Feb 1, 2018 - Fei Long. Rob Nicholls. Gideon Davies. Kevin Cowtan. Eleanor Dodson. Keith Wilson. Stuart McNicholas. Scott Hoh. Paul Bond. Wendy Offen.
Privateer So, what does it do?
Dr Jon Agirre Royal Society University Research Fellow @alwaysonthejazz
A bit of history Needed to mine PDB for carbohydrates Conformational analysis was critical → wanted nice, low-energy chairs We knew there were issues with nomenclature of monosaccharides and glycosidic linkages
Existing tools didn’t do what we wanted we = Gideon Davies, Keith Wilson, Kevin Cowtan & I at York Structural Biology Laboratory
Let us produce a simple program to leave out the 0.5% of structures that we expect will contain high-energy conformations…
A bit of history Needed to mine PDB for carbohydrates Conformational analysis was critical → wanted nice, low-energy chairs We knew there were issues with nomenclature of monosaccharides and glycosidic linkages
Existing tools didn’t do what we wanted we = Gideon Davies, Keith Wilson, Kevin Cowtan & I at York Structural Biology Laboratory
Let us produce a simple program to leave out the 0.5% of structures that we expect will contain high-energy conformations…
🤣
A look at ring conformation in N-glycosylation
Agirre, Davies, Wilson & Cowtan. Nature Chemical Biology, 11, 2015
A look at ring conformation in N-glycosylation
Agirre, Davies, Wilson & Cowtan. Nature Chemical Biology, 11, 2015
Agirre. Acta Cryst D, CCP4sw special issue, 2017
Privateer Tools for spotting errors Model vs data Omit mFo-DFc maps using model and original data Real space correlation for each monosaccharide
Model Ring puckering and conformation, anomericity, absolute stereochemistry, nomenclature checks 2D diagrams of glycosylation (SVG, vector goodness!), easier to spot funky linkages
Support for fixing errors Keywords for Refmac, scripts for Coot, dictionaries with aperiodic torsions…
Graphical interfaces CCP4i2 (me), CCP4mg (with Stuart McNicholas), Coot (Paul Emsley) CCP-EM (me, coming soon-ish)
Use cases Wrong anomer or absolute stereochemistry Built as beta, but used alpha 3-letter code: rename Used correct code, but linked it wrong: rebuild
Unlikely puckering amplitude (Q) Too puckered? check linkage distances Flat ring? check chiral volume signs, look out for 0º torsions in dictionaries - these are most certainly wrong
High-energy conformations At high resolution: rebuild unless in -1 subsite of an enzyme At low resolution: correct automatically with aperiodic torsions (privateerlib.cif), keep them on in subsequent iterations of Coot and Refmac5
Agirre. Acta Cryst D, CCP4sw special issue, 2017
Stereochemistry Same chemical formula! C6 H12 O6
beta-d-glucose ‘BGC’ in the PDB beta-d-mannose ‘BMA’ in the PDB
Stereochemistry Same chemical formula! C6 H12 O6 Anomeric carbon
⍺-D-mannose ‘MAN’ in the PDB
β-D-mannose ‘BMA’ in the PDB
“About 30% of all carbohydrate containing PDB entries comprise one or several errors” Lütteke, Frank and von der Lieth. Carb Res, 339(5), 2004
GLC
BGC
(alpha anomer)
(beta anomer)
demo
Use cases Wrong anomer or absolute stereochemistry Built as beta, but used alpha 3-letter code: rename Used correct code, but linked it wrong: rebuild
Unlikely puckering amplitude (Q) Too puckered? check linkage distances Flat ring? check chiral volume signs, look out for 0º torsions in dictionaries - these are most certainly wrong
High-energy conformations At high resolution: rebuild unless in -1 subsite of an enzyme At low resolution: correct automatically with aperiodic torsions (privateerlib.cif), keep them on in subsequent iterations of Coot and Refmac5
Cremer-Pople analysis 𝛩 and Φ tell us which atoms move away from the mean ring plane, thus describing the conformation
Q tells us how much:
