Cryo grids quality control policy
For users sending in ready-to-image cryo grids, we need to see 1) a couple of high magnification images to show how the sample look like, particle distribution and presence/absence of aggregation, degradation and contaminants; 2) a few low magnification images covering multiple grid squares to show ice thickness and enough usable grid squares on one grid for high throughput data collection. This is to ensure that suitable cryo grids with proper ice thickness, good particle distribution and plenty of usable area on a single grid are available to best use valuable microscope time.
To collect data on one sample, the cryoEM facility should only load up to 8 cryo grids all during one load among which one good grid will be chosen to set up data collection (one good cryo grids can easily last for more than 1 week’s data collection). No significant amount of Titan Krios time or staff hours should be used for screening grids to make the best use of valuable resources here and to maximize throughput.
Instruction on sending in cryo grids
For a scheduled data collection session, the cryo grids should arrive at the facility 1-5 business day before the start of the session if the user is shipping the grids. If the user is bringing the grids with him/her, it is fine for the grid to arrive on the morning on the first day of the scheduled session.
For one sample we typically load 6-8 cryo grids. To save sample handling and loading time, the 6-8 grids that will be loaded into the Krios should be stored in two cryo boxes instead of being scattered in multiple boxes. The two cryo boxes should be clearly labeled and tightened properly. User should choose cryoboxes that can be loosened with a commonly available tool such as a small Philip or slot screw driver.
For users with access to autogrids clipping tool (these are users with access to Titan Krios or Talos Arctica at their local cryoEM facility), they should clip the grids into autogrids (also called cartridges) before shipping. This will save sample loading time on the first day of the scheduled data collection session and thus the user gets more data collected instead.
Frozen cryo grids can be shipped to Janelia using a dry shipper that has been cooled with liquid nitrogen for extended period of time. The frozen grids stored in the so-called cryoboxes (or buttons) should be contained in a Falcon tube in the dry shipper. Holes should be made on the Falcon tube (see attached pictures). A string needs to be tied to the tube and one end left outside the dewar.
It is easy to use the dry shipper to transport frozen grids. Below is a rough guideline which can be used as a reference.
1, Cooled down the dry shipper with liquid nitrogen 3 days before shipment. May need to add more liquid nitrogen periodically.
2, Before shipping, lay the inner dewar of the shipper level on the side to pour out most of LN2 as FedEx does not ship a container filled with LN2. It is OK to leave a little bit LN2 in (it is also fine to completely empty the dewar. The dewar will still remain cold for days). Then put the inner dewar back in the shipper.
3, Transfer the falcon tube with cryoboxes filled with LN2 (which should have been stored in liquid nitrogen in a long term storage dewar) into the shipper. Leave one end of the string outside the inner dewar, secure the lid of the inner dewar.
4, Close the lid of the transfer case of the dry shipper (please use the lock mechanism if present) and the shipper is ready for shipping.
Instruction on sending in solution samples
We would like to start with the sample in relatively high concentration, 1-2 mg/ml if feasible. This is sample dependent. For small complex, say 200 kDa, 0.2-0.5 mg/ml might be enough. But for large sample, say 3 MDa, 5 mg/ml might be needed. Also samples with similar molecular weights may behave very differently on a cryo grid. There are samples which tend to stay on carbon film and do not get into holes. We will find the optimal concentration and conditions for making cryo grids for each sample here.
A couple of 20 microliter aliquots will be sufficient. Please also include 1ml dilution buffer.
The sample can be sent frozen on dry ice if applicable. This is preferred as it gives us flexibility on when to work on the sample. If we can not work on the sample immediately upon receiving, we can store it in -80C freezer and work on it at the earliest time when both equipment and staff hours are available.
If the sample is not stable with freeze/thaw cycle and it is stable at 4C, it can be sent on ice pack or wet ice. In this case, please coordinate with us on when to send the sample. Since we need to work on such sample immediately after receiving we need to plan ahead so that both equipment and staff hour will be available. Generally, it is preferred to receive the sample on Tuesday or Wednesday in a week as this gives us multiple consecutive weekdays to work on the sample if needed.
Ideally there should be no glycerol or sugar in the buffer and salt concentration is not very high (under 500 mM). But in special cases (such as glycerol must be present to keep structural integrity during freeze/thaw cycle), it is OK to have up to 30% glycerol in the buffer. We will try to remove the glycerol if feasible here before making cryo grids. So please include more buffer for this purpose. For some samples, a low concentration of glycerol (say 2%) might be helpful to keep the sample stable or dispersed on a grid. We can leave such low concentration of glycerol in the buffer while making cryo grids.
HHMI Janelia Research Campus
19700 Helix Drive
Ashburn, VA 20147
phone: 571-209-4000 x 1106
Any projects where Janelia CryoEM staff prepare samples, grids, or do analysis of resulting images are considered to be collaborations. For collaboration work with the CryoEM facility that results in publication, CryoEM staff members should be listed as co-authors.
For data collection-only services, on user-provided ready-to-image cryo grids, if CryoEM staff enabled customization of approaches or made significant intellectual input or overcame extra difficulties (such as multiple loads of grids to find a usable one instead of a single load of up to 8 grids for one sample) to make the work possible, they should be listed as co-authors. Otherwise, the following acknowledgement should be included in the publication: 1, When describing data collection in the Materials and Methods or equivalent section, please mention that data was collected on a 300 kV FEI Titan Krios microscope located at the HHMI Janelia Research Campus. 2, In the Acknowledgement section of the paper, please acknowledge help from the Janelia CryoEM facility staff. Here is one possible example:
We thank *** and *** at the HHMI Janelia CryoEM Facility for help in microscope operation and data collection.
After the paper is published, please send a link to the paper as well as one representative figure to the director of the Janelia CryoEM facility.