Events

Talks & symposia

CryoEM Current Practices Webinar series

This monthly seminar series is jointly hosted by NCCAT, PNCC, & S2C2. Talks feature highlights and strategies from users of the centers with a heavy focus on cryoEM methods. Q&A after the seminar is meant to stimulate discussion of best practices and interesting challenges that will be helpful to researchers new to the field. Representatives from all three service centers will be on hand to answer questions about the cryoEM resources available to biomedical researchers and how to access them.

Talks take place on zoom the last Thursday of the month at 9 AM pacific/12 PM eastern and are are open to all at no-cost. Registration is required.

See our upcoming lineup and register via the links here:

  • 02/29/2024: Roger Castells-Graells, Ph.D.Engineered Protein Cages for Imaging of Small Proteins by Cryo-EM

Past talks are available on our YouTube channel or can be viewed below.

Meet the CryoET Centers Webinar series

Cryo-electron tomography (cryo-ET) has revolutionized biological research by providing 3D reconstructions of biological structures with nanometer scale resolution. The NIH has funded a national network for cryo-electron tomography whose mission is to provide access to cryo-ET. Come join us for this webinar series on cryo-ET and learn about how you can access this network in support of your research.

Past Series Webinar Topics:

  • An introduction to cryo-ET and overview of the National Network for Cryo-ET, July 8, 2021, Elizabeth R. Wright, MCCET
  • High pressure freezing, the waffle method and cryo-FIB, July 15, 2021, Mykhailo Kopylov, NCITU
  • Correlative cryogenic light and electron microscopy and tomography, July 22, 2021, Gong-Her Wu, SCSC
  • Preparation of select murine brain sections for HPF, cryo-CLEM, and cryo-ET, July 29, 2021, Michael H.B. Stowell, CCET
  • Structural studies of virus infection using CLEM, cryo-FIB, and cryo-ET, August 12, 2021, Jie E. Yang, MCCET

Previous webinars

Some tips & tricks of note:

Sample preparation: Tag locations on heterologously expressed protein constructs. Time resolved cryo-EM from on-grid enzymatic reactions.

Data processing: Handling heterogeneous oligimerization. 3D Variability Analysis (3DVA). Ensemble generation from 3DVA. Assigning small ligand densities.

Complementary techniques: Crosslinking to validate oligomerization interfaces. Enzyme kinetics. Molecular Dynamics Ensemble Refinement. Using cryoEM results to drive development of Undergraduate CURE lab courses.

 


Some tips & tricks of note:

Sample preparation: Tips for dealing with low sample concentration, samples that stick to carbon, recognizing and troubleshooting strange types of ice.

Data collection: Using information from different magnifications to characterize grids and optimize data collection.

Data processing: How to do gain correction when you don’t have gain references. Helical processing challenges.

 


Tips & tricks of note:

Sample preparation: membrane protein purification using GDN & nano-disks, expression of leaky ion channels.

Data processing: multi-software workflows, masking and local refinement, symmetry expansion, variability analysis.

Complimentary techniques: patch clamping, single amino acid substitution to verify binding sites.

 


Tips & tricks of note (coming soon):

Sample preparation:

Data processing: 

Complimentary techniques: 

 


Tips & tricks of note (coming soon):

Sample preparation:

Data processing: 

Complimentary techniques: 

 


Tips & tricks of note:

Sample preparation: Continuous thin carbon substrates to reduce aggregation.

Data processing: Local refinement to improve map quality in regions of interest.

Complimentary techniques: 

 


Tips & tricks of note:

 


Tips & tricks of note:

Sample preparation: Anaerobic Grid preparation.

Data processing: “In silico purification” of heterogeneous sample (enzyme in turnover conditions). Determination of multiple structures from one grid. Local resolution analysis to understand dyamics. High resolution to analyze single amino acid side-chain movement.

Complimentary techniques: Protein design. Metal-mediated protein oligomerization to generate extended molecular assemblies. Analytical ultracentrifugation. Electron diffraction.

 


Tips & tricks of note:

Sample preparation: Using NMR analysis of point-mutants to choose an ideal cryoEM sample. Ideas for overcoming preferred orientation.

Data processing: Understanding substrate specificity by solving multiple structures. 

