LCLS 10 Year Bioscience Highlights

Axel T. BrungerDept. of Molecular and Cellular Physiology Stanford University/HHMI

April 10, 2019

4/10/2019

Today’s and Future Challenges of Structural Biology

Primary Imaging Methods:–X-ray crystallography –single particle electron microscopy–electron cryo-tomography

Today’s and Future Challenges:–super-molecular complexes–membrane proteins–cellular assemblies

Warkentin,...,Thorne, J. Sync. Rad., 2012

biologically relevant perturbations (Moffat, 1989, 2001;Schotte et al., 2003, 2004; Ihee et al., 2005; Henzler-Wildman& Kern, 2007; Bourgeois & Weik, 2009). Room-temperaturecrystallographic data can provide highly complementaryinformation to NMR and assist in, for example, identificationof allosteric networks (Fraser et al., 2009).

Crystallographers thus need robust techniques for datacollection at or near room temperature, and these mustinclude methods for managing radiation damage in thepresent era of third- and fourth-generation synchrotronsources. Here we summarize a range of recent studies thatenhance our understanding of radiation damage. Our discus-sion culminates in the exciting possibility that a large fractionof global damage to unfrozen crystals can be outrun by rapiddata collection using synchrotron source [not just free-elec-tron laser (FEL) source] intensities.

2. Radiation damage processes and timescales

The processes that lead to global radiation damage to proteincrystals, schematically illustrated in Fig. 1, take place on anenormous range of timescales. X-ray–electron and electron–electron interactions (Fig. 1a) occur in femtoseconds, and leadto the generation of hundreds of electrons with energies in the10–100 eV range (Nave & Hill, 2005; Finfrock et al., 2010;Sanishvili et al., 2011) (Fig. 1b). These interact with water andprotein, breaking bonds and creating radicals (Dertinger &Jung, 1970; Coggle, 1983) (Fig. 1c). These primary damageprocesses are at most weakly temperature dependent. Atsufficiently high temperature, atomic and molecular radicalscan diffuse and react with the protein, breaking additionalbonds (Fig. 1d); near room temperature, most radical reactionsshould be complete within a few microseconds (Pryor, 1986).X-ray- and radical-induced bond breaking increases averageatomic separations, creating internal stress that drives latticeexpansion. Local bond breaking and other chemical damagetrigger local conformational relaxation of, for example, sidechains, flexible loops and other more weakly constrainedregions (Fig. 1e). The larger of these local structural relaxa-tions likely take place on the microsecond to millisecondtimescale of conformation changes observed in undamagedprotein (Henzler-Wildman & Kern, 2007; Bourgeois & Weik,2009). As radiochemistry and local relaxation cause everlarger changes to individual protein molecules, the moleculeswill displace and rotate (Fig. 1f), the local lattice may deformand reorient (Fig. 1g), and the build-up of stress may even-tually cause plastic failure and cracking of the crystal (Fig. 1h).These larger motions will proceed on timescales of micro-seconds to hours or even days. In part because they involvesubstantial motions of solvent as the protein molecules movefrom their ideal lattice positions, these timescales will increaseas the temperature is decreased toward the glass transition.

Much of the literature on radiation damage to proteins hasfocused on radiochemistry, but a survey of the broaderradiation damage literature [especially for inorganic materials(Billington, 1962; Dupuy, 1975)] indicates the importance ofstructural relaxations of molecules and the crystal lattice

following chemical damage. Recent crystallographic analysesof the distribution of damage by residue within the unit cell(Juers & Weik, 2011; Warkentin et al., 2012b) suggest that,while solvent-exposed residues are more sensitive than buriedresidues at higher temperature, half of the damage is mani-fested uniformly over the entire structure (Warkentin et al.,2012b) (i.e. it affects surface and buried residues equally),suggesting the importance of lattice scale rather than bond-scale disorder.

