RECOORD
REcalculated COORdinates Database
Jurgen Doreleijers
Center for Eukaryotic Structural Genomics
University of Madison-Wisconsin
jurgen@bmrb.wisc.edu
Aart Nederveen
Bijvoet Center for Biomolecular Research
Utrecht University
a.j.nederveen@chem.uu.nl
Wim Vranken
Macromolecular Structure Database
European Bioinformatics Institute
wim@ebi.ac.uk
Aim
• Recalculation of protein structures based on
deposited NMR restraints using state of the art
methods
• Goals:
•
•
•
•
decrease user- and software-dependent biases
allow a better comparison between structures
comparison between different structure calculation programs
provide a database for the development and assessments of
validation tools and calculation protocols
Overview recalculation project
restraint
manipulation
PDB:
-coordinates
-restraints
recalculation
design of RECOORD
analysis
1
BMRB:
2
EBI/UU: 3
Generation of
consistent
STAR files
STAR files
Doreleijers et al. 2003
CNS
-topology
-MD SA
-refinement
4
CYANA 5
-sequence
-MD SA
-…
6
analysis
-improvement?
-correlations?
-…
Databases now publicly available
• DOCR/FRED (BMRB)
databases containing converted and filtered restraints
http://www.bmrb.wisc.edu/servlets/MRGridServlet
• RECOORD (EBI)
database containing recalculated coordinates
http://www.ebi.ac.uk/msd/recoord
PDB:
Selection
-coordinates
-restraints
• Formats
•
•
•
(if distance restraints available):
CNS/XPLOR
DIANA/DYANA/CYANA
DISCOVER/MSI
• PDB entries selected:
•
1
• only proteins
• no HET atoms
• multimers allowed (not yet re-calculated)
• at least 20 residues
Finally 545 monomers were selected
BMRB:
2
STAR files
Doreleijers et al. 2003
EBI/UU: 3
Conversion issues
Generation of
consistent
STAR files
• Data is converted to formats readable by calculation
software (e.g. XPLOR/CNS and CYANA) by the
FormatConverter available within CCPN software (Wim
Vranken, EBI).
Problems:
• Differences between coordinate and restraint data:
•
• e.g. 1 chain in pdb entry, 2 chains in restraint list
• residue numbering can differ in PDB entry and restraint
• restraints for residues not present in PDB entry…
Nomenclature in restraint list
list
CNS
Building topology
4
-topology
-MD SA
-refinement
CYANA
-sequence
-MD SA
-…
• Starting script: generate_easy.inp from CNS
• Automated detection in original ensemble of:
•
•
•
Disulfide bridges (<3Å S-S distance in original first models)
CIS peptides (if |w|<25º in original first models)
Protonation state of histidines (use CNS patches HISD, HISE)
• CYANA: sequence based on CNS topology
•
•
Add CYSS, HIST, HIST+, cPRO in sequence
Automated generation of disulfide restraints
5
CNS
CONDOR computer cluster CS
University Madison
-topology
-MD SA
-refinement
4
CYANA
-sequence
-MD SA
-…
• More than 800 processor used
• Total CPU time: 31,169 hours (3.5 years on single
workstation)
• Example 2EZM, calculation of 1 model
(101 a.a. & 2.2 GHz P4 computer)
CYANA
CNS
31 seconds
340 seconds
5
Evaluation of structure quality
6
analysis
-improvement?
-correlations?
-…
• Agreement with experimental restraints
• Improvement?
• Comparison CNS and CYANA
• Relation NMR data quality and structural quality
Distance restraints violations
6
analysis
-improvement?
-correlations?
-…
ORG: 0.08 Å (0.14 Å)
frequency
original entries
CNW: 0.04 Å (0.05 Å)
recalculated in CNS
and refined in water
RMS distance restraints violations (Å)
Dihedral restraints violations
6
analysis
-improvement?
-correlations?
-…
ORG: 1.6° (4.6°)
frequency
original entries
CNW: 0.5° (0.5°)
recalculated in CNS
and refined in water
RMS dihedral restraints violations (degrees)
Results: quality indicators
performance CNS vs. CYANA (no water refinement yet)
6
analysis
-improvement?
-correlations?
-…
Average value over 545
entries
Original PDB
CNS
recalculation
CYANA
recalculation
RMS distance restraints
violations (Å)
0.08 ± 0.14
0.04 ± 0.06
0.04 ± 0.05
RMS dihedral restraints
violations (degrees)
1.6 ± 4.6
0.5 ± 0.7
0.5 ± 0.7
Packing quality (Z-score)
WHATCHECK
-3.5 ± 1.9
-4.1 ± 1.9
-4.3 ± 1.8
Bumps per 100 residues
73 ± 63
11 ± 9
86 ± 37
% most favoured
PROCHECK
69 ± 14
69 ± 13
61 ± 14
Results: quality indicators
performance CNS before and after water refinement
6
analysis
-improvement?
-correlations?
