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SPHIRE – The Package

SPHIRE is a new software suite designed for easy access to cryo electron microscopy with the clear goal of quality assessment and result reproducibility by statistical resampling. While being well suited for cryo-EM novices, experienced users will find comfort in the accessibility of almost every possible variable in advanced option tabs and the transparent, easily customizable Python-based framework for non-standard processing pipelines. In a visually appealing and easy-to-use graphical user interface (GUI) the user will find an array of programs which will guide through the complete process of high-resolution cryo-EM. This begins with movie frame alignments (movie), CTF estimation of raw electron micrographs (cter) and picking/stack creation (window) and continues with reproducible 2-D classification (isac), reproducible initial model generation (viper), automatic gold-standard 3-D refinement (meridien), local resolution estimation and filtering (localres), up to the 3-D sorting of different conformational states based on the statistical 3-D variability of the data (sort3D).


Movie

Movie lets you align movie frames by using a parallel version of Unblur (https://grigoriefflab.janelia.org/unblur) in the framework of our graphical user interface. After processing the images you can assess the drift rates and have a look at the micrographs. Check our Wiki for details.


Cter

Cter (Penczek et al. (2014) Ultramicroscopy 140:9-19) contains a CTF estimation tool and an extensive, GUI-based CTF evaluation toolbox which make it easy to analyse and discard images based on a multitude of different parameters. The CTF evaluation toolbox is recommended for advanced users and is described in detail on our Wiki page. Check our Wiki for details.


Window

With Window you can use the boxing tool of EMAN2 (Tang et al. (2007) Journal of Structural Biology 157(1):38-46) to select your particles, import them into SPHIRE with a click of a button and create a stack for further processing. We decided to include e2boxer since it is one of the most versatile particle selection tools available at the moment. Check our Wiki for details.


Isac

With ISAC (Yang et al. (2012) Structure 20(2):237-247) you can reliably calculate 2-D class averages of your dataset. It is based on reproducible K-means classification and yields trustable averages of the data. ISAC is computationally demanding and thus scales images down to a box size of 72 pixels. With the new post-processing step, however, we introduce the possibility of obtaining high-resolution class averages for publication. Check our Wiki for details.


Viper

Initial model generation can be tricky. VIPER uses a stochastic hill-climbing algorithm to calculate initial models and has the ability of attempting the calculation of reproducible models. With this option, only results which are reproducibly obtained are used as reference for the subsequent refinement step, making false inital model estimations a little bit less probable. Of course you can also use a PDB model as initial reference for refinement by converting it with our Utilities. Check our Wiki for details.


Meridien

Reliable 3-D refinements which minimize the over-estimation of noise while preserving the possibility of changing most of the refinement parameters as an advanced user. This is Meridien. Calculate refinements with minimal user interaction and let Meridien intelligently estimate most of the variables according to the data in each iteration, or choose to define your own values and userfunctions (e.g. for specific filtering and/or masking after each iteration). With Meridien you have the flexibility of going into specifics whenever a structure is not easily obtained with standard values. Check our Wiki for details.


Sort3D

One of the nice properties of cryo-EM is the ability to sort different conformational states after the actual experiment has taken place, handing the researcher an extra degree of freedom for structure ellucidation and the physiological interpretation of the results. Sort3D takes the results obtained from Meridien and Localres and statistically analyses the 3-D variability of the data. This analysis is then used to run our 3-D sorting algorithm. As in other instances of SPHIRE you will have the choice of more and less user interaction. Check our Wiki for details.


LocalRes

3-D reconstructions never show a homogeneous resolution throughout the molecule. With the LocalRes program you can calculate the local resolutions of your reconstruction and filter the structure accordingly. The locally filtered structure is a much better representation of your data and can be used for your publications. Check our Wiki for details.


Utilities

We also packed some utility programs into SPHIRE which may come in handy during processing. You can analyize the structure which was calculated in Meridien for projection consistency, display data using EMAN2's e2display (Tang et al. (2007) Journal of Structural Biology 157(1):38-46), convert PDB data into reference structures or calculate adaptive 3-D masks and 3-D angle maps. Don't forget to check Utilities if you run into an issue where you would need this extra piece of script that you don't have! Check our Wiki for details.