“total puckering amplitude”
demo Cremer and Pople. JACS 97(6), 1975
Cremer-Pople analysis 𝛩 and Φ tell us which atoms move away from the mean ring plane, thus describing the conformation
Q tells us how much:
Z4
Z5 Z3
Z2
Z0 Z1
“total puckering amplitude”
demo Cremer and Pople. JACS 97(6), 1975
Use cases Wrong anomer or absolute stereochemistry Built as beta, but used alpha 3-letter code: rename Used correct code, but linked it wrong: rebuild
Unlikely puckering amplitude (Q) Too puckered? check linkage distances Flat ring? check chiral volume signs, look out for 0º torsions in dictionaries - these are most certainly wrong
High-energy conformations At high resolution: rebuild unless in -1 subsite of an enzyme At low resolution: correct automatically with aperiodic torsions (privateerlib.cif), keep them on in subsequent iterations of Coot and Refmac5
Cremer-Pople analysis
Pyranoses (Q,Φ,𝛩)
Agirre & Cowtan. Computational Crystallography Newsletter Jan:10-12, 2015
Cremer-Pople analysis
Furanoses (Q,Φ)
Agirre & Cowtan. Computational Crystallography Newsletter Jan:10-12, 2015
Conformations 0.0
beta-D-mannopyranose
beta-D-glucopyranose
20.0 (kcal/mol)
alpha-L-fucopyranose
Ardevol, Biarnés, Planas & Rovira. JACS 132(45), 2010
cost of breaking 3 or 4 H-bonds Sheu, Yang, Selzle and Schlag. PNAS 100(22), 2002
0.0
beta-D-mannopyranose
beta-D-glucopyranose
20.0 (kcal/mol)
alpha-L-fucopyranose
Ardevol, Biarnés, Planas & Rovira. JACS 132(45), 2010
Geometric restraints With weak/incomplete density, conformation should be imposed L(p)=wLX(p)+LG(p)
Geometric restraints With weak/incomplete density, conformation should be imposed L(p)=wLX(p)+LG(p) bond lengths
? ?
bond angles
harmonic torsions
NCS, etc
Geometric restraints With weak/incomplete density, conformation should be imposed L(p)=wLX(p)+LG(p) bond lengths
Eclipsed substituents
? ?
bond angles
NCS, etc
harmonic torsions
Staggered substituents
(…) publication and deposition of incorrect structures informs subsequent statistical analyses that suggest the deposited structures are normal. Agirre, Davies, Wilson and Cowtan. Nature Chemical Biology, 11, 2015
We can now use Privateer to detect and prevent these and other anomalies
How to validate and correct structures with Privateer, Coot and Refmac5 At lower resolution, even a correct starting model can result in a higher-energy conformation after refinement
Doing sphere refine with harmonic torsions
Doing sphere refine with aperiodic torsions
Doing sphere refine with harmonic torsions
Doing sphere refine with aperiodic torsions
Doing sphere refine with harmonic torsions
Doing sphere refine with aperiodic torsions
‘TM9’ in 4K3T
demo
Graphical analysis
The Standard Symbol Nomenclature
McNicholas & Agirre. Acta Cryst D, CCP4sw special issue, 2017
The Standard Symbol Nomenclature 4A5T (human)
4BYH (human)
5FJI (fungal) 5AOG (plant)
demo
Agirre, Davies, Wilson and Cowtan. Curr Op Str Biol, 2017
Got a structure to report?
Acta Crystallographica section F STRUCTURAL BIOLOGY COMMUNICATIONS Special issue: Crystallography of glycoproteins and protein-carbohydrate complexes Edited by Mark van Raaij & Jon Agirre
Submit: https://tinyurl.com/glycoissue-acta-f Final deadline: 1st of February 2018
Acknowledgements Paul Emsley Garib Murshudov Fei Long Rob Nicholls
Robbie Joosten
Tristan Croll
Supported by
Gideon Davies Kevin Cowtan Eleanor Dodson Keith Wilson Stuart McNicholas Scott Hoh Paul Bond Wendy Offen
Carme Rovira
Martyn Winn Tom Burnley Colin Palmer
Acknowledgements Paul Emsley Garib Murshudov Fei Long Rob Nicholls
Robbie Joosten
Tristan Croll
Supported by
Gideon Davies Kevin Cowtan Eleanor Dodson Keith Wilson Stuart McNicholas Scott Hoh Paul Bond Wendy Offen
Carme Rovira
Martyn Winn Tom Burnley Colin Palmer
