Perelman School of Medicine at the University of Pennsylvania

Electron Microscopy Resource Lab

Beckman Foundation
Electron Microscopy Resource Lab

Cryo-EM Training and Screening Facility


New User:

All New Users are required to email (SCREEN_SERV@lists.upenn.edu) for Cryo-EM services. Once you send the email with your request, we will get back to you about the project feasibility and goals.

Internal User

Penn and CHOP

External User

Institutions near and around Penn

(Wistar, Temple, Drexel, Jefferson, Swarthmore, etc.,)

Corporate User

Pharma or Scientific Industry

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Lab Access:

EMRL doors are locked 24x7. A new user will need to provide the Entire Name and Entire Penn ID number to activate the Penn Card and gain access to the facility.

External Users can request for a Guest PennCard at the facility and a charge of $30 is accessed by Penn Card services.

Equipment Availability for Sign-up

New User

9:00 AM to 5:00 PM

Independent User

7:00 AM to 11:00 PM

Staff Availability

9:00 AM to 5:00 PM

For after-hours assistance, Independent users can call core director 7:00 AM to 11:00 PM.

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iLab Access:

The new user will need to communicate with Katie Heer (Business Administrator of the cores) @ kheer@pennmedicine.upenn.edu and set up access to EMRL lab on iLab website. Internal users will have CAMS connected to iLab and External users will create a PO and upload on to the iLab with the help of Katie.


Instrument Rates:

 

Instrument

(Name)

Internal

(Hourly)

External

(Hourly)

Corporate

(Hourly)

Vitrobot Mark IV

$25.00

$35.00

$50.00

Leica EM GP2

$25.00

$35.00

$50.00

Leica Manual

$25.00

$35.00

$50.00

T12

$50.00

$70.00

$100.00

TF20

$50.00

$70.00

$100.00

 

Training Fees

Instrument

(Name)

Internal

(One-Time Fee)

External

(One-Time Fee)

Corporate

(One-Time Fee)

T12

$200

$280

$400

Tf20 Training for Cryo-EM*

$300

$420

$600

*CryoEM training Fee includes training Tf20, Vitrobot, Cryo Holders, Pumping station.

Pay-Per-Service: Imaging performed by staff (Internal Users only)

Negative Stain Imaging

(Performed by staff)

Internal

$350

External

N/A

Corporate

N/A

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EMRL Cryo-EM Listserv:

Subscribe to the EMRL-Cryo-EM user group (Click Here) and be updated on the status of the microscopes and the facility.

3DEM Listserv:

This listserv is highly recommended for cryo-EM. Most of the posts are related to learning, exploring and discussing cryo-EM. Click here to navigate to the subscription page.


Cryo-EM in brief:

Cryo-Electron Microscopy (Cryo-EM) begins with vitrification, in which the protein solution is cooled so rapidly that water molecules do not have time to crystallize, forming an amorphous solid that does little or no damage to the sample structure (a process known as vitrification). The sample is then screened for particle concentration, distribution, and orientation. Next, a series of images is acquired, and two-dimensional classes are computationally extracted. In the final step, the data is processed by reconstruction software, yielding accurate, detailed, 3D models of intricate biological structures at the sub-cellular and molecular scales. These models can reveal interactions that were impossible to visualize previously, a key to scientific results.

 

To prepare a cryo-EM specimen suitable for high-resolution data collection, the following steps are typically followed:

Biochemistry

Sample Preparation

Before a biological specimen such as protein macromolecule is examined using the electron microscope, the sample has to be purified to homogeneity using biochemical techniques. Once purified, it's often useful to check the polydispersity index of the sample in solution using Dynamic Light Scattering technique. This method is quick and easy to evaluate if the protein tends to aggregate dimerize or if the complex is falling apart. The structural integrity of the protein macromolecule is then confirmed by negatively staining the protein and observing for the suitability of the protein for solving the structure of the protein using cryo-EM. 

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Negative Staining

The freshly purified sample is diluted to ~0.05 mg/ml and applied on to a glow discharged copper grid coated with a thin layer of carbon. The sample is then stained using a freshly prepared 2% stain solution (uranyl acetate or formate or tungstate stain solutions) and the excess liquid is blotted away using a filter paper. The sample is now adsorbed on the carbon and covered with a thin layer of stain. The sample grid is now observed using T12 TEM microscope. If the sample is intact and free of contaminants, the sample is then vitrified for cryoEM.

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Cryo-EM Sample Preparation

 

A variety of conditions has to be screened in order to get the best condition for high-quality data collection on a Titan Krios microscope. The FEI Vitrobot and TF20 microscope in EMRL will be used for freezing and screening the samples for proper optimizations. Once the right condition is obtained, the sample will then be sent to Singh Center for data collection on Titan Krios microscope. 

 

 

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High quality cryo-EM Data collection on Titan Krios

Once the right condition is obtained, high-quality cryoEM data will then be collected on Titan Krios microscope at Beckman Center for Cryo-EM

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Computational Resources for Cryo-EM data processing

A number of software suites are available to process cryo-EM data:
Amazon

(AWS) cloud computing services by Amazon is providing Cryo-EM in the cloud tool for analyzing as well as processing data all on the cloud. Follow the link below and learn more about it.

Electron microscopy image analysis in the cloud

Also, use the link below to subscribe for the mailing list for AWS cloud computing for Cryo-EM:

https://groups.google.com/forum/#!forum/cryo-em-in-the-cloud


CCP-EM
(Collaborative Computational Project for Electron cryo-Microscopy)
http://www.ccpem.ac.uk/index.php

Subscribe to this mailing list and learn more from the updates: https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCPEM&A=1


EMAN 2.0      http://blake.bcm.edu/emanwiki/EMAN2

EMAN software is fairly easy to install and run on a PC or Mac. Its worth practicing the software of your interest by practicing the example data provided by the author. click on the above link and learn more about the software and the steps involved in the cryo-EM data processing.


RELION:       https://www2.mrc-lmb.cam.ac.uk/relion/index.php?title=Main_Page

SIMPLE:        http://simplecryoem.com/

SPHIRE:        http://sphire.mpg.de/

IMAGIC:       http://imagescience.de/imagic/

Xmipp:           http://xmipp.cnb.csic.es/

Appion:          http://appion.org

Scipion:          http://scipion.cnb.csic.es/

Sparx :         http://sparx-em.org/sparxwiki 

Spider:        http://www.wadsworth.org/spider_doc/

Suprim:          http://emg.nysbc.org/redmine/projects/suprim

Frealign:        http://emlab.rose2.brandeis.edu/

Jspr:            http://jiang.bio.purdue.edu/jspr

Some of the new noteworthy software suites that are highly used are as follows:
Gautomatch:          http://www.mrc-lmb.cam.ac.uk/kzhang/Gautomatch
bfactor:                   http://emlab.rose2.brandeis.edu/software
UCSF Chimera:    http://www.cgl.ucsf.edu/chimera
VMD:                     http://www.ks.uiuc.edu/Research/vmd/
em2em:                  http://www.imagescience.de/em2em.html
DireX:                    http://www.schroderlab.org/software/direx/index.html
Rosetta on Amazon Cloud computing:  http://cryoem-tools.cloud/rosetta-aws/

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