5.1K Views
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13:01 min
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June 10th, 2021
DOI :
June 10th, 2021
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Introduction
0:56
Protocol: Configuring gP2S
1:39
Results: Registering cryoEM Experiments in a Configured Instance of gP2S
12:27
Conclusion
Transcribir
To log into gP2S, type in your username and password. On the left-hand side, you can see the navigation bar that consists of a project selector and a set of navigation items that lists the experimental entity types of the CryoEMEM workflow, samples that are composed of proteins or ligands or combination of these two, grids, microscopy sessions, processing sessions, maps, and models. The last element in the navigation bar is a link to the settings section of the application.
Here, you will be able to add a number of entities that will allow you to record experiments in gP2S. Filling it in will provide information for various dropdown lists to use throughout the system. The accompanying manuscript includes detailed information on how to install and configure gP2S so that it can be used to log experiments.
In these video, we only illustrate one of the configuration steps, how to register a cryogenic sample holder, and then to illustrate in more detail how users can register experiments in gP2S. Sample holders can be configured once microscopes have been registered by navigating to the sample holder section. When the registering a new sample holder, you must specify which of the configured microscopes it can be used with, and whether it can be used to cryo specimens.
The gP2S application is project-oriented. It means that the workflow entity can only be created in the context of a project. The relevant project has to be selected from a dropdown.
When you select a project, the number of entities of each type which are associated with this project is displayed in the workflow section. You can see that in this particular case, 89 samples have been registered, 203 grids, 80 microscopy sessions, five processing sessions, seven maps, and two models. When you click on any of the workflow entity types, for example microscopy sessions, a list of those entities is shown.
This list consists of a label. There may be also a flag, as in this case, to show if it was a cryo or negative stain microscopy session, and up to six key metadata fields. In case of microscopy session, you can see which grid has been imaged, the number of images that were collected, the start and finish date times of the session, and what microscope and detector were used.
Selecting one of the listed entities opens a details page that lists all the information available for this item, which in case of the microscopy sessions is a lot. Below the microscopy details, you can see a summary list of all ancestor entities such as grids and samples. This allows for very quick navigation through the lineage of the displayed entity.
Let's jump to models to better visualize this. Finally, any entity in gP2S can be commented on by selecting comments. As you can see for this particular model, one comment already exists.
Clicking on it shows who and when created it. You may add another comment by entering a free text and optionally attaching one or more files. Now that we have toured the main part of the application, we will demonstrate how to register experimental entities during a CryoEM experiment.
In the first step of the workflow, you will be asked to describe your sample. In order to do so, you have to 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, you can also add a purification identifier.
This field can 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, 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 the stock concentration are mandatory and two other fields are optional, concept and batch lot identifier.
A sample is defined by any combination of proteins and ligands and their final concentrations. In our case, we will create a sample that contains one protein that has been previously registered in gP2S. Components are easy to find thanks to the searchable dropdown.
If you don't find your component, you may create one from scratch at this step. Optionally, you may also specify other experimental details of your sample, such as incubation time and temperature, buffer, and a free text protocol description. When your sample is ready and you are making grids, navigate to grids and create a new cryo one.
Select the grid type and the surface treatment protocol used, for example a glow discharge protocol. Then indicate whether you are preparing a cryo or a negative stain grid and select one of the pre-configured vitrification protocols from the dropdown list. Next, select from the dropdown list the sample you applied to the grid.
If you choose to dilute or concentrate the selected sample, use the toggle and specify the relevant dilution or concentration factor. You must also specify the volume applied onto the grid and optionally record an incubation time. Finally, you have to define the grid storage location.
If you like, change the default label and save the grid. Once you have registered your grids, you will be able to register data collection experiments by creating a microscopy session. The form consists of four sections:basic information, microscope settings, exposure settings, and microscope control.
The first section contains basic information, a label, change the default value if you want. Notice that the system automatically fills in the starting date and time. Finishing date and time are optional because you will probably be registering the microscopy session while your experiment is still ongoing.
If you do know the finish date and time, you can type it manually or use now button to enter the current one. Then select what grid was imaged and which microscope was used. By default, the microscope most recently used in the current project is pre-selected.
Select a detector and optionally, how many images were collected. The third section of the microscopy session contains information about exposure settings. In this section, the following metadata are recorded:magnification, spot size, diameter of illuminated area, exposure rate, exposure duration, and number of frames.
You should indicate whether nanoprobe, counting mode, dose fractionation, and super resolution were used. They are only enabled if the selected microscope and detector have these features. After entering the pixel binning factor, if any, a number of experimentally important parameters are calculated on the fly and displayed.
Image processing work is recorded in a processing session. Each processing session is related to at least one microscopy session, which you select from a dropdown list. You may add more microscopy sessions if you merge data from several sessions during processing.
You must also indicate which software packages were used by selecting the software label and its version. There is also a place for some related notes. You have to provide the number of micrographs and picked particles.
You may also record the name of the directory or the full path where you did the processing. 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.
gP2S allows for any file type. Enter key metadata such as size of the pixel, recommended is a contour level for surface rendering, what symmetry you applied, number of images used to create the map, and the estimated resolution in its best parts, the average global resolution, and resolution in worst parts. As in the case, in many parts of gP2S, validation rules check your input for obvious mistakes.
Maps may be associated with each other using different types of relationships. When registering such an association, you must select the type of relationship and related map. Once an atomic model has been obtained, it can be deposited in gP2S.
Add a model file, resolution, and the map or a list of maps from which the model was derived. Additionally, it is possible to indicate that a model is a refined version of a previously deposited model. Change the label and save the record.
If you collaborate with other researchers who don't have access to the application, it might be necessary to generate summary documents that you can then share. For this purpose, gP2S provides a report functionality. 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 sessions and grids are available in the single document. But a PDF can be generated from any details view page. As you can see, gP2S enables you to track various entities that are well-structured and easy to navigate to.
As shown earlier, data validation when registering particular entities contributes to higher quality metadata than might be achievable with classic spreadsheets or notebooks. Thank you for watching. We hope this video will encourage you to try gP2S as the laboratory information management system for your CryoEM lab.
gP2S is a web application for the tracking of cryoEM experiments. Its main features are described, as are the steps required to install and configure the application. Once configured, the application allows one to accurately record metadata associated with negative stain and cryoEM experiments.
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