NYSBC - New York Structural Biology Center

Best Usage

Scheduling Microscope Time

Krios Operations

Instrument Status
Krios01: Normal Operations
Krios02: Open beta - accepting applications. Time on Krios02 will be by request and applications awarded on a rolling basis rotating through all member institutions.
Krios03: Under testing

    General Krios information
    1) Please remind your users that grids will be due at the center no later than Wednesday at 2pm the week before the scheduled session and are encouraged to be dropped off well before then.

    To open the Krios Drop-off Checklist, click here.

    2) Each institution has pre-allocated days on instrument under normal operations, but are not limited to this time. There is rapid access time or NYSBC maintenance time each cycle that every user may apply for.

    3) We have two (2) types of rapid access, both of which require an application:
    A) Moment's notice
    Time available due to an unexpected opening in the schedule. Grids should be pre-clipped and stored at the center, or the user should be able to bring it to the center at a moment's notice for immediate cassette loading.
    B) Scheduled rapid access
    In between cycles we have system time for maintenance and development. Unused time during this period may be re-allocated as pre-scheduled rapid access time.

    4) The preallocated days are meant to be similar to an airline/train schedule. If time is lost or unassigned then the session is gone. However, there is at least 1 possible rapid access session each month to act as an emergency buffer.

    5) These dates are the allocation to the institution not an individual lab. Please contact your institution representative for more information.

General Krios Application

Email emg@nysbc.org the following:
    1) Title of the email should be: "[SEMC Krios1] (instrument number)] C(cycle number) - 2017: (Institution) Krios application", e.g., [SEMC Krios1] C5 - 2017: SEMC Krios application

    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:

[SEMC Krios1] C5 - 2017: SEMC Krios 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. In our submitted manuscript we have a sub-4Å structure, but the reviewers wanted additional evidence of the ßK382-ΔE15 salt bridge that was an important point in the paper, as well as a ligand-binding interface that impacts activity. Biochemical experiments are underway and we have 4 cryo grids ready to go (we froze 4 grids with the same protocol and screened 1 of them). Included are: a representative micrograph, 2D class averages and initial reconstruction with 25 micrographs from this screen. We are currently around 7-8Å on the TF20/4K CMOS and have saved this grid. 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:

Standard Acknowledgement:
"Some of this work was performed at the Simons Electron Microscopy Center and National Resource for Automated Molecular Microscopy located at the New York Structural Biology Center, supported by grants from the Simons Foundation (349247), NYSTAR, and the NIH National Institute of General Medical Sciences (GM103310) with additional support from [copy and paste depending on equipment]"

Krios1/K2: Agouron Institute [Grant Number: F00316]
Krios1/Falcon : NIH S10 OD019994-01
Helios: NIH S10 RR029300-01


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