3. Temperature dependence of global damage

At T = 100 K, studies to date suggest that all protein crystalsare comparably radiation sensitive, as quantified by how anappropriate metric such as relative B factor or integratedintensity varies with dose (energy deposited by ionizingparticles per kg) (Sliz et al., 2003; Kmetko et al., 2006; Leiros et

radiation damage

8 Matthew Warkentin et al. ! Global radiation damage J. Synchrotron Rad. (2013). 20, 7–13

Figure 1Illustration of some processes involved in the radiation damage cascade.(a) X-ray-induced ejection of a primary photoelectron. (b) Generation ofseveral hundred relatively low-energy ("100 eV) electrons. (c) Bondbreaking leading to internal stress and radical formation. (d) Radicalattack of the protein. (e) Conformation changes of side chains andflexible loops in response to chemical damage. ( f ) Displacement andreorientation of individual damaged molecules. (g) Deformation andreorientation of local lattice domains. (h) Plastic failure and crystalcracking.

electron

femtoseconds

biologically relevant perturbations (Moffat, 1989, 2001;Schotte et al., 2003, 2004; Ihee et al., 2005; Henzler-Wildman& Kern, 2007; Bourgeois & Weik, 2009). Room-temperaturecrystallographic data can provide highly complementaryinformation to NMR and assist in, for example, identificationof allosteric networks (Fraser et al., 2009).

Crystallographers thus need robust techniques for datacollection at or near room temperature, and these mustinclude methods for managing radiation damage in thepresent era of third- and fourth-generation synchrotronsources. Here we summarize a range of recent studies thatenhance our understanding of radiation damage. Our discus-sion culminates in the exciting possibility that a large fractionof global damage to unfrozen crystals can be outrun by rapiddata collection using synchrotron source [not just free-elec-tron laser (FEL) source] intensities.

2. Radiation damage processes and timescales

The processes that lead to global radiation damage to proteincrystals, schematically illustrated in Fig. 1, take place on anenormous range of timescales. X-ray–electron and electron–electron interactions (Fig. 1a) occur in femtoseconds, and leadto the generation of hundreds of electrons with energies in the10–100 eV range (Nave & Hill, 2005; Finfrock et al., 2010;Sanishvili et al., 2011) (Fig. 1b). These interact with water andprotein, breaking bonds and creating radicals (Dertinger &Jung, 1970; Coggle, 1983) (Fig. 1c). These primary damageprocesses are at most weakly temperature dependent. Atsufficiently high temperature, atomic and molecular radicalscan diffuse and react with the protein, breaking additionalbonds (Fig. 1d); near room temperature, most radical reactionsshould be complete within a few microseconds (Pryor, 1986).X-ray- and radical-induced bond breaking increases averageatomic separations, creating internal stress that drives latticeexpansion. Local bond breaking and other chemical damagetrigger local conformational relaxation of, for example, sidechains, flexible loops and other more weakly constrainedregions (Fig. 1e). The larger of these local structural relaxa-tions likely take place on the microsecond to millisecondtimescale of conformation changes observed in undamagedprotein (Henzler-Wildman & Kern, 2007; Bourgeois & Weik,2009). As radiochemistry and local relaxation cause everlarger changes to individual protein molecules, the moleculeswill displace and rotate (Fig. 1f), the local lattice may deformand reorient (Fig. 1g), and the build-up of stress may even-tually cause plastic failure and cracking of the crystal (Fig. 1h).These larger motions will proceed on timescales of micro-seconds to hours or even days. In part because they involvesubstantial motions of solvent as the protein molecules movefrom their ideal lattice positions, these timescales will increaseas the temperature is decreased toward the glass transition.

Much of the literature on radiation damage to proteins hasfocused on radiochemistry, but a survey of the broaderradiation damage literature [especially for inorganic materials(Billington, 1962; Dupuy, 1975)] indicates the importance ofstructural relaxations of molecules and the crystal lattice

following chemical damage. Recent crystallographic analysesof the distribution of damage by residue within the unit cell(Juers & Weik, 2011; Warkentin et al., 2012b) suggest that,while solvent-exposed residues are more sensitive than buriedresidues at higher temperature, half of the damage is mani-fested uniformly over the entire structure (Warkentin et al.,2012b) (i.e. it affects surface and buried residues equally),suggesting the importance of lattice scale rather than bond-scale disorder.

