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