NCCAT embedded cross-training workflow. What to expect.
Embedded Cross-training Modules
Objectives:
Learn about different grid preparation parameters that affect sample quality and ice thickness.
Milestones:
• Reproducible sample preparation with the Vitrobot Mark IV, Leica GP, and/or chameleon.
• Identify sample preparation conditions for the driving biological project.
Objectives:
Learn about the operation of screening microscopes and Krios microscopes. Learn which alignments are important for obtaining high-quality data on an EM, how to check if it is aligned correctly and how to do the alignments.
Milestones:
• Check the alignments on an EM and be able to adjust them if necessary.
• Know the most common errors occurring on an EM during operation and how to deal with them.
Objectives:
Learn to assess if a sample is ready for data collection, what to check on the microscope before starting a long data collection, and how to operate the Leginon acquisition software. Furthermore, judging how many micrographs would be necessary for a reasonably good structure by looking at the micrographs and pre-processed data.
Milestones:
• Set up a data collection from a test sample and obtain enough data to get a high-resolution structure.
• Determine data acquisition parameters for applicant’s sample and collect enough data for an initial 3D model.
Objectives:
Learn to use on-the-fly processing through Appion to optimize a data collection experiment in real time. After data collection is complete learn to process data with commonly used software packages and pipelines (cryoSPARC and RELION) from motion correction to 3D model refinement.
Milestones:
• Obtain a high-resolution structure from a test data set with commonly used software packages.
• Process a data set from the applicant’s own sample and obtain a 3D model.
Core Cross-training Components
NCCAT courses and workshops
• NCCAT Workshops and Short courses
• CryoEM Merit Badges
Curriculum development programs
• Getting Started in cryoEM
• cryoEM 101
• cryo VR
• Principles of cryoEM
Sample preparation:
• Vitrobot Mark IV
• Leica GP
• chameleon
• Refeyn Mass Photometer
Screening microscopes:
• Basic operations
• Independent use
Krios microscopes:
• Autoloader systems
• Basic operations
• Independent use
Image assessment:
• Pre-processing pipeline (Leginon & Appion)
Stand alone packages:
• Motion correction
• CTFestimation
• Object selection
3D refinement packages:
• RELION
• cryoSPARC
Single particle analysis:
• CryoEM tool kit
• Cryo grid handling
• Sample assessment
• Image assessment
• Reconstruction assessment
Additional benefits of embedding:
• Journal clubs/group meetings
• Interaction with other EM users
• Weekly meetings to follow up on milestones and training targets
Our trainees
Trainees come from across the nation, broadening the reach of new technologies, and building a large community of NCCAT friends.