3. Temperature dependence of global damage

At T = 100 K, studies to date suggest that all protein crystalsare comparably radiation sensitive, as quantified by how anappropriate metric such as relative B factor or integratedintensity varies with dose (energy deposited by ionizingparticles per kg) (Sliz et al., 2003; Kmetko et al., 2006; Leiros et

radiation damage

8 Matthew Warkentin et al. ! Global radiation damage J. Synchrotron Rad. (2013). 20, 7–13

Figure 1Illustration of some processes involved in the radiation damage cascade.(a) X-ray-induced ejection of a primary photoelectron. (b) Generation ofseveral hundred relatively low-energy ("100 eV) electrons. (c) Bondbreaking leading to internal stress and radical formation. (d) Radicalattack of the protein. (e) Conformation changes of side chains andflexible loops in response to chemical damage. ( f ) Displacement andreorientation of individual damaged molecules. (g) Deformation andreorientation of local lattice domains. (h) Plastic failure and crystalcracking.

microseconds

bond breakage, ionization, radical formation

biologically relevant perturbations (Moffat, 1989, 2001;Schotte et al., 2003, 2004; Ihee et al., 2005; Henzler-Wildman& Kern, 2007; Bourgeois & Weik, 2009). Room-temperaturecrystallographic data can provide highly complementaryinformation to NMR and assist in, for example, identificationof allosteric networks (Fraser et al., 2009).

Crystallographers thus need robust techniques for datacollection at or near room temperature, and these mustinclude methods for managing radiation damage in thepresent era of third- and fourth-generation synchrotronsources. Here we summarize a range of recent studies thatenhance our understanding of radiation damage. Our discus-sion culminates in the exciting possibility that a large fractionof global damage to unfrozen crystals can be outrun by rapiddata collection using synchrotron source [not just free-elec-tron laser (FEL) source] intensities.

2. Radiation damage processes and timescales

The processes that lead to global radiation damage to proteincrystals, schematically illustrated in Fig. 1, take place on anenormous range of timescales. X-ray–electron and electron–electron interactions (Fig. 1a) occur in femtoseconds, and leadto the generation of hundreds of electrons with energies in the10–100 eV range (Nave & Hill, 2005; Finfrock et al., 2010;Sanishvili et al., 2011) (Fig. 1b). These interact with water andprotein, breaking bonds and creating radicals (Dertinger &Jung, 1970; Coggle, 1983) (Fig. 1c). These primary damageprocesses are at most weakly temperature dependent. Atsufficiently high temperature, atomic and molecular radicalscan diffuse and react with the protein, breaking additionalbonds (Fig. 1d); near room temperature, most radical reactionsshould be complete within a few microseconds (Pryor, 1986).X-ray- and radical-induced bond breaking increases averageatomic separations, creating internal stress that drives latticeexpansion. Local bond breaking and other chemical damagetrigger local conformational relaxation of, for example, sidechains, flexible loops and other more weakly constrainedregions (Fig. 1e). The larger of these local structural relaxa-tions likely take place on the microsecond to millisecondtimescale of conformation changes observed in undamagedprotein (Henzler-Wildman & Kern, 2007; Bourgeois & Weik,2009). As radiochemistry and local relaxation cause everlarger changes to individual protein molecules, the moleculeswill displace and rotate (Fig. 1f), the local lattice may deformand reorient (Fig. 1g), and the build-up of stress may even-tually cause plastic failure and cracking of the crystal (Fig. 1h).These larger motions will proceed on timescales of micro-seconds to hours or even days. In part because they involvesubstantial motions of solvent as the protein molecules movefrom their ideal lattice positions, these timescales will increaseas the temperature is decreased toward the glass transition.

Much of the literature on radiation damage to proteins hasfocused on radiochemistry, but a survey of the broaderradiation damage literature [especially for inorganic materials(Billington, 1962; Dupuy, 1975)] indicates the importance ofstructural relaxations of molecules and the crystal lattice

following chemical damage. Recent crystallographic analysesof the distribution of damage by residue within the unit cell(Juers & Weik, 2011; Warkentin et al., 2012b) suggest that,while solvent-exposed residues are more sensitive than buriedresidues at higher temperature, half of the damage is mani-fested uniformly over the entire structure (Warkentin et al.,2012b) (i.e. it affects surface and buried residues equally),suggesting the importance of lattice scale rather than bond-scale disorder.

