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

Division of Molecular and Cellular Biosciences (MCB) Virtual Office Hours 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/ 8/12/2020 National Science Foundation

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 other disciplines Clusters • Molecular Biophysics • Genetic Mechanisms • Cellular Dynamics and Function • Systems and Synthetic Biology 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

MCB Virtual Office Hour Question and Answers Session: Click on the Q&A icon on the bottom of your Zoom screen, shown here: Submit your A Q&A box should appear on your screen. Please enter your questions via the question or comment in the box. You may select to submit Q&A function. your question anonymously. *For specific questions about your project, please contact a Program Director. 8/12/2020 National Science Foundation

Current and Future Opportunities in Structural Dynamics of Macromolecules at LCLS Mark S Hunter Sample Environment and Delivery Department Linac Coherent Light Source

SLAC National Accelerator Laboratory San Francisco SLAC 6

SLAC is home to many great tools for structural biology https://www-ssrl.slac.stanford.edu/smb/index.html https://cryoem.slac.stanford.edu/ 7

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 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 • Differences observed in cryogenic and room Liu et al . Science, 342 , 1521 (2013) temp structures of G-protein coupled receptors ~40% of drugs target GPCRs 9

Time scales for select chemical and biochemical processes LCLS pulse duration Bond vibrations Chemical & Physical time electron Time scales taken scales transitions from Allen M. Orville Membrane Ion transport, signal cascades Enzyme Protein folding Domain motion Biochemical Transition time scales States Pro isomerization Photo-isomerization, charge separation, H+ / e- transfer Amino acid Enzyme catalysis Protein synthesis sidechain mot. t 0 1 fs 100 fs 10 fs t ∞ 1 s 1 ps 1 ns 1 ms 1 µs • Many interesting biochemical processes and dynamics occur after 10+ ps • Initial photo-excitation events on proteins with chromophores now accessible 10

Time scales for select chemical and biochemical processes LCLS temporal resolving power Bond vibrations Chemical & Physical time electron Time scales taken scales transitions from Allen M. Orville Membrane Ion transport, signal cascades Enzyme Protein folding Domain motion Biochemical Transition time scales States Pro isomerization Photo-isomerization, charge separation, H+ / e- transfer Amino acid Enzyme catalysis Protein synthesis sidechain mot. LCLS provides the spatiotemporal resolving power to follow these processes. t 0 1 fs 100 fs 10 fs t ∞ 1 s 1 ps 1 ns 1 ms 1 µs Many experiments have made use of this unique combination • 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) 11

Photosystem II: Simultaneous X-ray Crystallography and Spectroscopy S 1 Crystallography Spin state Structural dynamics of protein, cofactors S 2 1F ( ) S 3 2F 3F S 0 X-ray spectroscopy Chemical changes at the catalytic site Kern et al. Nature (2018) 563, 421. Taguchi et al. JACS 134, 1996 (2012)

Fluorescent protein design based on chromophore twisting in the excited state V151 V151 • Picosecond time-resolved crystallography on reversible photoswitching fluorescent protein rsEGFP • Found bulky valine side chain (V151) interfered with twisted intermediate Models showing the off (white), on • Twisted intermediate not (cyan), and twisted (pink) structures of detected via TA the chromophore Model of on-off switching: I* T • Doubled the quantum efficiency (twisted intermediate) was of this fluorescent protein via not detected by previous V151A mutation Transient Absorption (TA) measurements Coquelle, N., Sliwa, M., Woodhouse, J. et al. Nature Chem 10, 31–37 (2018). https://doi.org/10.1038/nchem.2853 13

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). 14

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) 15

Where do we go from here? LCLS temporal resolving power Bond vibrations Chemical & Physical time Mid and Far IR and THz electron scales transitions Membrane Ion transport, signal cascades Enzyme Protein folding Domain motion Biochemical Transition time scales Pro isomerization States Photo-isomerization, charge separation, H+ / e- transfer Amino acid Enzyme catalysis Protein synthesis sidechain mot. t 0 1 fs 100 fs 10 fs t ∞ 1 s 1 ps 1 ns 1 ms 1 µs • THz and M/FIR can trigger dynamics throughout the timeline shown • Allows for measurements on samples grown in extremely viscous media Thompson, M.C. et al., Nat. Chem. 11, 1058–1066 (2019) 16

How can you get access to LCLS? • Protein Crystal Screening Program https://lcls.slac.stanford.edu/proposals/protein-crystal- screening-proposals • Test experiments for sample quality • Rapid Access Program https://lcls.slac.stanford.edu/rapid-access-program • Access for samples that are high scientific interest and ready to be measured • Regular Program https://lcls.slac.stanford.edu/proposals/run18-regular • Access for scientifically interesting but constrained scope experiments • Campaign Program https://lcls.slac.stanford.edu/proposals/run19-campaign • Ambitious research programs of high scientific interest that need multiple experiments at LCLS 17

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 18

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.

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

CHEXS / MacCHESS Beamtimeallocations through peer-reviewed proposal process. Fundamental biology and biomedicine -Characterizing Provide a resource for structural biology biomolecular interactions, conformational changes, and under ambient or high-pressure flexibility of biomolecules under physiological and extreme conditions 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!