EOM analysis of IgG antibodies

Linear (OLIGOMER), and non-linear (MIXTURE) analysis, singular value decomposition (SVDPLOT), addition of missing fragments (BUNCH, CORAL), analysis of flexible systems (EOM/RANCH & GAJOE), flexible refinement of high-resolution models (SREFLEX)
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li_fubin
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EOM analysis of IgG antibodies

#1 Post by li_fubin » 2019.03.06 14:39

Hi, all,

We are trying to run EOM to analyze our SAXS data on IgG antibodies as reported in (Tria G, Mertens HD, Kachala M, Svergun DI. Advanced ensemble modeling of flexible macromolecules using X-ray solution scattering. IUCrJ. 2015 Feb 26;2(Pt 2):207-17). We encountered two problems:

1. In one case, the following message was printed in EOM:

Loading values and configuration ...
Number of residues per chain: 659
--------------------- ATTENTION! ---------------------
- The domains specified as fixed may be too far away -
- to be connected by the linker extracted from the -
- sequence. EOM 2.1 may not be able to make the pool.-
------------------------------------------------------
... starts making models ...
[ 1%]
... completed making models
ERROR: the pool cannot be constructed. Please check the input files

2, I need to define a contact point (due to disulfide bonds between two homodimer chains) in addition to a PDB containing multiple chains. But it appears that EOM no longer asks for Contact file name once a PDB containing multiple chains is provided,

I wonder what could be the causes/solutions for these problems. Please see below and the attachment “Problem.pdf” for more details.
Problem.pdf
EOM problem
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Our goal and problems: Each IgG antibody molecule has two identical heavy chains (N-terminus-FabH-linker-CPPC-linker-Fc -C-terminus) and two identical light chains (N-terminus-FabL-C-terminus). These four chains form three rigid bodies and one contact point in the following way: each FabH pairs with a FabL to form a FabH:FabL rigid body (Fab.pdb, 2 in total); two Fcs form an Fc: Fc rigid body (Fc.pdb, 1 in total); "CPPC" sequences of two heavy chains form disulfide bonds (CPPC.pdb, 1 in total). We intend to run an EOM analysis with three defined domains (Fab.pdb, CPPC.pdb, Fc.pdb). But problem #1 was encountered when we run EOM in this way. If we run EOM without the CPPC.pdb, it does run and give reasonable models. But these models are built without the CPPC-CPPC contact constraints, which raises problem #2.

(Files for our problems (Problem.pdf), PDB files for FabH:FabL (Fab.pdb), CPPC (CPPC.pdb) and Fc:Fc (Fc.pdb) rigid bodies, and SAXS data (IgG.dat) are attached in this and the following posts).
Problem.pdf
EOM problem
(224.5 KiB) Downloaded 54 times


Thank you for your help. Any suggestions are appreciated.

Fubin

li_fubin
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Posts: 6
Joined: 2019.03.06 13:15

Re: EOM analysis of IgG antibodies

#2 Post by li_fubin » 2019.03.06 14:58

EOM input file 1:
IgG.seq
EOM input file 1
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li_fubin
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Posts: 6
Joined: 2019.03.06 13:15

Re: EOM analysis of IgG antibodies

#3 Post by li_fubin » 2019.03.06 14:59

EOM input file 2:
Fab.pdb
EOM input file 2
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li_fubin
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Posts: 6
Joined: 2019.03.06 13:15

Re: EOM analysis of IgG antibodies

#4 Post by li_fubin » 2019.03.06 15:00

EOM input file 3:
CPPC.pdb
EOM input file 3
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li_fubin
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Posts: 6
Joined: 2019.03.06 13:15

Re: EOM analysis of IgG antibodies

#5 Post by li_fubin » 2019.03.06 15:01

EOM input file 4:
Fc.pdb
EOM input file 4
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li_fubin
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Posts: 6
Joined: 2019.03.06 13:15

Re: EOM analysis of IgG antibodies

#6 Post by li_fubin » 2019.03.06 15:01

EOM input file 5:
IgG.dat
EOM input file 5
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hf3g13
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Re: EOM analysis of IgG antibodies

#7 Post by hf3g13 » 2019.06.21 17:11

Also interested in this topic, been attempting to use EOM with antibody fragments following the idea of Tian et al 2015 without any success.

A previous thread (Attempting to Use EOM with Ig Fragments - 2015.04.15) mentioned using alpraxin to align domains, then reversing the sequence of the second F(ab) + Fc to create a single chain and finally defining contacts between Fc and the CPPC.

I haven't been able to get this to work with my system but may help with yours?

Best wishes,

Hayden

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