From the Browse Sessions page, create and configure new sessions and view a summary of existing sessions.
On the Configuration Tab, enter required parameters (or load a saved Configuration Profile) and select compute resources.
Preprocessing Lane: Select a Preprocessing Lane and a Number of Preprocessing GPU Workers. This is effectively the number of GPU workers that will carry out motion correction, CTF estimation, particle picking and extraction in parallel concurrently.
Reconstruction Lane: Select a Reconstruction Lane where Streaming 2D classification and Streaming Refinement jobs will be launched.
Auxiliary Lane: Select an Auxiliary Lane where Ab-initio Reconstruction and Generate Templates jobs will be launched.
Use SSD: If the disk you are using for the project directory is already an SSD, you don't need to copy the files to another SSD. Turn off
Use SSDif you do not wish to copy files over (for larger datasets, it can take some time to write all files over to the SSD before processing can stream in new collected particles).
Priority: Unless specified, all jobs in the Live Session will run according to the Session-level priority. For details on NEW Priority Queuing in cryoSPARC, please see: Priority Queuing Tutorial
Raw pixel size (A): The raw pixel size of the input movie data. For super-resolution data, this should be the super-resolution pixel size (i.e. not the camera native pixel size). For EER data, this should be the camera native pixel size, and you should also modify the corresponding EER parameters for upsampling and dose fractionation.
Accelerating voltage (kV): The accelerating voltage of the microscope collecting the data.
Spherical abberation (mm): The spherical aberration of the microscope collecting the data. This should be zero if there is a Cs-corrector.
Total exposure dose (e/A^2): This should be the total electron dose across each movie (i.e. not the per frame dose).
Minimum particle diameter (A): This can initially be set as the minimum dimension of the particle expected. This can be fine-tuned later on the Picking tab.
Maximum particle diameter (A): This should be set equal to or slightly larger than the maximum dimension of the particle expected. This can be fine-tuned later on the Picking tab.
Extraction box size (pix): This should be a box size in pixels for extracting particles. Generally, this should be set to about twice the particle diameter, and should be a set to a number that has 2,3,5,7 as its prime factors. Typical box sizes are between 256 and 640 pixels. Note that the box size is in pixels after Fourier-cropping (if enabled in motion correction). Mathematically good numbers are:
Apply to Allto save your parameter entries for all exposures (existing and incoming) in the Session. This is the recommended course of action when starting a New Session.
Apply to Future.
Enable continuous import: Toggling this on will allow new exposures added to the directory, to be processed as they are found. Be default, this is enabled. If you are creating more than one exposure group and the initial exposure groups are not expected to contain any new images that Live has not already found, you can disable continuous import from the older exposure groups to save disk operations needed to check for new files periodically.
Ignore exposures from group: This is useful only if you have multiple Exposure Groups configured, and you would like to ignore all exposures from a particular Group. This might be used if you find that all the images from a group are of poor quality, for example.
Directory to watch: The file system location where the exposures are being written or are already saved. This is a directory on the filesystem, not a wildcard path.
File name wildcard filter: You can optionally filter by wildcard to select files within the Directory to watch, that match a specific extension, e.g.,
*.mrc. This is important if multiple types of files will be saved in the same directory.
Search recursively: This option will traverse all subdirectories within the directory specified in
Directory to watchthat matches the
File name wildcard filtervalue. This can be helpful if your data collection software writes files to multiple sub-folders, for example. Leaving this option turned off will tell the file engine to only search for files matching the
File name wildcard filtervalue in the top level directory. If using Search Recursively, then in
Directory to watch, ensure you choose the highest-level folder to which movies are being written for the data collection session. For example, for EPU, specifying
Directory to watchvalue and
FoilHole_*_Data_*_*_*_*.mrcfor the filter value when
Search Recursivelyis enabled allows you to get around creating an exposure group for every grid square.
Gain Reference Path: Enter or select an absolute path to the gain reference, if the gain reference file is available. The gain reference should be in
.gain(for EER data) format.
Defect File Path: Enter or select an absolute path to the defect file, if the defect file is available. Note that zeros in the gain reference are also treated as defects and corrected in the same way.
As the session progresses, view diagnostic plots and metadata for each exposure. Start Manual Picking anytime, and optionally Reject or Reprocess individual exposures.
The Overview Tab contains time-series plots depicting the evolution of computed data attributes. Use these to optionally exclude groups of exposures based on various thresholds.
The Browse Tab contains various exposure statistics that can be plotted, compared and downloaded.
.csvfile containing all of the data.
Perform manual, blob or template-based particle picking from the Picking Tab as the session progresses, with the option to set new particle score thresholds and re-extract particles during preprocessing, which will be fed into downstream steps.
Minimum particle diameter (A),
Maximum particle diameter (A)and
Extraction box size (pix)that you provided on when configuring the Session, until and unless Blob Picker settings are adjusted or the active picker is changed.
Minimum particle diameter (A),
Maximum particle diameter (A), blob shape (circular, elliptical, blob, or a combination of them),
Lowpass filter to apply (A)and the
Min. separation distance (diameters)at any time during a session.
Activate for All: Applies the thresholds and. any changed parameters to all exposures (i.e., will cause all exposures to be re-processed with the changes)
Activate for Future: Applies the thresholds and any changed parameters to all exposures that have not yet been processed. Useful if you do not want your changes to trigger reprocessing.
Inspect Picksin cryoSPARC, to adjust and exclude poor quality picks.
Inspect Picksin cryoSPARC, to adjust and exclude poor quality picks. See the similar section for Blob picking here.
2D classification is performed seamlessly while a session is in progress. Newly available particles can be classified into existing classes as they are extracted, or 2D classification can be re-started at any time.
On the Ab-Initio Tab, generate an initial model from available particles or load an initial model to use for refinement.
Number of Classesif you wish to resolve more than 1 class,
Symmetry(optional, we recommend using the default C1 as it is not necessary/recommended to enforce symmetry during ab-initio reconstruction), and the number of
Particlesyou wish to use for Ab-Initio (also optional, default 100,000). Note that if you use multiple classes at this stage, you can select one as the initial model for streaming 3D refinement, but all particles will be used for refinement. Streaming heterogeneous refinement is not currently available in Live.
Refinement in cryoSPARC Live operates in a streaming manner, taking into account new particles that become available after preprocessing.
What to do once you have finished processing in Live.
Interacting with outputs and performing advanced processing.
CryoSPARC Live Exposure ExportJob: This job will run in the cryoSPARC Project where the Live Session is housed and will separately output accepted, rejected, and manually rejected exposures for each Exposure Group in the Session.
CryoSPARC Live Particle ExportJob: This job will export all particles from the Live session that pass the threshold tests in picking and have been extracted. These are the same particles that would be seen by streaming 2D classification. These particles will not come with alignment or class assignment information, but do contain
The Details Tab contains session history and a handy notes features.