A Perspective on Molecular Structure and Bond-Breaking in Radiation Damage in Serial Femtosecond Crystallography

Round 1

Reviewer 1 Report

Caleman et al described radiation damage that can occur in time-reslved SFX using extream intense XFEL. They discussed experimental evidence for local damge and molecular damage effectors. A reviewer of recent research results, as well as general radiation damage, will provide useful information to researchers conducting serial femtosecond crystallography studies. This review has been well organized overall, and it is easy for readers to follow. This manuscript  is recommended to publish a paper after improving some of the items below.

1. In Abstract, “Certain classes of sample are higher risk for radiation damage.” It should be clearly stated for the “Certain classes of sample”

2. The author used the expression "di-sulphide bonding" in the abstract and main text. In general, the expression "disulphide" is appropriate.

3. In Section 2.1: “Heavier elements that are common in proteins such as sulphur and phosphorous, or the metals contained by metalloproteins, have higher ionization rates.” In the case of phosphorus, it is essential for nucleic acid, not protein. The author will need to add the expression "nucleic acid" to the statement.

4. References to the following sentences should be added to improve reader readability and understanding.

In Introduction

- “Local damage is not independent of the spatial arrangement of the atoms. It occurs when there is significant variation in the ionization rates of different elements or when certain ions exhibit reproducible, non-thermal motion.”

-“Local damage is less well-understood because it is element specific and depends on the local molecular environment around each atom.”

- “Time-resolved experiments are particularly sensitive because changes as small as a few tenths of an Angstrom can be interpreted as significant chemical changes.”

-“It is precisely at these very small length scales that we can not yet rule out the impact of local damage.”

-“Local damage is also of importance for anomalous dispersion methods of structure determination.”

Section 2.1.

-“At the wavelengths typically used in XFEL experiments, the most likely interaction between x-rays and matter is photoabsorption.”

-“Photoabsorption rates are highly dependent on atomic number. Light elements such as carbon, nitrogen and oxygen have similar photoabsorption rates. When few heavier elements are present, the photoionization of these light elements can be treated globally with an average scaling the atomic scattering factor.”

Section 2.2.

-“Photoabsorption initiates a chain of subsequent ionization processes that ultimately leads to the destruction of the sample.”

-“Photoabsorption is more likely from the core shells than the valence shells, so that the resulting ions are in excited states.”

 

Section 2.3.

-“Photoelectrons and Auger electrons further ionize the sample, initiating a cascade of low energy electrons (called secondary electrons).”

-“The result is a plasma-like state of ions and a trapped electron gas. The thermalized electron gas is characterized by macroscopic variables such as the density and temperature.”

Section 2.4.

-“There are two types of ion motion: i) thermal motions and ii) motion due to electrostatic repulsion.”

-“Thermal motion of ions increases during the pulse-sample interaction as the trapped electron gas collides with the ions and heats them.”

Section 3.3.

-“The simulation of XFEL damage processes divide into to two main approaches: i) molecular dynamics (MD) and ii) rate equations.”

-“The molecular dynamics approach simulates the motion of each ion and unbound electron in the sample, subject to Coulomb forces.”

-“The rates of ionization are required as input to the simulation to determine the time-dependent ionic charges, the number of unbound electrons and the energy of the unbound electrons.”

Section 4.2.

-“In conventional crystallography, photoionization from the x-rays is relatively sparse and the damage that it generates to the structure occurs on picosecond timescales. In SFX, however, the situation is very different due to the high photon ux. Atoms can be ionized several times during the short x-ray exposure.” - “When a molecule is suddenly ionized several times, it is pushed far from its electronic ground state.

The electronic rearrangement only alters the position of the nuclei as a secondary effect due to the aforementioned influence of electronic states on bonding. However, immediately upon ionization it affects the scattering of the x-rays, and the intensity recorded at the detector. In an XFEL experiment, an average ionization of above 1 per atom is commonly reached.”

-“Carbon, oxygen and nitrogen all have similar photon ionization cross sections and, along with hydrogen, are the most common elements in proteins.”

