Resources · Data-Processing Pipeline

Model Building & Validation Workflow

The standardized steps our trainees use to turn a refined cryo-EM density map into a validated, deposition-ready atomic model — picking up where the Cryo-EM Processing Pipeline leaves off.

Fitting & refinement

Geometry & map validation

ChimeraX-based

Workflow steps

Once the final map is in hand, model building and validation follow a consistent, iterative sequence — taught in our Cryo-EM Workshop's "Model Building & Validation in ChimeraX" session.

Starting model selection

Choose a starting point: a homology model, an AlphaFold prediction, or a related deposited structure to rigid-body dock into the map.

AlphaFold ChimeraX

Interactive fitting

Rigid-body and flexible fit the starting model into the density, adjusting domain placement to match the observed map features.

ChimeraX Coot

Manual rebuilding

Correct side-chain rotamers, backbone register, and loop conformations residue-by-residue against the map.

Coot ISOLDE

Real-space refinement

Refine coordinates and B-factors directly against the map using restrained real-space refinement to optimize the fit while preserving good geometry.

Phenix Rosetta

Iterate fitting & refinement

Cycle between manual rebuilding and refinement until the model converges on a stable, well-fit conformation with no major outliers remaining.

Coot Phenix

Geometry validation

Check bond lengths/angles, Ramachandran outliers, rotamer outliers, and clashes to confirm the model is stereochemically sound.

MolProbity

Map-model validation

Assess how well the model explains the experimental density using map-model FSC, EMRinger side-chain fit scoring, and local resolution cross-checks.

Phenix EMRinger

Figure generation & deposition

Generate publication figures in ChimeraX and deposit the final coordinates and map to the PDB and EMDB with full validation reports.

ChimeraX PDB EMDB

Worked training example

PDB 7UL6 / EMD‑26593 — full-length dimeric ClbP

The same ClbP reconstruction produced by the Cryo-EM Processing Pipeline (EMPIAR‑11422) carries through to this stage: the deposited atomic model (PDB 7UL6) and map (EMD‑26593) reflect a completed round of fitting, refinement, and validation, and can be opened side-by-side in ChimeraX to see the workflow's end result.

References

Primary sources for this workflow. Each link goes to the original resource so you can confirm details directly.

SBGrid NIH R25 Training — CryoEM & CryoET Data Processing WorkshopsProgram context for the companion image-processing curriculum this workflow builds on.

PDB 7UL6Deposited, validated atomic model used as the worked example above.

EMD‑26593Corresponding deposited density map.

Velilla et al., 2022, Nature Chemical BiologyOriginal publication of the ClbP structure.

UCSF ChimeraXVisualization, interactive fitting, and figure generation.

CootManual model building and rebuilding against density.

Phenix real_space_refineRestrained real-space refinement against the map.

MolProbityGeometry and clash validation.

Barad et al., 2015, Nature Methods — EMRingerMap-model side-chain fit validation metric.