Division of Molecular and Cellular Biosciences (MCB) Virtual Office - - PowerPoint PPT Presentation

8/12/2020 National Science Foundation

Welcome to the MCB Virtual Office Hours, we will begin at 2pm EDT! Please submit questions by selecting the Q&A function available to you on Zoom. Previous office hours: https://mcbblog.nsfbio.com/office-hours/

Division of Molecular and Cellular Biosciences (MCB) Virtual Office Hours

8/12/2020 National Science Foundation

Clusters

• Molecular Biophysics
• Genetic Mechanisms
• Cellular Dynamics and

Function

• Systems and Synthetic Biology

Molecular and Cellular Biosciences (MCB)

Supports quantitative, predictive and theory-driven research to understand complex living systems at the molecular, subcellular, and cellular levels Encourages use of approaches at intersections of biology with

• ther disciplines

8/12/2020 National Science Foundation

MCB Virtual Office Hour Topic NSF Supported Facilities of Interest

Mark Hunter Linac Coherent Light Source (LCLS), Stanford MCB Program Director Engin Serpersu Cornell High Energy Synchrotron Source (CHESS) MCB Program Director Jarek Majewski ChemMatCars at Advanced Photon Source Moderator: Marcia Newcomer

8/12/2020 National Science Foundation

Submit your questions via the Q&A function.

MCB Virtual Office Hour Question and Answers Session:

*For specific questions about your project, please contact a Program Director.

Click on the Q&A icon on the bottom of your Zoom screen, shown here: A Q&A box should appear on your screen. Please enter your question or comment in the

• box. You may select to submit

your question anonymously.

Current and Future Opportunities in Structural Dynamics of Macromolecules at LCLS

Mark S Hunter Sample Environment and Delivery Department Linac Coherent Light Source

6

SLAC National Accelerator Laboratory

San Francisco SLAC

7

SLAC is home to many great tools for structural biology

https://cryoem.slac.stanford.edu/ https://www-ssrl.slac.stanford.edu/smb/index.html

8

Damage-free room temperature structures of Biomolecules

• Outrunning radiation damage allows room

temperature measurements

• Avoid site specific and global radiation damage
• LCLS allows crystals too small for

conventional high-resolution structural analysis

• Could save months to years in optimization

9

Damage-free room temperature structures of Biomolecules

• Outrunning radiation damage allows room

temperature measurements

• Avoid site specific and global radiation damage
• LCLS allows crystals too small for

conventional high-resolution structural analysis

• Could save months to years in optimization

Liu et al. Science, 342, 1521 (2013)

• Differences observed in cryogenic and room

temp structures of G-protein coupled receptors

~40% of drugs target GPCRs

t01 fs 1 ps 1 ns 1 µs 1 ms 1 s

10 fs 100 fs

t∞

Enzyme catalysis Protein synthesis Photo-isomerization, charge separation, H+ / e- transfer Amino acid sidechain mot. Domain motion Protein folding Enzyme Transition States Pro isomerization Membrane Ion transport, signal cascades

Biochemical time scales Chemical & Physical time scales

electron transitions Bond vibrations

10

Time scales for select chemical and biochemical processes

• Many interesting biochemical processes and dynamics occur after 10+ ps
• Initial photo-excitation events on proteins with chromophores now accessible

Time scales taken from Allen M. Orville

LCLS pulse duration

t01 fs 1 ps 1 ns 1 µs 1 ms 1 s

10 fs 100 fs

t∞

Enzyme catalysis Protein synthesis Photo-isomerization, charge separation, H+ / e- transfer Amino acid sidechain mot. Domain motion Protein folding Enzyme Transition States Pro isomerization Membrane Ion transport, signal cascades

Biochemical time scales Chemical & Physical time scales

electron transitions Bond vibrations

11

Time scales for select chemical and biochemical processes

• Unique properties of LCLS that will facilitate studying these dynamic processes:
• Short, intense pulses  Diffraction before destruction  Room/Physiological Temperature Studies
• Short pulses  Unprecedented Temporal Resolving Power  Follow time series of reactions

