Yu Chen

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Yu Chen Shoichet Lab Dept of Pharm. Chem. UCSF
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PDB Content Growth
www.pdb.org
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Resolution determines the map quality Atomic
resolution 1.2 Å 400 structures
James Holton, ALS
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Resolution determines the map quality
James Holton, ALS
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3-1 Å
Information vs Resolution
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Structures less biased to library stereochemistry
parameters
Multiple conformations for flexible residues
Direct visualization of atom types and hydrogen
atoms
The Joy of High-Resolution Structures
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penicillin
cephalosporin
H2O
E S
??-lactamase hydrolyzing ?-lactam antibiotics
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Classes A, C and D serine-active
enzymes best studied examples Class A TEM,
SHV Class C AmpC
Class A TEM-1 (286aa)
Classe B metallo-enzymes
Class C AmpC (357aa)
Four classes of ?-lactamases
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Acylation transition state
De-acylation transition state
? Reaction cycle of TEM-1?-lactamase
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Acylation transition state
De-acylation transition state
? Reaction cycle of TEM-1?-lactamase
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Acylation transition state
De-acylation transition state
? Reaction cycle of TEM-1?-lactamase
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Acylation transition state
De-acylation transition state
? Reaction cycle of TEM-1?-lactamase
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Acylation transition state
De-acylation transition state
? Reaction cycle of TEM-1?-lactamase
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Acylation transition state
De-acylation transition state
? Reaction cycle of TEM-1?-lactamase
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Acylation transition state
De-acylation transition state
? Reaction cycle of TEM-1?-lactamase
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Acylation transition state
De-acylation transition state
? Transition State analog Boronic Acid
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Space group P212121
Rwork 9.10 Rfree 11.17
60 residues modeled in multiple
conformations 70 hydrogen atoms observed in
Fo-Fc difference map at 1.5 sigma level
0.85 Å structure of TEM-1 M182T ?-lactamase
Minasov, Wang Shoichet, JACS, 1245333-40
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(a) Asp273 atom types
(b) Met69Hydrogen atoms
Structural details revealed by high-res data
Blue density 2Fo-Fc electron density map used to
fit the model Red density positive peaks in
Fo-Fc map used to show the difference between
data and model (Fo, Fobserved Fc, Fcalculated)
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(a) 1.7 Å
(b) 0.85 Å
Glu 58 Structural details revealed by high-res
data
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? General base for acylation step
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(a) Glu 166 vs (b) Lys 73
? General base for acylation step
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Before proton transfer
After proton transfer
Seeing is believing Glu166 as general base
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? General base for acylation step
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Space group C2
Rwork 13.2 Rfree 16.4
15 residues currently modeled in multiple
conformations
1.07 Å structure of AmpC ?-lactamase (under
refinement)
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Thr 316 (1.07 Å)
Thr 316 (1.8 Å)
Double conformations in the active site
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Asp 10
Glu 228
Radiation Damage de-carboxylation of acidic side
chains
Magenta density negative peaks in Fo-Fc map
used to show the difference between data and
model
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Tyr 150
Lys 67
Asn 152
Lys 315
Ser64
Boronic acid
Thr 316
Active site of AmpC ?-lactamase deacylation
transition state
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Tyr150
B
Lys315
Ser64
Y150 protonating Ser64?
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Conclusions
Visualization of active site residues
protonation state help identify their catalytic
roles
A less-biased and detailed ultra-high resolution
structure can provide a better template for drug
discovery, with more information on side-chain
comformations and less ambiguity in atom types or
hydrogen positions.
Experimental data to compare with computational
predictions.
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Acknowledgment
Brian Shoichet George Minasov Xiaojun
Wang Richard Bonnet John Irwin Sandri
Soelaiman Ruth Brenk Binqing Wei Alan
Graves Federica Morandi Veena Thomas Brian
Feng OpenEye