Rigid Body and Flexible Fitting

Greg Pintilie

Wah Chiu

S2C2 Workshop, July 10, 2019

Rigid Body and Flexible Fitting

• Segmentation• Segger – Segment Map, EMAN (e2segment3d)

• ‘Rigid-body Fitting• Chimera, Segger - Fit to Segments, Situs

• Flexible Fitting/Refinement• MDFF (Molecular Dynamics Flexible Fitting), Phenix, RefMac, FlexEM, Direx

• Model Assessment• Molprobity, Probability/uncertainty modeling (ProMod)

CryoEM Map Segmentation - Segger

Grouping• Map is smoothed by Gaussian filter (red

line)• Regions are grouped based on which

peak is reached after uphill climb (black lines)

• Result – 2 regions

Example: 1-dimensional map• Height proportional to density• Watershed method• Peak = region, result 5 regions

CryoEM Map Segmentation - Segger

Cryo-EM mapEMDB:5001GroEL @ 4Å

2124 watershed regions (too many)

After smoothing and grouping 1 step (42

regions)

After smoothing and grouping 3 steps (14

regions)

Segger – Smoothing and Grouping

Watershed regions

1 region per protein

Density Peaks

InputMap Smoothed Maps

Segger – Interactive Modification

Segger – Smoothing and Grouping

Segger – Smoothing and Grouping

Segger – Grouping by Connectivity

Segger – Grouping by Connectivity

Rigid-body Fitting

The model docked inside the density map

A known model(from PDB)

Map-Model Scores

• Average Density at all atom positions

• Blur model coordinates to a particular resolution• Cross-correlation

• Fourier Shell Correlation

r1

r2

r3

r4

r5

b1

b2

b3

b4

b5

b4

r1

r2r3

r4 r5b1

b2

b3 b5

Cross-correlation

Segger – Fit to Segment

Principal Axes of Model

Principal Axes of Segment

4 possible alignments in 3D

• Local rotational search with structure around a region• Align centers, rotate structure

around the center

• Compute correlation score at each rotation

• Sort scores from highest to lowest

• Is top score the correct fit?

• Z-score:

0.74

0.76

0.78

0.8

0.82

1 4 7 10 13 16 19 22 25

Cro

ss -

Co

rrel

atio

n

Fit #

S1

S2..N

Rigid Fit: Confidence

Flexible Fitting - MDFF

https://www.ks.uiuc.edu/Research/mdff/

Flexible Fitting – MDFF

After MDFF

Initial Modelhttps://www.ks.uiuc.edu/Research/mdff/

Model assessment - Molprobity

http://molprobity.biochem.duke.edu

MDFF – Example

•P22 Portal Protein• Gray: initial structure

• From X-ray model

• Green: flexibly fitted• Corresponds to Cryo-EM state

Flexible Fitting – Validation & Test for Over-fitting

Segment

Flexible Fitting

FSCA

FSCBSegment

Rigid docking

Crystal structure

Compare model to

map B

Map A(1 portal protein)

Map B(1 portal protein)

Masked Portal from Independent Map 1

Masked Portal from Independent Map 2

Pintilie, Chiu, Biophysical J., 2015

Initial Model

Flexible Fitting – Validation & Test for Over-Fitting

Gradient Scale = 0.3 Gradient Scale = 500

0 0.5 1.0

24.010.3

MapA - M

apB

MapA - Initial M

odel

MapB - Initial M

odel

0 0.5 1.0

24.09.3

MapA - M

apB

MapA - Initial M

odel

MapB - Initial M

odel

MapA - M

DFF dw=500

MapB - M

DFF dw=500Fou

rier

Sh

ell C

orr

elat

ion

Resolution (Å) Resolution (Å) Resolution (Å)

0 0.5 1.0

24.016.0

MapA - M

apB

MapA - Initial M

odel

MapB - Initial M

odel

MapA - M

DFF dw=0.3

MapB - M

DFF dw=0.3

Probabilistic Modeling

Results from flexible fitting

3Å

13Å

Probabilistic model(mean and st.devat each residue)

Rigid Body and Flexible Fitting

• Segmentation• Segger – Segment Map, EMAN (e2segment3d)

• ‘Rigid-body Fitting• Chimera, Segger - Fit to Segments, Situs

• Flexible Fitting/Refinement• MDFF (Molecular Dynamics Flexible Fitting), Phenix, RefMac, FlexEM, Direx

• Model Assessment• Molprobity, Probabilistic modeling (ProMod)

https://cryoem.slac.stanford.edu/ncmi/resources/software/segger