(enzymes) or dynamics (general biomacromolecules)

LCLS temporal resolving power

Time scales taken from Allen M. Orville

LCLS provides the spatiotemporal resolving power to follow these processes. Many experiments have made use of this unique combination

Photosystem II: Simultaneous X-ray Crystallography and Spectroscopy

Structural dynamics of protein, cofactors

Crystallography

Chemical changes at the catalytic site

X-ray spectroscopy

1F 2F 3F

( )

S3 S2 S1 S0

Spin state

Taguchi et al. JACS 134, 1996 (2012) Kern et al. Nature (2018) 563, 421.

13

Fluorescent protein design based on chromophore twisting in the excited state

Model of on-off switching: I*T (twisted intermediate) was not detected by previous Transient Absorption (TA) measurements

• Picosecond time-resolved

crystallography on reversible photoswitching fluorescent protein rsEGFP

• Found bulky valine side chain

(V151) interfered with twisted intermediate

• Twisted intermediate not

detected via TA

• Doubled the quantum efficiency
• f this fluorescent protein via

V151A mutation Models showing the off (white), on (cyan), and twisted (pink) structures of the chromophore

Coquelle, N., Sliwa, M., Woodhouse, J. et al. Nature Chem 10, 31–37 (2018). https://doi.org/10.1038/nchem.2853

V151 V151

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Sub-Picosecond dynamics in bacteriorhodopsin

• Time-series on photoactivation of bR captured the

cis-trans isomerization of retinal (100s of fs)

• Retinal isomerization followed with ~200 fs temporal

resolution

• Absorbed energy kinetically dissipated due to conical

intersection!

• Observed twisted geometry of retinal
• Most biological macromolecules don’t have a native

chromophore!

Nogly, P., et al., (2018) Science, 361(6398).

15

Riboswitches, genetic control, and mixing experiments,

• MISC used to study the reaction of large RNA molecule that

participates in genetic control (riboswitch)

• MISC at LCLS allowed dynamics of riboswitch to be observed

with long time scales

• Need better temporal resolution in the ms range to resolve some

large-scale dynamics

• Small Crystals can allow MISC to measure reactions in the µs

time scale (challenging)

Stagno, J.R., et al., (2017) Nature, 541(7636)

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Where do we go from here?

• THz and M/FIR can trigger dynamics throughout the timeline shown
• Allows for measurements on samples grown in extremely viscous media

LCLS temporal resolving power

Mid and Far IR and THz

t01 fs 1 ps 1 ns 1 µs 1 ms 1 s

10 fs 100 fs

t∞

Enzyme catalysis Protein synthesis Photo-isomerization, charge separation, H+ / e- transfer Amino acid sidechain mot. Domain motion Protein folding Enzyme Transition States Pro isomerization Membrane Ion transport, signal cascades

Biochemical time scales Chemical & Physical time scales

electron transitions Bond vibrations

Thompson, M.C. et al., Nat. Chem. 11, 1058–1066 (2019)

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How can you get access to LCLS?

• Protein Crystal Screening Program
• Test experiments for sample quality
• Rapid Access Program
• Access for samples that are high scientific

interest and ready to be measured

• Regular Program
• Access for scientifically interesting but

constrained scope experiments

• Campaign Program
• Ambitious research programs of high

scientific interest that need multiple experiments at LCLS

https://lcls.slac.stanford.edu/proposals/run18-regular https://lcls.slac.stanford.edu/rapid-access-program https://lcls.slac.stanford.edu/proposals/protein-crystal- screening-proposals https://lcls.slac.stanford.edu/proposals/run19-campaign

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General Information and Inquiries

https://biology-lcls.slac.stanford.edu Contact Mark Hunter (mhunter2@slac.stanford.edu) Upcoming LCLS Webinar Townhall: Science Campaign in Structural Biology – Function & Dynamics Aug 18, 2020 10am PT https://lcls.slac.stanford.edu/news/l cls-virtual-town-hall-run-19- campaign-proposals

CHE HEXS@CHE HESS

Engin Serpersu Program Director Office Hour August 12,2020

The Cornell High Energy Synchrotron Source (CHESS) is located on the Cornell Ithaca campus underneath the Upper Alumni Athletic field. CHESS is one of five 3rd generation, high-energy X-ray facilities in the world providing extremely intense beams of polarized X-rays for users from around the world.

