gP2S, an Information Management System for CryoEM Experiments

Overall design and navigation pattern
The gP2S application is project oriented, such that an entity can only be created in the context of a project. The relevant project is first selected from the dropdown located near the top left corner of the application. For convenience, the list of projects is filterable and it is sorted with the recently used projects shown at the top. When selecting a project, the number of entities of each type which are associated with this project is displayed in the workflow section of the left-hand-side navigation bar. The user can then click on any of the workflow entity types (e.g., Microscopy Sessions) to display a list of those entities within the selected project (Figure 4). This list consists, for each entity, of a label, date and time of creation, the name of the user who created it, an indication of whether any comments have been made about this entity, and up to six key metadata fields (for example, for each Microscopy Session: Grid, number of images, starting and finishing times, and what Microscope and Detector were used). Selecting one of the listed entities opens a details page listing all the information available for this item, including a summary list of all ancestor entities (for example, for a Microscopy Session, its parent Grid and Sample are listed). This allows for very quick navigation through the "lineage" of an entity, for example enabling single-click navigation from an atomic Model to the details of the Sample (Figure 5). In addition, any entity in gP2S can be commented on, by selecting "Comments" in the upper right part of its details page, entering a free-text comment, and optionally attaching one or more files.

Sample preparation
In the first step of the workflow describe the Sample. To do so, first define at least one component: Protein or Ligand.

Adding a new Protein requires only a protein label, but to help in better describing the protein add a PUR ID (for purification identifier). This field accepts any text and can for example contain a lot/batch number or serve as a place for a barcode label. If gP2S has been customized to integrate with a protein registration system (see Discussion), the PUR ID can be validated automatically and used to retrieve and display detailed information about this lot of protein. For Ligands, a label and stock concentration are mandatory information. All other fields are optional, and include: concept (barcode, common name or other ligand identifier) and batch/lot identifier. Again, if gP2S has been configured to integrate with a ligand registration system, the concept and lot identifiers can be used to fetch and display externally-stored data describing the ligand (e.g. its chemical structure, assay results).

A Sample is defined by any combination of Proteins and Ligands and their final concentrations. Optionally, specify other experimental details of the sample such as incubation time and temperature, buffer and a free-text protocol description.

Grid preparation
When the Sample is ready, navigate to Grids. In the list, under each Grid's label find one or two colored tags that indicate the grid type (cryo or stain) and whether that grid is available for use. To create a new Grid, select Create New Grid. Type in a label, select the Grid Type and the Surface Treatment Protocol (e.g., glow discharge) used. Then, indicate whether preparing a cryo or negative stain grid, and select one of the pre-configured preparation protocols from the dropdown list, which is populated with Negative Stain Protocols or Vitrification Protocols, depending on the grid preparation type selected earlier. Next, select the appropriate Sample from the dropdown list and use a toggle switch to indicate if the sample remains available (described in more detail below). If choosing to dilute or concentrate the selected sample, indicate this using the "diluted/concentrated?" toggle and specify the relevant dilution or concentration factor. Specify the volume applied onto the grid (in µL) and optionally can also record an incubation time. Finally, define the Grid's storage location. For negative stain grids, record the storage box label/number and the Grid's position within the box. For cryo grids, first select a storage device from the list and then provide information for the available and appropriate fields (cylinder, tube, and/or box, depending on the Cryo Storage Device properties previously defined in the Settings).

The parts of the workflow that were described above, Samples and Grids, are part of an inventory management system. This feature keeps track of whether the components are still available for use.

1. A Protein or Ligand can be made unavailable from the Sample level. When creating a Sample, selecting "last drop" for any of that Sample's components marks those components as unavailable for future use: they will no longer be available in the drop down when creating Sample, and they will not be marked by the "Available" tag in the list view.
2. A selected Sample can be marked as unavailable by using one of the two toggle switches – "Available for grid-making?" (under Samples) or "Sample is available for further use?" (under Grids).
3. To manage the grid's availability, use the "Grid returned to storage?" toggle (under Microscopy Sessions). By default, this value is set to "Yes" for all negative stain grids and to "No" for cryoEM grids.

Data collection
Once the grids are registered, register data collection experiments by creating Microscopy Sessions in gP2S. Microscopy Session is the most complex experimental entity tracked by the application and it is organized into four sections: basic information, microscope settings, exposure settings and microscope control.

The first section contains basic information: a Microscopy Session label, its start and finish dates and times, what Grid was imaged, which Microscope, Detector and Sample Holder (if applicable) were used, and how many images were collected. When creating a new Microscopy Session, the system automatically fills in the starting date and time. Finishing date and time are optional. This is because a Session may be registered in the system while the experiment is still ongoing and therefore its ending time would not be precisely known. If the finish date and time are not known, type it in manually or use the "now" button to enter the current date and time. Another way is to take advantage of the fact that gP2S does not allow more than one unfinished Microscopy Sessions on any given Microscope. Starting a new Microscopy Session on the same Microscope automatically marks any previously-started Session as finished.

In the next step, choose the Grid. The dropdown list will have all available Grids in the current project. After choosing a Grid, some of its basic information will be seen: who created it and when, and what Sample was applied to it. Depending on what type of grid is selected, the Microscopy Session will be marked as "stain" or "cryo" on the list view.

