NYSBC - New York Structural Biology Center

Best Usage

Scheduling Microscope Time During The Renovation Period

SEMC Rapid Access

Krios#1 - Summer 2016 (during facility upgrades)

Details of Rapid Access Time:


During this rapid access phase all active users from our member institutions will be allowed to apply for EM time. The minimum requirement would be for users to have screened similar cryo sample conditions (including preliminary data processing) for the grids that they will be loading into the instrument. We caution users that there is a risk to use the instrumentation at this time, however the reward is a chance of collecting data on a Titan Krios. If users would prefer to wait until after the renovations are complete that is understandable and recommended.

    1) Interested users should email emg@nysbc.org. They must state they are applying for rapid access time acknowledging that the center is undergoing renovations that may impact their data collection and submit an application. The application should include: a) Leginon project ID (if you do not have one please create one), b) Leginon IDs of the personnel that will be using the instrumentation, and c) a brief update of the progress of the project including why it is ready for the Krios (please include preliminary cryo data). See page 2 for details.

    2) All Krios1 users should have a project registered with the center and must fill out a survey at the end of their session.

    3) Rapid access user's grids must be dropped off and clipped before entering the rapid access queue.

    4) We will fill a cassette with multiple rapid access users (2-3 grids from each user) based upon the queue and rotate through the member institutions as conditions will allow. If more than 1-person from the institution applies, we will consult with the IR.

    5) We may not cycle through every user or institution during this rapid access phase because there are limited slots available. In addition, we hope that the construction will finish allowing us to resume our beta-test schedule.

    6) The cassette will be kept in the autoloader for the week and we will arrange for sessions as the contractor's schedules allow us to work. The contractor's work and any FEI engineer maintenance required during this time have first priority over the session and user's samples. The session length may vary or be terminated if the contractors deem it unsafe for SEMC staff to enter the Krios1 room.

    7) At the end of the week any rapid access user that has not had an opportunity will have their grids loaded into the next cassette.

    8) Rapid access users should leave contact information including a phone number, such that we may notify the user at a moment's notice. We will make every effort to give the user as much lead time before their session. If the user is unavailable to make use of their opportunity they may lose their spot in the queue and we will skip to the next user. We will be as accommodating as possible, but the second priority is to ensure maximum use of the instrumentation.

    9) We will support use of the Falcon II and K2 counting for single-particle data collection in nano-probe using Leginon. You may opt to use the instrumentation for other types of data collection, however the necessary time for setup will come from your session.

    10) Again the use of this rapid access time is at the user's risk. The staff will help as much as we can, but given the potential impact from the renovations recommend users with limited time, EM experience and samples to wait until we are out of beta-testing.?

Email emg@nysbc.org the following:
    1) Title of the email should be: "[SEMC Krios1] Rapid Access Application - summer 2016"

    2) State the Leginon Project ID(s). If you do not have one please register a project at http://emg.nysbc.org/semc_content/submitProject.html.

    3) State the Leginon User ID(s) of the personnel that will be using the Krios.

    4) Provide a brief update (300 word limit) on the progress of the project with why it is ready for the Krios (e.g. representative micrographs, 2D class averages and, if available, a preliminary 3D reconstruction). Links to the Appion session ID(s) may also be used.


Example Application:
Leginon Project: T20S proteasome dynamic studies
Leginon Project ID: 153
Leginon User ID: Careful User (cuser)

We are interested in the domain interactions between the subunits of the T20S proteasome. Initial negative-stain characterization of the protein went well and we moved onto cryo experiments. After extensive cryo screening we have been able to optimize freezing conditions to consistently obtain good quality grids. We have 7 grids ready to go (we froze 8 grids with the same protocol and screened 1 of them). Included are: a representative micrograph, 2D class averages and initial reconstruction with 50 micrographs from this screen. We are currently around 7-8Å and would like to use the Krios to be able to move the project forward. This processing may also be found in Appion experiment ID=1094.
Preliminary Cryodata

Waitlist:

You may request to be put on the waitlist to access unused time or last minute cancellations for your institution’s time. However, you must have grids ready to go and be available to come to the center at a moment’s notice (less than 24 h advanced notice).

Consecutive Sessions:

You may not request 3 consecutive sessions during weekday-daytime (9am-5pm) hours. It is OK and encouraged to do this with evening and weekend sessions or in combination with a daytime session (e.g. 9am to midnight on weekdays or 8am to 8am the following day on weekends) assuming you have All-Hours access.

Special Bookings:

Special case sign-up is available with the help of the SEMC Staff.

1) If you have a special need for more microscope time (e.g. you have to get a paper out quickly), please talk to the your Institutional Representative and email emg@nysbc.org to submit a request for extra time.

