NSLS-II User Workshop · – Fast access – Workshop, training • Online courses Perspectives....
Transcript of NSLS-II User Workshop · – Fast access – Workshop, training • Online courses Perspectives....
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NSLS-II User Workshopa user point of view
Pierre-Damien COUREUXBrandeis University
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Macromolecular crystallographyStructure ⇔ function
your proteinyour protein(substate A)
your protein(substate C)(substate B)
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Enzyme vs photoreceptor
Enzyme Photoreceptor
I1 I2
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Photoreceptors
- six families : rhodopsins, phytochromes, xanthopsins, cryptochromes, phototropins and BLUF-proteins
- membrane/soluble, single domain/multidomains proteins
- modulate gene expression, enzyme activity and/or motility
Structure Function
PYP
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dark dataset
dark adapt flash freeze
light
light dataset
dark structure light structuredifference map
Typical experiment
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Phe 96Chr
Tyr 42
Arg 52
Tyr 98Pro 68
Glu 46
LN2
~70 % ~30 %
+
100 %Ground state Ground state I1 intermediate
Activation results
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Why is photo-conversion limited?
Not enough light
High optical density of crystal
Dynamic equilibrium
Photochemical equilibrium
Conformational heterogeneity
0
10
20
30
40
50
60
70
80
90
100
0 10 20 30 40 50 60 70 80
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Crystal glowing
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Fluorescence scan
Setup: Single-crystal fluorescence spectrometer
- PYP crystal frozen in the dark- excitation light- 100K- fluorescence detector
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00.10.20.30.40.50.60.70.80.9
1
375 400 425 450 475 500Wavelength (nm)
Predicted photochemicalequilibrium
Measured intermediate fraction
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Why is photo-conversion limited?
Not enough light
High optical density of crystal
Dynamic equilibrium
Photochemical equilibrium
Conformational heterogeneity
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Conformational heterogeneity
dark intermediate
Res 0.85 ÅP63
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Res 0.85 ÅP63
Res 1.15 ÅP65
Res 1.2 ÅP65
S atom of the chromophore
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Met 109 as a control
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O1 atom in chromophoredark intermediate
Res 0.85 ÅP63
Top view
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Conclusion
Subatomic conformational change ↔ drastic activity change
Crystallographic model corresponds to the inactive form of PYP
P2
P1
70%
30%
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Perspectives• What I would like to see on a beamline
– Robot for mounting crystals (limiting time is crystal screening: open door, move detector, mount crystal, close door, shoot crystal)
– Higher flux and larger detectors• Ultra-high resolution• Mainly for large complexes
– Spectroscopic equipment• Tunable light source, optic fibers and lenses, laser…• Fluorescence detection
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– Microfocus beamline• Different experiments on the same crystal
– Cryotemperature cooling control• Characterize transition states• Better control on annealing
– Better dorms• (please!!!)
Perspectives
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• What I still would like to see on a beamline
– Remote data collection
– Mail-in crystals • Live interaction with collaborator
– Fast access
– Workshop, training• Online courses
Perspectives
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Acknoledgments
Genick labUlrich K. Genick
Zi Peng FAN
Nikolaus Grigorieff lab(cluster)
NSLS X6A Beamline teamVivian Stojanoff
HFSP funding