Alternative methods: Site-directed mutagenesis, NMR as a complementary technique, RMSD analysis of substrate bound/unbound structures.


Tips & tricks of note:

Sample preparation: Blot-free vitrification to overcome preferred orientations

Data collection: Tilted data collection to overcome preferred orientations 

Data processing: Using neural network particle pickers to find harder-to-pick views of non-spherical particles

Tips & tricks of note:

Sample preparation: Use of C. elegans for native source expression of a mechanosensing protein. Purification of samples from native sources (including tag choice). Fluorescence-detection size-exclusion chromatography (FSEC) to optimize buffer conditions. Continuous-carbon support to increase sample concentration for precious samples. Amylamine treatment of cryoEM grids to alleviate preferred orientation and improve ice thickness. 

Data processing: Heterogeneous refinement to reconstruct multiple conformations from a single data set. Masked local refinement to improve resolution a weak binding partner. Observation of phospholipids in reconstrution.  

Complementary techniques: Mass spectrometry to characterize sample composition. Molecular dynamics to probe role of membrane deformation in mechanism.

Tips & tricks of note:

Data processing: Practicalities of neural network picker software installation and usage. How to choose a particle picker & validation. Neural network picking for single particle analysis, filaments, & tomography. Interplay of particle picking and preferred orientations. Particle centering.

Tips & tricks of note:

Sample preparation: Synthetic G-quadruplex design. Screening of different grid types. Micrograph evaluation for a small (sub-30 kDa) particle.

Data processing: Elliptical blob picking. Heterogenenous refinement and 3D variability to separate conformational heterogeneity. Non-uniform refinement.

Complementary techniques: Analytical Ultracentrifugation. 1H-NMR. Small angle X-ray Scattering (SAXS) for size and flexibility estimation. Molecular dynamics with cryoEM map derived restraints. Molecular dynamics of a cryoEM derived model. Computational drugability analysis.

Tips & tricks of note:

Sample preparation: Transmembrane proteins, HEK293 freestyle cell expression, purification using FLAG-tag, pH-dependent enzyme activity, thermal denaturation, golgi apparatus, muscular dystrophy, IEX, SEC-MALS, SAXS 

Data processing: 2-D classification, template-based picking, unbinned repicking to eliminate bias and improve resolution, preferred orientation.

Tips & tricks of note:

Sample preparation: Protein purification from red blood cells using glycerol gradients & gel filtration, detergent solubilization & purification, detergent screening, digitoxin solubilization, mass photometry.

Data collection: Single particle analysis & Cryo-ET

Data processing: Particle heterogeneity, non-uniform refinement, class-based processing. Tomogram 2-D slices, Sub-tomgram averaging.

 

Tips & tricks of note:

Sample preparation: Fibril extraction from heart tissue,  mutation-specific perturbations, amyloid seeding, use of structure-based inhibitors.

Data processing: Manual picking, conformational heterogeneity, local resolution analysis, featureless cylinder, helical processing, twist and local maxima.

Complimentary techniques: X-ray microcrystallography.

Tips & tricks of note:

Sample preparation: Screening of: detergents, nano disc size, temperatures, grid type, and plunge freezer type. Use of pre-heated tools for temperature sensitive samples.

Tips & tricks of note:

Sample preparation: Protein prep and grid freezing optimization, glutaraldehyde cross-linking

Data collection: Tilted data collection to overcome preferred orientations 

Data processing: Tips for processing tilted data, particle picking tips for tricky particles, helical filament processing 

A copy of this presentation is available on the Open Science Framework (OSF) repository: https://osf.io/x7dv8/  

Tips & tricks of note:

Data processing: computational infrastructure and resources

Tips & tricks of note:

Sample preparation: Optimization of grid freezing conditions  

Data processing: Tips for dynamic membrane proteins

Tips & tricks of note:

Sample preparation: Testing detergents and nanodisks for integral membrane proteins.

Data collection: Optimizing pixel size and data collection rate.

Data processing: Using symmetry during 3D reconstruction; cleaning particle stacks with 3D classification.

 

Tips & tricks of note:

Sample preparation: Sample QC using differential scanning calorimetry & negative stain EM. The effects of temperature on protein quality and grid preparation.