X-ray Footprinting at NSLSII ray Footprinting at NSLSII Sayan Gupta Sayan Gupta Case Western Reserve University Case Western Reserve University Case Western Reserve University Case Western Reserve University
XX--ray Footprinting at NSLSIIray Footprinting at NSLSII
Sayan GuptaSayan GuptaCase Western Reserve UniversityCase Western Reserve UniversityCase Western Reserve UniversityCase Western Reserve University
What is done in Beamline X28C What is done in Beamline X28C
― X-ray mediated •OH radical Footprinting: Structure and dynamicsof macromolecular complexes in solution.
40% i bli ti h Hi h Q lit R h― ~ 40% premier publication each year: High Quality Research.
― Support user from US and foreign universities – collaboration andservice project.p j
― Core research programs to develop radiolysis methods, detectionand analysis of various biological systems.
― Core research programs to develop beam line components, mixingdevice and automation.
― Importance of Footprinting technique availability at NSLSII
Scientific Needs and Current Limitations - 1Scientific Needs and Current Limitations - 1
Macromolecular Interactions In Vivo and Sub-cellular Components
Ribosome Assembly in CellJohns Hopkins University, MD
Rhodopsin Photoactivation in ROSCase Western Reserve University,OH
16S rRNA 5S rRNAtRNA
Disks
Cell
hν
30S 50S23S rRNA
CellularResponse
Increase in the flux density of XIncrease in the flux density of X--ray allows ray allows μμs to ms exposure times. s to ms exposure times. Shorter exposure results low perturbation in the cell and large assemblies. Shorter exposure results low perturbation in the cell and large assemblies.
Membrane Protein Dynamics
Scientific Needs and Current Limitations - 2Scientific Needs and Current Limitations - 2
G-protein Coupled ReceptorCase Western Reserve University
Protein
Micro-fabricated Flow Cell Mixer
Activated
Cytoplasm side
Cell ResponseG-prot
Activated
Ligand /Buffer <1μM
diameter
GPCR
Mem
b
Movable X-ray windowHigh Flux
X-rayDecrease
GPCR Activation
raneDecreaseNo Change< 2 fold increase> 2 fold increaseNot oxidizedNot detected
Rhodopsin
Increase in flux density and smaller beam size will allow Increase in flux density and smaller beam size will allow μμs exposure. s exposure. UltraUltra--fast kinetic studies can be developed on the biological time scale.fast kinetic studies can be developed on the biological time scale.
Macromolecular AssembliesScientific Needs and Current Limitation - 3Scientific Needs and Current Limitation - 3
ClpAP Protease, >1300kDaCase Western and MIT
Arp 2/3 Complex, >300kDaCase Western Reserve University
100Å
6 ClpA
14 ClpP 100Å
6 ClpA
Kiselar et al. PNAS (2007)
Increase in flux density of XIncrease in flux density of X--ray will allow shorter exposure. Shorter ray will allow shorter exposure. Shorter exposure results first order dose and high S/N exposure results first order dose and high S/N -- High Quality DataHigh Quality Data
New NSLS II beamline for X-ray FootprintingNew NSLS II beamline for X-ray Footprinting
• Insertion device suitable for X-ray Footprinting: Damping Wiggler (DW)• Insertion device suitable for X-ray Footprinting: Damping Wiggler (DW)
• DW will provide high flux with a broad energy range (<10eV - ~100keV).
C t l l t f th Lif S i t f X F t i ti
• Current thinking: either one 7m long device or two canted 3.5m devices (see the conceptual layout below)
Conceptual layout of the Life Science sector for X-ray Footprinting, EXAFS, SAXS
Footprinting beamlineFootprinting beamline in DW or canted DW
Canted DW for EXAFS or SAXSor SAXS
Phased ConstructionPhased Construction
Phase 1- A
− Construction of DW beamline.
− Incorporation of vertical collimating mirror within the ring tunnel, upstream of the ring wall.
− Construction of front optic enclosure for future upgrades.
− Construction of experimental end station and sample exposure set-ups.
Development of sample exposure cells for NSLSII− Development of sample exposure cells for NSLSII.− Development of ultra fast mixing device for time resolved studies.
Phase 1 - B
− Transfer of existing beamline end-station components.
Year 2008 2009 2010 2011 2012 2013 2014 2015
NSLS-X28C OperationPhase 1 - A
Exposure Cell and
X28C Shut down
Beamline
Phase 1 - Bμs mixer development
X28C transfer
construction
Phase II – Beamline Development and UpgradesPhase II – Beamline Development and Upgrades
Phase 2 - A− Incorporate horizontal focusing mirror in FOE.− Continued development of sample exposure cell for the focused beam andultra fast mixing device for time resolved studies.− Precise control of beam size and shape, sample positioning and alignment.− Automation of sample handling and exposure (including live cell samples)
Phase 2 - B− Incorporation of SAXS or EXAFS on the second canted DW beamline.− Development of joint facility for biological SAXS and Footprinting.p j y g p g− Incorporation of other techniques compatible with footprinting set-up.
Year 2015 2016 2017
Phase 2SAXS
2nd CDW
Automation
MirrorExposure Cell & μs mixer development (cont.)
NSLSII Operation Start from
day 1
FundingFunding
NIH-NIBIB current funding ends, renewal begins
Year 2008 2009 2010 2011 2012 2013 2014 2015
NSLS-X28C
Phase I
NSLS-X28C OperationPhase I - A
Ph I B
Exposure Cell and μs mixer development
X28C Shut down
Beamlineconstruction
Phase I - BX28C
transfer
Supplemental Funding for Phase I - A and II
Phase II
Year 2015 2016 2017SAXSExposure Cell & μs
pp g
Phase II
NSLSII Operation
SAXS2nd CDW
Automation
MirrorExposure Cell & μs mixer development (cont.)
Start from day 1
Renewed NIH-NIBIB Funding
SAXS and Footprinting : Concordant measurement of Global & Local StructureSAXS and Footprinting : Concordant measurement of Global & Local Structure
Tetrahymena Ribozyme Gelsolin activation by Ca2+
Kwok et al. JMB (2006) Ashish et al. JBC (2007)
Kiselar et al. PNAS (2003)
Footprinting and SAXS are complementary techniques
Proposal for a Joint SAXS and Footprinting FacilityProposal for a Joint SAXS and Footprinting Facility
― Several footprinting users use SAXS on the same biomolecularsystem (> 50% in past five years )
― A joint facility where user can determine both global and localstructural information could boost user demand significantly.
― Footprinting requires much less sample concentration thanSAXS, so a consecutive/simultaneous data collection facility will beuseful.
― The second CDW beamline can be built for biological SAXS (orthe footprinting users need collaboration with another NSLSII SAXSfacility).
― Efficient utilization of sample preparation time and beamtimeusage.
SummarySummary
― Significant need for a footprinting beamline at NSLSII
― Footprinting is a flux driven experiment, thus the DW isthe appropriate source
― Construction timeline for the development of beamlineand transfer.
― Footprinting is a technique well suited to complementother techniques (SAXS, MX) and should be included in abiological sector at NSLSIIg