomes often aggregate when the ribosome pellet is not fully suspended ( Figure 5C). Sufficient time is needed for careful resuspension of ribosome pellets. Fully suspended ribosomes at the optimal concentration should resemble those shown in Figure 3B. Fully suspended ribosomes at low and high concentrations should be resemble those shown like in Figures 5D and 5E, respectively.

Problem 4
Cracked grids and wrinkled carbon film. Potential solution Cracked grids are often observed during cryo-EM data collection ( Figures 6A and 6B). This occurs during blotting ribosome samples to grid. Usually, cracked girds can be discarded at the final screening step. However, if this occurs to all grids with no useful squares, blotting force should be reduced. Wrinkled carbon film is frequently observed when the grid is coated by graphene oxide ( Figure 6C). Alternatively, carbon coating can be performed using plasma deposition. Either method is appropriate if it results in a single layer of carbon on the grid.

RESOURCE AVAILABILITY
Lead contact Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Yumi Kim (yumikim@ibs.re.kr).

Materials availability
This protocol did not generate new materials.

Data and code availability
The data from this study are available from the corresponding author upon reasonable request.
This study did not generate any new codes.

ACKNOWLEDGMENTS
This work was supported by the Institute for Basic Science (IBS-R013-D1 and IBS-R022-D1), Republic of Korea. EM grid preparation and transmission electron microscopes were performed at the DGIST Center for Core Research Facilities.