-…
Average value over 545
entries
Original PDB
CNS
recalculation
CNS + water
refinement
RMS distance restraints
violations (Å)
0.08 ± 0.14
0.04 ± 0.06
0.04 ± 0.05
RMS dihedral restraints
violations (degrees)
1.6 ± 4.6
0.5 ± 0.7
0.5 ± 0.5
Packing quality (Z-score)
WHATCHECK
-3.5 ± 1.9
-4.1 ± 1.9
-2.5 ± 2.0
Bumps per 100 residues
73 ± 63
11 ± 9
10 ± 7
% most favoured
PROCHECK
69 ± 14
69 ± 13
76 ± 11
Improvement:
packing and Ramachandran Z-scores
6
analysis
-improvement?
-correlations?
-…
improvement Ramachandran
Improvent Z-score:
DZ=Zrefined - Zoriginal
For ~ 5 % of entries no
improvement possible
because of missing NMR
data compared to authors
missing data
improvement packing
6
analysis
-improvement?
-correlations?
-…
In search of correlations
(Pearson coefficient)
(correlations higher)
data
density
data density
refined
RMS
violations
circular
variance
packing
Ramachandran
(Z score)
(Z score)
-0.23
-0.46
0.35
0.31
-0.03
0.22
-0.25
-0.37
0.58
-0.60
-0.67
0.25
0.69
-0.39
RMS
violations
-0.11
circular
variance
-0.32
0.00
packing
0.32
-0.06
-0.49
0.16
-0.11
-0.48
0.48
0.04
0.04
0.07
-0.21
bumps
(Z-score)
Ramachandran
-0.51
(Z-score)
bumps
original
(correlations lower)
-0.47
6
analysis
-improvement?
-correlations?
-…
In search of correlations
(Bumps)
refined
data
density
data density
RMS
violations
circular
variance
packing
Ramachandran
(Z score)
(Z score)
-0.23
-0.46
0.35
0.31
-0.03
0.22
-0.25
-0.37
0.58
-0.60
-0.67
0.25
0.69
-0.39
RMS
violations
-0.11
circular
variance
-0.32
0.00
packing
0.32
-0.06
-0.49
0.16
-0.11
-0.48
0.48
0.04
0.04
0.07
-0.21
bumps
(Z-score)
Ramachandran
-0.51
(Z-score)
bumps
original
-0.47
6
analysis
-improvement?
-correlations?
-…
In search of correlations
(NMR data density)
refined
data
density
data density
RMS
violations
circular
variance
packing
Ramachandran
(Z score)
(Z score)
-0.23
-0.46
0.35
0.31
-0.03
0.22
-0.25
-0.37
0.58
-0.60
-0.67
0.25
0.69
-0.39
RMS
violations
-0.11
circular
variance
-0.32
0.00
packing
0.32
-0.06
-0.49
0.16
-0.11
-0.48
0.48
0.04
0.04
0.07
-0.21
bumps
(Z-score)
Ramachandran
-0.51
(Z-score)
bumps
original
-0.47
6
analysis
-improvement?
-correlations?
-…
Correlation NMR data density
Ramachandran Z-score
Ramachandran Z-score
r=0.31
NMR data density
Correlation NOE completeness and
packing Z-score
6
analysis
-improvement?
-correlations?
-…
r=0.20
packing Z-score
NMR data-based indicators
cannot yield any indication of
the normality of the
structures
NOE completeness
6
analysis
-improvement?
-correlations?
-…
In search of correlations
(Precision)
refined
data
density
data density
RMS
violations
circular
variance
packing
Ramachandran
(Z score)
(Z score)
-0.23
-0.46
0.35
0.31
-0.03
0.22
-0.25
-0.37
0.58
-0.60
-0.67
0.25
0.69
-0.39
RMS
violations
-0.11
circular
variance
-0.32
0.00
packing
0.32
-0.06
-0.49
0.16
-0.11
-0.48
0.48
0.04
0.04
0.07
-0.21
bumps
(Z-score)
Ramachandran
-0.51
(Z-score)
bumps
original
-0.47
Correlation between precision and data
density
circular variance
r=-0.46
NMR data density
6
analysis
-improvement?
-correlations?
-…
Correlation between precision and
Ramachandran
6
analysis
-improvement?
-correlations?
-…
circular variance
r=-0.67
Protein with high
Ramachandran normality
will have small circular
variance
1SUT
Ramachandran plot appearance (Z-score)
6
analysis
-improvement?
-correlations?
-…
Correlation between RMSD and structural
uncertainty (QUEEN)
backbone RMSD (Å)
r=-0.69
Structural uncertainty
imposes lower limit to the
RMSD
structural uncertainty
Conclusions I
• NMR-STAR files made consistent for 545 out of ±1700
•
•
•
entries
Protocols and scripts available for recalculation in CYANA
and CNS
Validation database available for testing of new protocols
Improvement compared to original data: 1 standard
deviation closer to X-ray db
• violations in original data do no limit recalculation effort
• refinement in water required
• 5 % no improvement: data missing
Conclusions II
• Correlations higher after recalculation and
refinement, though most of them still weak
•
Highest correlation: precision vs. Ramachandran
score & structural uncertainty (QUEEN)
Acknowledgements
•
•
•
•
•
•
Utrecht University
Alexandre Bonvin
Rob Kaptein
EBI Cambridge
Wim Vranken
CESG/BMRB
Jurgen Doreleijers
Zachary Miller
Eldon Ulrich
John Markley
Radboud University Nijmegen Chris Spronk
Sander Nabuurs
RIKEN Japan
Peter Güntert
Institut Pasteur Paris
Michael Nilges