3. Temperature dependence of global damage

At T = 100 K, studies to date suggest that all protein crystalsare comparably radiation sensitive, as quantified by how anappropriate metric such as relative B factor or integratedintensity varies with dose (energy deposited by ionizingparticles per kg) (Sliz et al., 2003; Kmetko et al., 2006; Leiros et

radiation damage

8 Matthew Warkentin et al. ! Global radiation damage J. Synchrotron Rad. (2013). 20, 7–13

Figure 1Illustration of some processes involved in the radiation damage cascade.(a) X-ray-induced ejection of a primary photoelectron. (b) Generation ofseveral hundred relatively low-energy ("100 eV) electrons. (c) Bondbreaking leading to internal stress and radical formation. (d) Radicalattack of the protein. (e) Conformation changes of side chains andflexible loops in response to chemical damage. ( f ) Displacement andreorientation of individual damaged molecules. (g) Deformation andreorientation of local lattice domains. (h) Plastic failure and crystalcracking.

biologically relevant perturbations (Moffat, 1989, 2001;Schotte et al., 2003, 2004; Ihee et al., 2005; Henzler-Wildman& Kern, 2007; Bourgeois & Weik, 2009). Room-temperaturecrystallographic data can provide highly complementaryinformation to NMR and assist in, for example, identificationof allosteric networks (Fraser et al., 2009).

Crystallographers thus need robust techniques for datacollection at or near room temperature, and these mustinclude methods for managing radiation damage in thepresent era of third- and fourth-generation synchrotronsources. Here we summarize a range of recent studies thatenhance our understanding of radiation damage. Our discus-sion culminates in the exciting possibility that a large fractionof global damage to unfrozen crystals can be outrun by rapiddata collection using synchrotron source [not just free-elec-tron laser (FEL) source] intensities.

2. Radiation damage processes and timescales

The processes that lead to global radiation damage to proteincrystals, schematically illustrated in Fig. 1, take place on anenormous range of timescales. X-ray–electron and electron–electron interactions (Fig. 1a) occur in femtoseconds, and leadto the generation of hundreds of electrons with energies in the10–100 eV range (Nave & Hill, 2005; Finfrock et al., 2010;Sanishvili et al., 2011) (Fig. 1b). These interact with water andprotein, breaking bonds and creating radicals (Dertinger &Jung, 1970; Coggle, 1983) (Fig. 1c). These primary damageprocesses are at most weakly temperature dependent. Atsufficiently high temperature, atomic and molecular radicalscan diffuse and react with the protein, breaking additionalbonds (Fig. 1d); near room temperature, most radical reactionsshould be complete within a few microseconds (Pryor, 1986).X-ray- and radical-induced bond breaking increases averageatomic separations, creating internal stress that drives latticeexpansion. Local bond breaking and other chemical damagetrigger local conformational relaxation of, for example, sidechains, flexible loops and other more weakly constrainedregions (Fig. 1e). The larger of these local structural relaxa-tions likely take place on the microsecond to millisecondtimescale of conformation changes observed in undamagedprotein (Henzler-Wildman & Kern, 2007; Bourgeois & Weik,2009). As radiochemistry and local relaxation cause everlarger changes to individual protein molecules, the moleculeswill displace and rotate (Fig. 1f), the local lattice may deformand reorient (Fig. 1g), and the build-up of stress may even-tually cause plastic failure and cracking of the crystal (Fig. 1h).These larger motions will proceed on timescales of micro-seconds to hours or even days. In part because they involvesubstantial motions of solvent as the protein molecules movefrom their ideal lattice positions, these timescales will increaseas the temperature is decreased toward the glass transition.

Much of the literature on radiation damage to proteins hasfocused on radiochemistry, but a survey of the broaderradiation damage literature [especially for inorganic materials(Billington, 1962; Dupuy, 1975)] indicates the importance ofstructural relaxations of molecules and the crystal lattice

following chemical damage. Recent crystallographic analysesof the distribution of damage by residue within the unit cell(Juers & Weik, 2011; Warkentin et al., 2012b) suggest that,while solvent-exposed residues are more sensitive than buriedresidues at higher temperature, half of the damage is mani-fested uniformly over the entire structure (Warkentin et al.,2012b) (i.e. it affects surface and buried residues equally),suggesting the importance of lattice scale rather than bond-scale disorder.

