Rigid Body and Flexible Fitting

Greg Pintilie

Wah Chiu

S2C2 Workshop, Jan. 15, 2020

Rigid Body and Flexible Fitting

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

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

• Flexible Fitting/Refinement• MDFF, Phenix, RefMac, FlexEM, Direx, Coot, ISOLDE

• Model Assessment• Multi-model analysis, Molprobity

CryoEM Map Segmentation - Segger

Grouping• Map is smoothed with Gaussian filter (red

line)• Regions are grouped based on which peak is

reached after uphill climb (black lines)

Example: 1-dimensional map• Height proportional to density• Watershed method

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)

Pintilie et al, JSB, 2010https://github.com/gregdp/segger

Segger – Smoothing and Grouping

Input Map Atomic Model

https://github.com/gregdp/segger

Segger – Smoothing and Grouping

https://github.com/gregdp/segger

Segmented Map Atomic Model

Segger – Grouping by Connectivity

https://github.com/gregdp/segger

Watershed Regions Connections between Regions

Segger – Grouping by Connectivity

https://github.com/gregdp/segger

Segmented Map Atomic Model

Segmentation Example: Apoferritin

EMD:200261.75Å Resolution

Group by Connectivity(Threshold: 0.015) https://github.com/gregdp/segger

Rigid Fitting

• Manual placement followed by local search• Chimera, Fit in Map

• Exhaustive Search• SITUS

• https://situs.biomachina.org/• phenix.dock_in_map

• https://www.phenix-online.org/• Chimera

• fitmap #0 #1 search [N]• https://www.cgl.ucsf.edu/chimera/current/docs/

UsersGuide/midas/fitmap.html

• Using segmentation• Fit to Segments (Segger)

• https://github.com/gregdp/segger

Segger – Fit to Segment

Principal Axes of Model

Principal Axes of Segment

4 possible alignments in 3D

Pintilie et al, JSB, 2010https://github.com/gregdp/segger

• 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 Fitting - Confidence

Pintilie et al, Biopolymers, 2012, https://github.com/gregdp/segger

Fit 1 Fit 7

…

Flexible Fitting/Refinement

• Phenix• https://www.phenix-online.org/

• MDFF/NAMD• https://www.ks.uiuc.edu/Training/Tutorials/science/mdff/tutorial_mdff-html/

• Flex-EM• http://topf-group.ismb.lon.ac.uk/flex-em/

• Direx• http://schroderlab.org/software/direx/

• RefMac• http://www.ccpem.ac.uk/

• ISOLDE• https://isolde.cimr.cam.ac.uk/what-isolde/

• Coot• https://www2.mrc-lmb.cam.ac.uk/personal/pemsley/coot/

MDFF

• Molecular Dynamics Flexible Fitting• Run MD simulation

• Temperature of 300K simulates random motions of atoms

• Add forces in the direction of the gradient at each atom’s position• Parameter: gradient weight

• Use artificial restraints to maintain secondary structures

• Setup:• VMD to generate files• NAMD to run simulation• NAMDinator can help

• https://namdinator.au.dk/

MDFF – Example

Single ProteinRigid Fit

(PDB: 3lj5)

P22 PortalEMD:8005Segment Flexible Fitting

(MDFF)

12 Portal Proteins after Flexible Fitting

Deposited as PDB:3gai

Pintilie, Chiu, Biophysical J., 2015

Flexible Fitting – ‘Over-Fitting’

Initial Model Gradient Weight = 0.3 Gradient Weight = 500

Pintilie, Chiu, Biophysical J., 2015

Multi-model Analysis

3Å

13Å

10 Results from flexible fitting

Probabilistic model (ProMod)(mean and st.dev at each residue)

Pintilie, Chiu, Biophysical J., 2015

• Phyre2• http://www.sbg.bio.ic.ac.uk/phyre2

• I-TASSER• https://zhanglab.ccmb.med.umich.edu/I-TASSER/

• MODELLER• https://salilab.org/modeller/

Sequence

Similar Sequences which have Structures Available

(Templates)

Comparative/Homology Model

Sequence Alignment

‘Threading’Insertions/Deletions

Comparative/Homology Modeling

Comparative/Homology Modeling

MODELLER I-TASSER Phyre2Template (PDB:1m3y)

EMD:200918.6Å Resolution

Pintilie et. al, Structure, 2019

0.3Å

5.0Å

Flexible Fitting

Variability

Model assessment - Molprobity

http://molprobity.biochem.duke.edu

Rigid Body and Flexible Fitting

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

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

• Flexible Fitting/Refinement• MDFF, Phenix, RefMac, FlexEM, Direx, Coot, ISOLDE

• Model Assessment• Multi-model analysis, Molprobity