NSF supports the Center for High Energy X-ray Sciences, CHEXS, at CHESS, consisting of 4 beamlines. HPBio-SAXS/ BioSAXS HPBio-MX / FlexX

CHEXS / MacCHESS Beamtimeallocations through peer-reviewed proposal process.

Provide a resource for structural biology under ambient or high-pressure conditions Fundamental biology and biomedicine-Characterizing biomolecular interactions, conformational changes, and flexibility of biomolecules under physiological and extreme conditions

• High pressure is known to affect a vast number of biochemical

functions

• Most of the biosphere is at high pressure
• High pressure structural studies might help answer some of

the most fundamental questions in biology,

• And yet, with a world full of highly competitive biologists, the area of high

pressure structural biology is barely explored

Pressure-induced changes can reveal voids and imperfect packing High pressure influences structure and function. Anything a biomolecule does changes its volume!

Diffraction pattern of Beta- Lactoglobulinat 4.5 kbar

Glucose isomerase under pressure Chromatography linked SAXS at high pressure

RNA’s magic begins with base-specific ion interactions

Alex Plumridge, Kurt Andresen and Lois Pollack, Cornell

DOI: 10.1021/jacs.9b04461

Diffuse X-ray Scattering from Correlated Motions in a Protein Crystal

Steve P. Meisburger1,2, David A. Case3& Nozomi Ando1,2.

High Pressure Small Angle X-ray Scattering Workshop, Cathy Royer, royerc@rpi.edu

Sector 15 of the Advanced Photon Source X-rays

NSF’s Chem ChemMatCAR CARS – Ex Existing B g Beamline (at Argon

• nne N

National L Lab)

Liquid Surfaces and Liquid-Liquid Interfaces:

• Reflectivity
• Grazing-Incidence Diffraction (GID)
• Diffuse Scattering
• Fluorescence near Total Reflection
• Temporal resolution:

1-D Pinhole GID, GIXOS

• GISAXS
• Surface XAS New Initiative

Black: 2007 Red: post-2007 upgrades of instrumentation and X-ray techniques Beamline Parameters

• Energy range: 5.5 keV to 70 keV
• X-ray Flux: ~4x1014 @ 10 keV;

~1x1014 @ 30 keV

• X-ray Energy Resolution: ∆E/E ~ 10-4
• Detectors:
• Pilatus 200K ( E < 15 keV)
• Pilatus 1M CdTe (E> 16 keV)
• Vortex-EX (Energy dispersive)
• Q range @ 10 keV for Liquid Surface

Qz up to 3 Å-1; Qxy up to 4 Å-1

Liquid Interface Scattering Station at NSF’s ChemMatCARS

Pilatus3X CdTe 1M Bruker CCD Pilatus 100K

Beamline Accel (Bruker) Monochromator

• Cryogenic Si (111) and (311), parallel, ΔE/E = 1.3*10-4

(111), 2.8*10-5 (311)

• Energy range: 5-36 keV (for (111)), 9-70 keV (for (311))

Beamline Focusing Optics

• 1st mirror – 16-element bimorph: vertically focusing ~100 µm
• Upgrade with compound refractive lens system (CRL):

vertical focusing ~ 5 µm

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NSF’s C ChemMatCARS I S Instrumentation

• n a

and Sample En Envi viron

• nment

Liquid/liquid interface sample cells Liquid/vapor sample environment Langmuir trough Liquid Surface/Interface Spectrometer