 

Minors

1) “Figure”, “figure” and “Fig.” are used interchangeably. Should be unified in a journal format.

2) The spacing between sentences and [References] should be corrected.

3) The manuscript should be written in the journal format.

Author Response

Caleman et al described radiation damage that can occur in time-reslved SFX using extream intense XFEL. They discussed experimental evidence for local damge and molecular damage effectors. A reviewer of recent research results, as well as general radiation damage, will provide useful information to researchers conducting serial femtosecond crystallography studies. This review has been well organized overall, and it is easy for readers to follow. This manuscript  is recommended to publish a paper after improving some of the items below.

Answer: We thank the reviewer for their positive assessment of our manuscript.

1. In Abstract, “Certain classes of sample are higher risk for radiation damage.” It should be clearly stated for the “Certain classes of sample”

Answer: This sentence in the abstract has been rewritten to remove the term “certain classes”, as follows: “Proteins containing relatively heavy elements, such as sulfur or metals, have a higher risk for radiation damage.”

2. The author used the expression "di-sulphide bonding" in the abstract and main text. In general, the expression "disulphide" is appropriate.

Answer: This has been corrected.

3. In Section 2.1: “Heavier elements that are common in proteins such as sulphur and phosphorous, or the metals contained by metalloproteins, have higher ionization rates.” In the case of phosphorus, it is essential for nucleic acid, not protein. The author will need to add the expression "nucleic acid" to the statement.

Answer: The expression “nucleic acid” has been added to the sentence. It now reads “Heavier elements that are common in proteins and nucleic acids such as sulphur and phosphorous…”

4. References to the following sentences should be added to improve reader readability and understanding.

Answer: We agree with the reviewer that citations can be added to following sentences. In most cases, citations to articles in our previous bibliography are sufficient - the numbers are marked below. New additions to the bibliography are indicated.

In Introduction

- “Local damage is not independent of the spatial arrangement of the atoms. It occurs when there is significant variation in the ionization rates of different elements or when certain ions exhibit reproducible, non-thermal motion.”

Answer: Reference added. 

-“Local damage is less well-understood because it is element specific and depends on the local molecular environment around each atom.”

Answer: Reference added. 

- “Time-resolved experiments are particularly sensitive because changes as small as a few tenths of an Angstrom can be interpreted as significant chemical changes.”

-“It is precisely at these very small length scales that we can not yet rule out the impact of local damage.”

Answer: Reference added for both comments above. 

-“Local damage is also of importance for anomalous dispersion methods of structure determination.”

Answer: Reference added. 

Section 2.1.

-“At the wavelengths typically used in XFEL experiments, the most likely interaction between x-rays and matter is photoabsorption.” 

Answer: Reference added. 

-“Photoabsorption rates are highly dependent on atomic number. Light elements such as carbon, nitrogen and oxygen have similar photoabsorption rates. When few heavier elements are present, the photoionization of these light elements can be treated globally with an average scaling the atomic scattering factor.”

Answer: We added a reference to figure 1, as this can be seen from the ionization rates. 

 

Section 2.2.

-“Photoabsorption initiates a chain of subsequent ionization processes that ultimately leads to the destruction of the sample.”

Answer: Reference added. 

-“Photoabsorption is more likely from the core shells than the valence shells, so that the resulting ions are in excited states.”

 Answer: Reference added. 

Section 2.3.

-“Photoelectrons and Auger electrons further ionize the sample, initiating a cascade of low energy electrons (called secondary electrons).”

 Answer: Reference added. 

-“The result is a plasma-like state of ions and a trapped electron gas. The thermalized electron gas is characterized by macroscopic variables such as the density and temperature.”

 Answer: Reference added. 

Section 2.4.

-“There are two types of ion motion: i) thermal motions and ii) motion due to electrostatic repulsion.”

Answer: We have tried to clarify what we mean with this, instead of giving a reference. Now the text reads: “As the sample is interacting with the x-rays, the motion of the ions can be affected in two different ways. Either by thermal motion, caused by ion-ion and ion-electron collision, or by electrostatic repulsion, where particles of the same charge repel each other.” 