2) Should a multi-day session be needed users should contact the emg@nysbc.org and provide an explanation.

No Shows:

Failure to show up for sessions you booked in advance inconveniences everyone. If you find for any reason, that you will not use a session you have booked – YOU MUST CANCEL 24 HOURS IN ADVANCE.

Lateness:

If you are more than 30 minutes late, you will forfeit your session, if others wish to use it. If you know you will be late in advance notify the SEMC Staff 24 hours in advance.

Microscope down time/early termination of a session:

If there is an unexpected issue that leads to termination/cancellation of your session it is considered an act of nature/God. Though we will try to accommodate you, there should be no expectation of a makeup session.

An exception is if a pre-planned maintenance session must be rescheduled. We will work with you to reschedule your session for the next available time.

General Scheduling and Use Rules for all TEMs

Facility

The Simons Electron Microscopy Center provides expertise and resources for understanding both molecular and cellular structures. Molecular structure determination is enabled by high-end transmission electron microscopes (TEMs), direct detection cameras, and computational support for single particle analysis. Cellular structure determination is enabled by tomographic reconstructions using the TEMs and a focused ion beam (FIB) scanning electron microscope (SEM).

SEMC is supported by dues from member institutions as well as the Simons Foundation. The facility strives to provide service to our members, but has limited personnel and a major effort is made to provide training in the use of appropriate instruments to enable users to do their own microscopy.

All new users must register with the facility: http://semc.nysbc.org/start.html

Acknowledgement

Any use of the resources at SEMC requires acknowledgement of use of the resources in publications as follows:

"Some of this work was performed at the Simons Electron Microscopy Center at the New York Structural Biology Center that is supported by a grant from the Simons Foundation (Grant Number: 349247) with additional support from: NIH S10 OD019994-01, the Agouron Institute (Grant Number: F00316), NIH S10 RR029300-01, NIH S10 RR017291-01, NYSTAR, and NIH C06 RR017528-01-CEM."

Training

SEMC offers several training outreach programs: http://semc.nysbc.org/training.html

Project Discussion meetings [weekly]: Mon @11am and Thurs @3pm or @4pm
New user training [monthly]: Sample prep and TEM use
Appion workshop [quarterly]: Appion single particle data processing
EM course [yearly]: Theory of EM
All users of the center must be approved by their Institutional Representative prior to using any of the high-end instrumentation. This prior approval includes those who have been trained elsewhere in electron microscopy. The intent is not to limit access, but to ensure efficient usage of the EMs and protect the instruments from possible damage.

Scheduling

Once a user completes new user training and is checked out for a particular instrument they will be entered into the scheduling system. This is an on line scheduler that enables users to schedule time on the screening EMs without contacting Facility staff.

Time on the high-end EMs requires a project discussion meeting and preliminary cryo data. Users should notify their Institutional Representative and the SEMC staff to be placed on the queue. The intent of the following rules is to ensure access to the instruments for the largest number of users, not to limit access.

In general, users may sign up for up to 2 consecutive sessions (9am-1pm or 1pm-5pm on weekdays) or 1 day (9am-5pm on weekdays). Should a longer session be needed users should contact the emg@nysbc.org and provide an explanation. Users may sign up for no more than 4 sessions in total.

Signing up less than 24 hours before a session begins will not count against your bookings allotment. This is called a “waitlist” session.

Food and Drink:

No food is allowed in the microscope rooms, drinks must not be left on the counter or microscope tables.

Suggested “Best practices at NYSBC Simons Electron Microscopy Center:

• Arrive on time for your session!

• Try not to use microscope time for sample preparation. Whenever possible your grids should be ready to go at the start of your microscope session.

• Sign up to use any microscope, sample preparation equipment, or computer workstations prior to your arrival.

• Absolutely no food or drink in the lab.

• Be courteous to other users: finish your session on time and clean up after yourself.

• The dehumidified room often gets crowded; please leave any unnecessary belongings outside in the lab or control room. We will provide some lockers soon!

• Users who have not completed safety training and registered their C14s do not have 24 hour access and are only allowed to use the facility during business hours.

• If you ever have any doubts on equipment use, please seek help from an EMG staff member.

• Never use force on any equipment. If something is stuck leave it alone and call for help.

• We would suggest that when possible you use the instruments as follows:
JEOL1230: Negative stain screening
FEI Tecnai F20: CryoEM screening
JEOL 2100: High-resolution cryoEM single particle (once sample conditions have been optimized)

Emergency contact numbers:


Safety Officer:
William Rice
Normal Hours: x344
Second Contact:
Edward Eng
Normal Hours: x346
Lab Heads:
Clint Potter
Normal Hours: x301

Bridget Carragher
Normal Hours: x302