NSF’s Chem ChemMatCAR CARS – Sci Scientific c Focu

• cus i

in Life Proce

• cesses

Wide Range of Fundamental and Applied Research

Chemistry of Life Processes

Lipid-Enzyme Interaction on Water Surface

Soft Matter 14, 4068 (2019)

• Y. Liu, UIC

PNAS 111, E1463 (2014) KYC Lee, U of Chicago

Lipid-Protein Binding

ACS Nano 13, 8680, (2019)

• C. Chen, NCNT, China
• L. Wang, IHEP, China

Nanotoxicity Peptoid-membrane interactions

BBA-Biomembranes 1860, 1414 (2018)

• D. Gidalevitz, IIT

Experiment Type (New in Red) Station Comments Liquid Interface Scattering Current: Liquid-Vapor Interfaces C Established Community of Users Liquid-Liquid Interfaces C Established Community of Users New Possibilities: Liquid-Solid Interfaces B1 Biomembranes & Cell-Surface Scanning Transmission Probe B1 Heterogeneities – Bio-processes

Layout for

• r 2 Beamlines a

and New Initiatives i in Liquid Interface ce P Prog

• gram

New I Ini nitiatives es on t n the Can he Canted ed B1 S B1 Station

Liquid-Solid Interfaces Life Processes (biomembranes, membrane proteins, cell-surface interactions)

• J. K. Blasie et al., Langmuir 30, 4784 (2014)

Supported lipid membranes loosely tethered to a solid surface Mount samples here for horizontal scattering

NSF’s Chem ChemMatCAR CARS Co Contac acts f s for Liquid Interface ce Sc Scattering P g Prog

• gram
• Wei Bu: Beamline Scientist for Liquid Interface Scattering, University
• f Chicago (bu@cars.uchicago.edu)
• Binhua Lin: Beamline Scientist for Liquid Interface Scattering and

Executive Director of NSF’s ChemMatCARS, University of Chicago (lin@cars.uchicago.edu)

• Mark Schlossman: PI of NSF/CHE-1836674 for the construction of the

second beamline at NSF’s ChemMatCARS, University of Illinois at Chicago (schloss@uic.edu)

Neutrons in biology research @ NCNR

36

Length scales: 1-1000 nm Time scales: 0.1-1000 ns

https://www.nist.gov/industry-impacts/cancer- therapy-formulations

nSoft

Frank Heinrich / NIST Susan Krueger / NIST David Hoogerheide / NIST

37

Contributions from Lise Arleth, Peter Moody, Frank Heinrich

Neutrons in structural biology

vSANS uSANS NG7-SANS NGB-SANS 10m SANS CANDoR MAGIK PBR NG7REFL

NSE HFBS DCS

8/12/2020 National Science Foundation

Submit your questions via the Q&A function.

MCB Virtual Office Hour Question and Answers Session:

*For specific questions about your project, please contact a Program Director.

Click on the Q&A icon on the bottom of your Zoom screen, shown here: A Q&A box should appear on your screen. Please enter your question or comment in the

• box. You may select to submit

your question anonymously.

Backup slides

40

Simultaneous XRD and XES for Ribonucleotide reductase

with Krebs group, Penn State, and Hoegbom group, Stockholm Univ. Fuller et al., Nature Methods (2017) Mn/Fe RNR (oxidized) room T 2Fo-Fc (blue) and Fo-Fc (green) map

• Ribonucleotide reductase contains either Fe/Fe, Fe/Mn or Mn/Mn active site
• Changes oxidation state during O2 activation to form catalytically active form
• Chemical environment and structure can be tracked using LCLS

Structure of Rhodopsin-Arrestin Complex

• Rhodopsin is a GPCR involved in vision
• Arrestin binding to GPCRs blocks G

protein interaction

• Arrestin adopts a pre-activated

conformation, with a ~20º rotation between the N- and C- domains

• Opens up a cleft in arrestin to

accommodate the second intracellular loop of rhodopsin

• This structure provides a basis for

understanding GPCR-mediated arrestin- biased signaling

Kang, Y., Zhou, X., Gao, X. et al. Nature 523, 561–567 (2015). https://doi.org/10.1038/nature14656

