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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 lets you align movie frames by using a parallel version of 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 (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.


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.


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.


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.


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.


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.


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.


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.


In this section you can download the latest version of SPHIRE, crYOLO, and TranSPHIRE, 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 release:

Latest crYOLO release:

Latest TranSPHIRE release:

Older SPHIRE releases:


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 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!


Our next workshop will take place in Dormund, Germany, June 11th, 2018. Registration is now CLOSED.

Workshop outline

This workshop will provide hands-on and comprehensive training in all aspects of single particle cryo-EM image processing using SPHIRE. Attendees will be introduced to the SPHIRE workflow and process a high-resolution cryo-EM dataset with guidance provided by a team of instructors.

The exercises will cover the main phases of the SPHIRE processing pipeline:
    - Robust deep learning paricle picking (crYOLO)
    - CTF estimation (CTER) with error assessment
    - 2D classification (ISAC2)
    - 3D starting model generation (RVIPER)
    - High-resolution 3D refinement (MERIDIEN)
    - 3D Variability analysis
    - Reproducible Focused 3D Clustering (SORT3D) with statistical analysis of variance (ANOVA)
In addition to the practical sessions, short lectures will be given by SPHIRE developers focusing on crucial steps of the processing, possible pitfalls, as well as the theoretical background behind SPHIRE.
Registered participants will have the opportunity to present posters on their research work and are also strongly encouraged to bring their own data, since there will be ample time to interact with the instructors and process their own project with SPHIRE during dedicated hands-on sessions. Computing hardware will be provided in place.

Target group

The course is aimed at graduate students, faculty, and staff, that currently work in or plan to enter the 3DEM field and wish to use SPHIRE to process cryo-EM single particle data at their home laboratories for their own research projects.


The workshop will start on June 11, 2018 at 9 am and end on June 13, 2018 at ~1 pm. The exact schedule will be announced closer to the date of the workshop.

Registration fee

The registration is free and all meals are included. Participants are expected to cover their own travel and accommodation expenses.

Travel and accomodation

Travel and accommodation expenses are NOT included in the registration fee and must be arranged by the participants themselves.

Special rate hotel rooms in a nearby location will be reserved. We will send an info package with more detailed venue, travel and lodging information to all successful applicants. Participants are expected to attend the complete workshop.

The workshop is limited to 30 delegates. Application will close on Thursday, May 10, 2018. Applicants will be selected based on their motivation letter. Confirmation letters and invoices will be sent on Monday, May 14, 2018. 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.


Christos Gatsogiannis
Stefan Raunser
Pawel Penczek

Christos Gatsogiannis
Toshio Moriya
Markus Stabrin
Thorsten Wagner
Felipe Merino
Pawel Penczek
Stefan Raunser

Administration and Questions

If you have any questions, please feel free to contact Meike Schulte using the e-mail address below.

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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 ( as required by German law.


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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