By default, the Microscope most recently used in the current project is pre-selected. If a particular Microscope has a sample insertion mechanism defined as an autoloader, this is the information displayed as the Sample Holder. However, if the selected Microscope requires the use of side entry holders, select the holder used from the list of Sample Holders configured to work with this microscope (if the selected grid is a cryo grid, only cryo-capable holders are listed).

The second section of a Microscopy Session form contains information about Microscope settings such as extraction and acceleration voltages, gun lens, diameter of C2 aperture, objective aperture and energy filter slit width. During routine usage, these settings are rarely changed because users commonly do not have to deviate from default values.

The third section of the Microscopy Session contains information about exposure settings. In this section the following metadata are recorded: magnification (pixel size), spot size, diameter of illuminated area, exposure duration, and whether nanoprobe, counting mode, dose fractionation and super resolution were used (counting mode, dose fractionation and super resolution settings are only enabled if the selected Detector has these features). If dose fractionation was used, the number of frames and exposure rate are also recorded.

For convenience, a number of experimentally important parameters are calculated on the fly and displayed within the form: the final image pixel size (Å), exposure rate (electrons/Ų/s), total exposure (electrons/Ų), frame duration (s) and exposure per frame (electron/Ų).

The fourth and final section of the Microscopy Session can be used to record the minimum and maximum target underfocus, and the number of exposures per hole.

While Microscopy Sessions in gP2S can be used to register any type of microscopy work, be it for screening or data collection purposes, we have found that it is sufficient and more efficient to ask users to focus on registering data collection sessions, and that screening sessions, wherein a grid is only briefly inspected for quality control need not necessarily be registered as Microscopy Sessions.

Image processing
Image processing work is recorded in gP2S as Processing Session entities. Each Processing Session is related to one or more Microscopy Session, which must be selected from a dropdown list. Indicate which Software packages (programs and versions) were used, the number of micrographs and number of particles picked. Optionally, record the name of the directory of the processing.

Map deposition
Once one or more three-dimensional reconstructions have been obtained, the Maps can be deposited into gP2S. Each Map is associated with a Processing Session, and consists of the actual map file (typically an MRC-formatted file, but gP2S allows for any file type) and key metadata: size of the pixel (Å), recommended isocontour level for surface rendering, what symmetry is applied, number of images used to create the map, and the estimated resolution: in its best and worst parts as well as the average global resolution. Maps may be associated with each other using the following types of relationships: filtered, masked, resampled, or refined versions. When registering such an association, select the type of relationship (e.g., "is filtered version of '' or "has filtered version").

Model deposition
Once an atomic model has been obtained, it can be deposited into gP2S's Model section for the relevant project. The Model feature in the first release of gP2S is barebones: other than the actual model file (typically a PDB or mmCIF file), only the resolution (in Å) and the Map (or list of Maps) from which the model was derived, are required. Additionally, it is possible to indicate that a Model is a refined version of a previously-deposited Model. Additional features, including model validation, are under development and may be added to the open-source version of gP2S in future.

Reports
It may be necessary to generate summary documents to be distributed to collaborators, who may not have access to gP2S, or to be archived on a filesystem. gP2S provides a report functionality for this purpose, available via a printer icon at the top right of each entity details view page. This generates a printable PDF file that includes all metadata describing the entity and each of its ancestor entities, including all comments. This feature is particularly valuable following Model deposition, since all data and metadata tracing the lineage of the final atomic model all the way back to specific protein and small molecule ligand lots via Microscopy Session(s) and Grid(s) will be available in a single document.

Figure 1. gP2S running on an iPad at a vitrification lab bench. The user interface has been designed for operation using touch screens, which facilitates in-lab use and accurate metadata entry. Please click here to view a larger version of this figure.

Figure 2: gP2S system architecture. gP2S follows a classic three-tier organization and relies on two database servers for data storage and an LDAP server for user authentication. Please click here to view a larger version of this figure.

Figure 3: The gP2S data model. Entities are depicted as rectangles (dark orange for workflow entities, orange for equipment and protocols, yellow for other entity types), and their relationships are (one-to-one, one-to-many, many-to-many) denoted by continuous lines. Please click here to view a larger version of this figure.

Figure 4. Microscopy Session list view. In this view, all Microscopy Sessions registered under the selected project ("CARD9" in this screenshot) are listed. A green or purple tag differentiates between room-temperature (negative stain) and cryogenic Microscopy Sessions, and a few key metadata describing each session is listed (e.g. the user who registered it, at the far right). Clicking on the name of a Microscopy Session opens a detailed view of that Session (a detailed view of a Model is shown in Figure 5). Please click here to view a larger version of this figure.

Figure 5. Model detail view. The top part of the page shows available metadata for the selected model. The comment pane on the right can be hidden by clicking on the cross (top right) or the "Comments (1)" to its left. Below, a set of icons enables the generation of a PDF report (printer icon, see main text), editing of the entry (pencil icon), or duplicating it (double rectangles icon). The lower part of the page contains a structure list of all of the entities from which this Model is descended, from Samples to Maps. Please click here to view a larger version of this figure.

Table 1. Libraries and frameworks used by gP2S