3. Temperature dependence of global damage

At T = 100 K, studies to date suggest that all protein crystalsare comparably radiation sensitive, as quantified by how anappropriate metric such as relative B factor or integratedintensity varies with dose (energy deposited by ionizingparticles per kg) (Sliz et al., 2003; Kmetko et al., 2006; Leiros et

radiation damage

8 Matthew Warkentin et al. ! Global radiation damage J. Synchrotron Rad. (2013). 20, 7–13

Figure 1Illustration of some processes involved in the radiation damage cascade.(a) X-ray-induced ejection of a primary photoelectron. (b) Generation ofseveral hundred relatively low-energy ("100 eV) electrons. (c) Bondbreaking leading to internal stress and radical formation. (d) Radicalattack of the protein. (e) Conformation changes of side chains andflexible loops in response to chemical damage. ( f ) Displacement andreorientation of individual damaged molecules. (g) Deformation andreorientation of local lattice domains. (h) Plastic failure and crystalcracking.

biologically relevant perturbations (Moffat, 1989, 2001;Schotte et al., 2003, 2004; Ihee et al., 2005; Henzler-Wildman& Kern, 2007; Bourgeois & Weik, 2009). Room-temperaturecrystallographic data can provide highly complementaryinformation to NMR and assist in, for example, identificationof allosteric networks (Fraser et al., 2009).

Crystallographers thus need robust techniques for datacollection at or near room temperature, and these mustinclude methods for managing radiation damage in thepresent era of third- and fourth-generation synchrotronsources. Here we summarize a range of recent studies thatenhance our understanding of radiation damage. Our discus-sion culminates in the exciting possibility that a large fractionof global damage to unfrozen crystals can be outrun by rapiddata collection using synchrotron source [not just free-elec-tron laser (FEL) source] intensities.

2. Radiation damage processes and timescales

The processes that lead to global radiation damage to proteincrystals, schematically illustrated in Fig. 1, take place on anenormous range of timescales. X-ray–electron and electron–electron interactions (Fig. 1a) occur in femtoseconds, and leadto the generation of hundreds of electrons with energies in the10–100 eV range (Nave & Hill, 2005; Finfrock et al., 2010;Sanishvili et al., 2011) (Fig. 1b). These interact with water andprotein, breaking bonds and creating radicals (Dertinger &Jung, 1970; Coggle, 1983) (Fig. 1c). These primary damageprocesses are at most weakly temperature dependent. Atsufficiently high temperature, atomic and molecular radicalscan diffuse and react with the protein, breaking additionalbonds (Fig. 1d); near room temperature, most radical reactionsshould be complete within a few microseconds (Pryor, 1986).X-ray- and radical-induced bond breaking increases averageatomic separations, creating internal stress that drives latticeexpansion. Local bond breaking and other chemical damagetrigger local conformational relaxation of, for example, sidechains, flexible loops and other more weakly constrainedregions (Fig. 1e). The larger of these local structural relaxa-tions likely take place on the microsecond to millisecondtimescale of conformation changes observed in undamagedprotein (Henzler-Wildman & Kern, 2007; Bourgeois & Weik,2009). As radiochemistry and local relaxation cause everlarger changes to individual protein molecules, the moleculeswill displace and rotate (Fig. 1f), the local lattice may deformand reorient (Fig. 1g), and the build-up of stress may even-tually cause plastic failure and cracking of the crystal (Fig. 1h).These larger motions will proceed on timescales of micro-seconds to hours or even days. In part because they involvesubstantial motions of solvent as the protein molecules movefrom their ideal lattice positions, these timescales will increaseas the temperature is decreased toward the glass transition.

Much of the literature on radiation damage to proteins hasfocused on radiochemistry, but a survey of the broaderradiation damage literature [especially for inorganic materials(Billington, 1962; Dupuy, 1975)] indicates the importance ofstructural relaxations of molecules and the crystal lattice

following chemical damage. Recent crystallographic analysesof the distribution of damage by residue within the unit cell(Juers & Weik, 2011; Warkentin et al., 2012b) suggest that,while solvent-exposed residues are more sensitive than buriedresidues at higher temperature, half of the damage is mani-fested uniformly over the entire structure (Warkentin et al.,2012b) (i.e. it affects surface and buried residues equally),suggesting the importance of lattice scale rather than bond-scale disorder.