Downloads

In this section you can download the latest beta version of SPHIRE, patched with the latest bugfixes. Furthermore, we have created an extensive tutorial for you to have the best possible experience with the GUI and a smooth introduction into the SPHIRE world. The PDF tutorial and test data can be downloaded here as well. If you are interested in specific information about the algorithms of SPHIRE, please visit out Wiki page. Be up-to-date and subscribe to the SPHIRE mailing list (see Contact section)! We will post any important news there.


Latest SPHIRE beta release:


Older SPHIRE beta releases:








Contact

We will do everything in our power to get back to you whenever you have a problem. However, in order to prevent double and triple questions, please search our Wiki and our mailing list for information on your topic of interest before contacting us directly!


Mailing list

In order to subscribe to the SPHIRE mailing list, please go to https://listserv.gwdg.de/mailman/listinfo/sphire and subscribe. Once you have subscribed, you can use the address below to send e-mails to the list. Please do not send an e-mail to the list for subscription!

sphire@lists.mpg.de








Workshops

Our next workshop is going to take place in May 2017. Registration is now CLOSED. We are already planning a second workshop for this year and will inform over the standard channels (mailing lists, Twitter) once there is something concrete.






Workshop outline

This workshop will provide hands-on training in all practical aspects of single particle cryo-EM image processing using SPHIRE. Attendees will be introduced to the image processing workflow of SPHIRE and process a high-resolution cryo-EM dataset in real-time under close supervision.

The intensive hands-on practicals will cover all stages of the SPHIRE processing pipeline:
    - Micrograph movie alignment
    - CTF estimation
    - 2D classification
    - 3D starting model generation
    - High-resolution 3D refinement
    - 3D focused classification and heterogeneity analysis
In addition to the practical sessions, lectures will be given by SPHIRE developers (incl. Pawel Penczek, Christos Gatsogiannis, Toshio Moriya and Stefan Raunser) focusing on crucial steps of the processing, possible pitfalls, as well as the theoretical background behind SPHIRE.

Linux laptops and guest accounts to a remote linux cluster will be available for all delegates throughout the workshop. Processing of own data will not be possible, however, there will be ample time to discuss own projects with the SPHIRE developers.




Target group

The workshop is targeting students, postdocs, staff scientists and group leaders from academia and industry who have some experience in cryo-EM and wish to process their own data in the near future with SPHIRE.




Program

The workshop will start on May 8, 2017 at 9 am and end on May 10, 2017 at 12 pm. The exact schedule will be announced closer to the date of the workshop.




Registration fee

The registration is 150 Euro, and includes all meals and coffee breaks during the workshop.




Travel and accomodation

Travel and accommodation expenses are NOT included in the registration fee and must be arranged by the participants themselves. Participants are expected to attend the complete workshop.

Our administration reserved a sufficient number of rooms in a nearby hotel (price range of 70-90 Euro/night). We will send an info package with more detailed venue, travel and lodging information to all successful applicants on April 4, 2017.




The workshop is limited to 30 delegates. Application will close on Sat, April 1, 2017. Applicants will be selected based on their motivation letter. Confirmation letters and invoices will be sent on Tue, April 4, 2017. The motivation letter is limited to 1000 characters. Please briefly explain why you would like to attend, your previous experience in cryo-EM, and how you think your own research project will benefit from the workshop.


Organization

Christos Gatsogiannis
Stefan Raunser

Instructors
Christos Gatsogiannis
Felipe Merino
Toshio Moriya
Pawel Penczek
Stefan Raunser
Michael Saur
Markus Stabrin



Administration and Questions

If you have any questions, please feel free to contact Meike Schulte using the e-mail address below.
Email: sphire-workshop@mpi-dortmund.mpg.de









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The following provides mandatory data concerning the provider of this website, obligations with regard to data protection, as well as other important legal references involving the Internet site of SPHIRE (sphire.mpg.de) as required by German law.


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The provider of this Internet site within the legal meaning of the term is the registered association Max Planck Society for the Advancement of Science e.V.


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The value added tax identification number of the Max Planck Society is DE 129517720.


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Responsible editors for the contents of the website of SPHIRE (sphire.mpg.de) with regard to media law:

Max Planck Institute for Molecular Physiology
sphire.mpg.de
Prof. Dr. Stefan Raunser
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Technically responsible (Webmaster)

Technically responsible for the website of SPHIRE (sphire.mpg.de):

Markus Stabrin
markus.stabrin@mpi-dortmund.mpg.de


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SPHIRE