Combined Automated Assignment

1

• Combined Automated Assignment Structure
  Calculation
• CcpNmr Nexus

2
The CLOUDS Protocol

• Assignment free structure determination
• Per Kraulis, Thérèse Malliavin, Irvin D. Kuntz
• CLOUDS by Miguel Llinas, Alex Grishaev
• Spatial distribution of anonymous resonances
  generated with NOEs

H
H
H
H
A network of distance constraints between
anonymous atoms is sufficient to generate a low
resolution protein structure.
3
Original CLOUDS problems

• Few restraints given distance accuracy
• Protocols need clean 2D spectra
• Poor resonance disambiguation
• Distance calibration only for 2D NOESY
• Needs covalent information
• ABACUS

Example Yeast Hho1p GI Linker Histone H1
globular domain I J.O.Thomas, K.Stott (92
residues)
An ideal family of Clouds
Backbone of ideal Clouds
Backbone of REAL Clouds Globally inconsistent
4
The CcpNmr Nexus protocol
Experiments used 15N HSQC-NOESY, 13C
HSQC-NOESY, HNcoCACB, HNCACB, HNCA, HNcoCA
NOESY Peaks
Network Anchoring
Restraints
1H Structure
Assignments
Threader
Xplor NIH
Violation Analysis
Covalent Connections
5
Network Anchoring
Hy1
Hx1
NO support
Hx1
,Hy1
Hy1
Hx1
,Hy2
Hy2
Hy3
,Hy3
Hy3
Hx2
,Hy1
Hx2
,Hy2
Hx1
Hx2
Hx2
,Hy3
Ha
Hy3
Hx2
Hb
Dense initial network
Ha and Hb bridge Network SUPPORT
6
Nexus Threader

• Fully automated
• Accepts any initial typing and assignment
  information
• Bayesian approach generates assignments from
  clouds using Monte-Carlo sampling
• Works with any backbone triple-resonance
  experiments
• Works with side chains
• Fits side chains into clouds given backbone

Globally inconsistent structure Local
connectivity still threadable!
7
Growing Residue Templates
HN
HN

• Residue fragments
• ANY
• ALF
• BET
• BBR
• GAM
• DEL
• PHY
• IVT
• ASX
• GLX
• Fit from 1H structure
• Xplor topology files
• Grow through iterations
• ANY-gtALF-gtBBR-gtVAL
• Etc

C
CHCH2CH2CH2
OC
OC
HN
HN
CH
CHCH2CH2CO
OC
OC
H
H
HN
HN
CHCH2
CHCH2
OC
OC
H
H
HN
HN
CHCH2CH2
CHCHCH3
OC
OC
8
Iterative improvement
A
B
C

• Network anchoring
• Improves with bonds!
• Generate structure
• Thread assign
• Grow new bonds
• Repeat