3. Temperature dependence of global damage

At T = 100 K, studies to date suggest that all protein crystalsare comparably radiation sensitive, as quantified by how anappropriate metric such as relative B factor or integratedintensity varies with dose (energy deposited by ionizingparticles per kg) (Sliz et al., 2003; Kmetko et al., 2006; Leiros et

radiation damage

8 Matthew Warkentin et al. ! Global radiation damage J. Synchrotron Rad. (2013). 20, 7–13

Figure 1Illustration of some processes involved in the radiation damage cascade.(a) X-ray-induced ejection of a primary photoelectron. (b) Generation ofseveral hundred relatively low-energy ("100 eV) electrons. (c) Bondbreaking leading to internal stress and radical formation. (d) Radicalattack of the protein. (e) Conformation changes of side chains andflexible loops in response to chemical damage. ( f ) Displacement andreorientation of individual damaged molecules. (g) Deformation andreorientation of local lattice domains. (h) Plastic failure and crystalcracking.

milliseconds seconds to hours

local deformations global deformations, and crystal failure

Radiation Damage Interferes with Imaging

XFELs Can Circumvent Radiation Damage

Solem, J. Opt. Soc. Am B, 1986Neutze,…,Hajdu, Nature 2000

Diffraction before destruction

Feasibility: Imaging of Mimivirus (450 nm Diameter)

Seibert,…, Hajdu, Nature 2011Ekeberg,…,Hajdu, Phys. Rev. Lett. 2015

Assembly of 198 diffraction patterns

Reconstructed density at ~ 100 nm resolution

XFELs Enable Studies of Submicron and Radiation Sensitive Crystals

Chapman,…,Spence, Nature 2011

Development of Serial XFEL Crystallography at LCLS

Chapman,…,Spence, Nature 2011

X-ray

Photosystem I(~ 8.6 Å resolution)

Development of Serial XFEL Crystallography at LCLS

Lysozyme at 1.9 Å resolution. Boutet,…,Schlichting, Science 2012

• Mosquitos are vectors of malaria, Dengue fever, Filiariasis, Chikungunya, Zika• Some bacteria express their toxins in the form of submicron-crystals• Binary toxin BinAB produced by Bacillus sphaericus:

X

• The BinAB complex does not re-crystallize after extraction from the cell• Natural submicron-crystals are too small for current synchrotron data collection

XFEL Crystallography of Mosquito Larvicide

Colletier, …, Eisenberg, Nature, 2016

LCLS Enabled De Novo Structure of Mosquito Larvicide

Colletier, …, Eisenberg, Nature, 2016

2.25 Å resolution

LCLS Produced Improved Resolution for GPCR Structures

Structure of the human δ-opioid receptor in complex with a bi-functional DIPP-NH2 peptide at 2.7 Å resolution

Fenalti,…,Stevens, NSMB 2015

Limited Sample: Fixed-target Delivery and Post-Refinement

detector

sample

goniometer

XFEL beam

Cohen,…,Hodgson, PNAS 2014Uervirojnangkoorn,…,Weis, eLife 2015

post-refinementpartially recorded reflection

LCLS Produced Improved Resolution for a Neuronal Complex

• SSRL goniometer at LCLS-XPP• 3.5 Å resolution

Zhou,…,Brunger, Nature 2015

Structure of the SNARE-synaptotagmin Neuronal Complex

Zhou,…,Brunger, Nature 2015

Understanding the Brain in Space and Time

molecules

synapses

Electron cryo-tomography

Brunger et al, unpublished, 2019

X-ray crystallographyZhou,…,Brunger,

Nature, 2015, 2017

Single particle cryo-EMWhite,…,Brunger,

eLife 2018

neuronal networks

Light sheet expansion microscopy

Gao, …,Betzig, Science 2019

brain

Optical imaging of live brainsKim,…,Schnitzer, Cell Reports 2016

Crystal Structures are Still Essential

• Resolution of EM structures is often limited for large complexes

• Interfaces between molecules in large heterogeneous complexes are often poorly resolved in EM structures

• Well ordered crystal structures provide the highest achievable resolution

• Complete diffraction datasets can be collected in seconds

XFELs Enable Time-resolved Studies

Laser-Flash Time-Resolved XFEL Studies

Kupitz,…, Fromme, Nature 2014

flash laser excitation

Photosystem II

Kupitz,…, Fromme, Nature 2014

S0

S2

S3

S4

S1 1 2

34

H+ e- H+ e-

H+ e-H+ e-

O2H2O

H2O

DARK State

Mn4CaO5 cluster at ~ 5 Å resolution

Combined Time-Resolved XFEL Crystallography and XES

Kern,…,Yachandra , Nature 2018

X-ray diffraction(protein structure)

X-ray emission spectroscopy

(oxidation state of catalytic center)

multi-flash laser excitation

Structural Changes at the Photosystem II Catalytic Site

S2 S3 S0S1

X: substrate water?