• J. Ullrich, A. Rudenko, R. Moshammer Ann. Rev. Phys. Chem. 63, 635 (2012)

Pulsed X-ray Sources Provide Unique Capabilities

Storage ring sources

• 10-100 ps pulse length
• 0.25-500 MHz repetition

rate

• 1010 photons/pulse
• 107 photons/100 fs

LCLS X-ray FEL

• pulse lengths 0.5-300 fs
• 1012 – 1013 photons/pulse
• 480 eV-25 keV
• mJs per pulse!

42

Diffraction before destruction

Neutze, et al., Nature 406, 752-757 (2000).

43

LCLS Call for Scientific Campaigns

• Letters of Interest for new LCLS Scientific Campaigns are solicited in the

area of “Structural Biology – Function & Dynamics”

• https://lcls.slac.stanford.edu/proposals/run19-campaign
• Campaigns are meant to be challenging and ambitious research programs

needing multiple beamtimes at LCLS

• Joint projects with Cryo-EM, synchrotron, or other biophysical data are welcome!
• Examples:
• Determining how photo-active enzymes mediate and accelerate photochemical

reactions.

• Illuminating the structural dynamics that underpin a range of RNA functions
• Questions: contact Mark Hunter (mhunter2@slac.stanford.edu)

44

Structural Molecular Biology at the Stanford Synchrotron Radiation Lightsource

• The Structural Molecular Biology

(SMB) Group at the Stanford Synchrotron Radiation Lightsource (SSRL)

• Macromolecular

crystallography beamlines

• Small angle X-ray scattering

beamline

• X-ray spectroscopy beamlines

SMB: https://www- ssrl.slac.stanford.edu/smb/index.html SSRL: https://www-ssrl.slac.stanford.edu/

45

Stanford-SLAC Cryo-EM Initiative

• Stanford-SLAC cryo-EM center
• Stanford-SLAC cryo-EM facilities
• National Center for Macromolecular Imaging (NCMI)
• Regional Crryo-EM Consortium
• Seven Thermo Scientific™ Krios™ microscopes planned

in total

• Stanford-SLAC Cryo-EM:

https://cryoem.slac.stanford.edu/

Professor Wah Chiu near a Krios™ microscope

46

Time scales for select chemical and biochemical processes

LCLS temporal resolving power

Mix and Inject SFX

• MISC will allow for reaction of enzymes to be

studied (theoretically) in µs to ms time scales

• Shorter time points will be limited by mixing

before the diffusion

• M. Schmidt. (2013) Advances in Condensed Matter Physics 10.1155

t01 fs 1 ps 1 ns 1 µs 1 ms 1 s

10 fs 100 fs

t∞

Enzyme catalysis Protein synthesis Photo-isomerization, charge separation, H+ / e- transfer Amino acid sidechain mot. Domain motion Protein folding Enzyme Transition States Pro isomerization Membrane Ion transport, signal cascades

Biochemical time scales Chemical & Physical time scales

electron transitions Bond vibrations

47

Time scales for select chemical and biochemical processes

• MISC will allow for reaction of enzymes to be studied (theoretically) in µs to ms time scales
• Caged compounds and photoswitchable ligands can allow laser-driven reactions or dynamics
• Shorter time points can be accessed
• Utilize the great infrastructure at LCLS for laser-triggered experiments

LCLS temporal resolving power

MISC Caged compounds Photoswitchable compounds

t01 fs 1 ps 1 ns 1 µs 1 ms 1 s

10 fs 100 fs

t∞

Enzyme catalysis Protein synthesis Photo-isomerization, charge separation, H+ / e- transfer Amino acid sidechain mot. Domain motion Protein folding Enzyme Transition States Pro isomerization Membrane Ion transport, signal cascades

Biochemical time scales Chemical & Physical time scales

electron transitions Bond vibrations