2025A CryoTEMs for public use at SPring-8

Contents

1. Outline

CryoTEMs are offered for the contribution and the promotion of research and the enhancement of the competitiveness of users of the SPring-8 public beamline facilities in the field of structural biology and drug discovery. CryoTEMs will be available for use as ancillary facilities and will be treated in analogy to X-ray beamlines for research proposals in structural biology. Therefore, the expenses for CryoTEM use will be charged in the same way as it is for the use of SPring-8 public beamlines.

1.1 Mode of Operation

The following operation procedures will be applied to General Proposals and Proprietary Proposals. Graduate Student Proposals will be treated the same as General Proposals.

1. CryoTEMs are operated as a part of proposals in the field of Structural Biology (SB).
2. CryoTEMs are only available for research proposal that use both X-ray beamlines and CryoTEMs.
3. Available proposal types for CryoTEMs are General Proposals, Graduate Student Proposals, Long Term Graduate Student Proposals, Non-Proprietary Priority Proposals, Proprietary Proposals and Proprietary Time-Designated Proposals.
4. A questionnaire on the machine time allocation (to determine the number of shifts, date of use, desired microscope), termed “beamtime request survey”, will be completed by project leaders approx. 5 times a year. Prior consultation with CryoTEM staff regarding the schedule is necessary for time-designated use of CryoTEMs.

1.2 CryoTEMs Available to Users

The following four CryoTEMs are available to users.

• EM01CT: CRYO ARM 300 (K3)
• EM02CT: CRYO ARM 200 (K2)
• EM03CT: Titan Krios G1 (K3)
• EM04CT: Glacios (Falcon3/K2)

2. Application and Questionnaires for Machine Time Allocation

Applications of General Proposals, Graduate Student Proposals and Proprietary Proposals should include the statement that the use of both, beamlines as well as CryoTEMs, is planned. In addition, a description of how protein sample and protein crystals, even if protein crystals have not been obtained yet, are to be prepared for measurements must be provided. Every submitted questionnaire should also include a description of the status of protein or complex preparation. This can be done by using results from size exclusion chromatography (SEC), crystallization trials, native and denaturing PAGE, negative staining EM or CryoEM.
Since applications of Non-Proprietary Priority Proposals, Time-Designated Proposals and Long Term Graduate Student Proposals need to be accompanied by applications to other beamlines, please select the appropriate beamlines at application.
In addition, please note that prior consultation about the experimental schedule with staff of the beamlines and CryoTEMs is necessary for Time-Designated Proposals.

3. Notes on the Use of CryoTEMs

Participants in CryoTEM experiments are required to attend the pre-use training course*. It is free of charge.

• Users will carry out operation of CryoTEM during the allocated machine time (a unit of 3 shifts, starting at 10:00 a.m. and ending at 10:00 a.m. the next day) of the experiment.
• In general, the facility staff will handle the loading of samples into the CryoTEM. However, sample loading is performed at designated times as described below**.
• Vitrification equipment for cryo-specimen preparation is available for use in person at the facility.
• Together with automated data collection, we provide an environment for image processing (motion correction, and CTF estimation, which however, does not include full structural analysis, i.e. 3D reconstruction).

* This training course is intended for first-time users of CRYO ARM (JEOL) and teaches the minimum user requirements of operating the CryoTEMs in the facility.
Content: Software operation (JADAS: made by JEOL) for data acquisition after the sample has been loaded by the facility staff. This takes 1-3 days depending on prior experience.
Time period: In addition to the allocated machine time, courses are regularly held every other week during the operation period and can also be taken even before the approvals of the proposals.
** In principle, the timing of sample loading is assumed to be around 10:00, after 13:00, and before 17:00 during the allocated machine time. A maximum number of 12 grids can be loaded at one time. If you wish to perform independent sample loading and image acquisition, it is required to take the CryoTEM operation course for beginners (practical training for sample loading for independent on-site measurements) in addition to the pre-use training course described above.

4. Fees for Use of CryoTEMs

As with general proposals for other beamline usage, you are required to pay the following fees.

Machine Time Fee:
• General proposal, Graduate Student Proposal, and Long Term Graduate Student proposal: Exempted (Publication of results in peer-reviewed journal publications, etc. within 3 years after the end of the term of use is mandatory.)
• Non-Proprietary Priority proposal: 22,000 JPY/shift*
• Proprietary proposal (excluding Time-Designated proposal): 80,000 JPY/shift*
• Proprietary Time-Designated proposal: 120,000 JPY/shift*
※ The fees for CryoTEM use may be revised based on actual usage results of the trial operation.

User Fees:
• Fixed fee for consumables is 12,400 JPY/shift* (same as beamline use) for any types of applications.

* 1 shift = 8 hours operation

5. Contact

(1) Information Desk for CryoTEM

Center for Synchrotron Radiation Research
Structural Biology Division, JASRI
Director, Takashi Kumasaka
E-mail: kumasaka＠spring8.or.jp / TEL: +81-(0)791-58-0833
Or
Hideki Shigematsu
E-mail: shigematsu@spring8.or.jp / TEL: +81-(0)791-58-0833

(2) Information Desk for Procedure of Proposal Application