Kern,…,Yachandra , Nature 2018

Mn4CaO5 cluster

Structures at ~ 2 Å resolution

Picosecond Collective Motions in CO Myoglobin

Barends,…,Schlichting, Science 2015

F(light)-F(dark) difference maps:red: CO-boundgreen: photo dissociated CO

1.8 Å resolution

Bacteriorhodopsin (bR)

Light-driven Proton Pump Bacteriorhodopsin

Trp182

Lys216

Asp212

Asp85

Arg82Tyr57

Retinal

H+

H+

Nogly,…,Standfuss, Science 2018

1.5 Å resolution

Femtosecond Retinal Isomerization

Nogly,…,Standfuss, Science 2018~ 200 fsec time resolution

LCLS 10 Year Bioscience Highlights

• Submicron and radiation-sensitive crystals• Time-resolved structures

END

Optional Slides

IR Laser Perturbations to Study Protein Dynamics

Thompson,…, Fraser, bioRxiv 2018 and unpublished results

Time-resolved difference electron density, 3σ

Temperatureperturbation

Milestones of XFEL Crystallography at LCLS

• Diffraction data and electron density map at 8.7 Å resolution from sub-micron crystals of photosystem I using the GDVN liquid jet (Chapman et al., Nature 2011)

• 1.9 Å resolution XFEL structure of lysozyme (Boutet et al., Science, 2012)• Diffraction data and refinement of natively inhibited variant of cathepsin B at 2.1 Å

resolution reveals carbohydrate groups (Redecke, et al., Science 2013)• Simultaneous X-ray diffraction at 4.1 Å resolution and X-ray emission spectroscopy

study of photosystem II confirms radiation-damage free fsec structure, including Mn4CaO5 cluster (Kern et al., Science 2013)

• De novo phases for a lysozyme crystal structure at 2.1 Å resolution using a strong anomalous scatterer, gadolinium (Barends et al., Nature 2013)

• G Protein-Coupled Receptor (GPCR) XFEL structure at 2.8 Å resolution using a lipid cubic phase injector (Liu et al., Science 2013)

EM Structure of the SNARE/SNAP/NSF/ATP Complex

White,…, Brunger, eLife 2018

Table 1 continued

FL-20S-1 FL-20S-2 FL-20Sfocus-1 FL-20Sfocus-2

All-atom clashscore 4.75 6.63 6.69 5.27

EMRinger score 0.51 0.52 1.91 1.39

MolProbity score 1.53 1.55 1.37 1.38

DOI: https://doi.org/10.7554/eLife.38888.007

Figure 3. Architecture of the 20S complex, composed of NSF (N domains, salmon; D1 domains, cyan; D2 domains, purple), aSNAPs (gold), and the

neuronal SNARE complex (syntaxin-1A, red; synaptobrevin-2, blue; SNAP-25A, green). (A) Sharpened FL-20S-1 map contoured at 4.8 s; N domains for

NSF subunits A–D are visible at this threshold. (B) FL-20S-1 composite model, with nucleotides represented by yellow spheres. (C) The pattern of N

domain engagement with the aSNAP/SNARE complex varies between the FL-20S-1 and FL-20S-2 classes; in the second class, the pattern of

engagement shifts one protomer counter-clockwise about the hexamer axis. The bottom panels show schemas of the configurations. Despite changes

in spire architecture, the split in the D1 ring is found between protomers A and F in both classes, with protomer A furthest from the viewer.

DOI: https://doi.org/10.7554/eLife.38888.008

The following figure supplement is available for figure 3:

Figure supplement 1. Comparison of NSF N domain engagement with aSNAPs and different SNARE complexes.

DOI: https://doi.org/10.7554/eLife.38888.009

White et al. eLife 2018;7:e38888. DOI: https://doi.org/10.7554/eLife.38888 7 of 26

Research article Neuroscience Structural Biology and Molecular Biophysics

Interfaces still require higher resolution

New interaction discovered:

Crystal structures of individual parts were required

Room Temperature Experiments are Enabled by XFELs

Keedy,…,Fraser, eLife, 2015

• Model system: cyclophilin A• Synchrotron structures at

different temperatures• Dynamic features are

preserved in XFEL structure

Correlated X-ray Scattering

Azimuthal angular intensity correlations contain 3D structural information

Qiao,…,Doniach, submitted 2019

I1(φ)I1(φ2+φ)

Δφ

GDP-bound G-protein α subunit

openclosed