-“Thermal motion of ions increases during the pulse-sample interaction as the trapped electron gas collides with the ions and heats them.”

Answer:  Reference added. 

Section 3.3.
-“The simulation of XFEL damage processes divide into to two main approaches: i) molecular dynamics (MD) and ii) rate equations.”
-“The molecular dynamics approach simulates the motion of each ion and unbound electron in the sample, subject to Coulomb forces.”

Answer: Reference added for the two comments above. 

-“The rates of ionization are required as input to the simulation to determine the time-dependent ionic charges, the number of unbound electrons and the energy of the unbound electrons.”

 Answer: Reference added

Section 4.2.

-“In conventional crystallography, photoionization from the x-rays is relatively sparse and the damage that it generates to the structure occurs on picosecond timescales. In SFX, however, the situation is very different due to the high photon ux. Atoms can be ionized several times during the short x-ray exposure.”

Answer: We appended the text “, as seen in Figure 1” as our Figure illustrates that multiple ionizations that can occur in XFEL protein crystallography. We also added a refence to an experimental study. 

 - “When a molecule is suddenly ionized several times, it is pushed far from its electronic ground state. The electronic rearrangement only alters the position of the nuclei as a secondary effect due to the aforementioned influence of electronic states on bonding. However, immediately upon ionization it affects the scattering of the x-rays, and the intensity recorded at the detector. In an XFEL experiment, an average ionization of above 1 per atom is commonly reached.”

Answer: Reference added

-“Carbon, oxygen and nitrogen all have similar photon ionization cross sections and, along with hydrogen, are the most common elements in proteins.”

Answer: Reference added

Minors

1) “Figure”, “figure” and “Fig.” are used interchangeably. Should be unified in a journal format.

Answer: It has been changed to Figure everywhere.

2) The spacing between sentences and [References] should be corrected.

Answer: This has been corrected

3) The manuscript should be written in the journal format.

Answer: The manuscript has been transferred to the journal template.

 

Reviewer 2 Report

The manuscript by Coleman et al. takes a look at the types of local radiation damage that occurs during time-resolved XFEL experiments. Being one of the most interesting applications of XFEL in the future, this manuscript is a timely incorporation to these studies. The manuscript is well written, and the introduction presents an overview of radiation damage occurring in protein samples and protein crystals. The last two sections in particular are very informative to the type of atomic processes occurring during damage. Figures are clear and add to the overall clarity of the paper.  

Author Response

The manuscript by Coleman et al. takes a look at the types of local radiation damage that occurs during time-resolved XFEL experiments. Being one of the most interesting applications of XFEL in the future, this manuscript is a timely incorporation to these studies. The manuscript is well written, and the introduction presents an overview of radiation damage occurring in protein samples and protein crystals. The last two sections in particular are very informative to the type of atomic processes occurring during damage. Figures are clear and add to the overall clarity of the paper.  

 

Answer: We thank the reviewer for their positive assessment of our manuscript.

 

Reviewer 3 Report

(NOTE: I am an export of SFX data processing but not an expert on physics; so I reviewed this manuscript from a perspective of a structural biologist) For a protein crystallographer, a practical concern is whether obtained atomic coordinates are affected by radiation damage or not. In early days of SFX, people had believed that SFX structures were completely damage free. This has turned out to be false but many people still believe the damage free myth. This manuscript is a good opportunity to debunk this problem. I enjoyed reading this manuscript and can recommend publication with minor changes to improve readability for structure biologists. In the current order of narrative, protein crystallographers might lose interests in reading further, because it is unclear until later in the manuscript whether damages actually change the refined structure or not. Note that many people wrongly believe atoms cannot move significantly within few tens of femtoseconds of an XFEL pulse. If I understand your manuscript correctly, many things happen: changes in electronic states (1, 3) and movement of atoms (2). (1) The global change in scattering factors is of smaller concern, because the refined atomic coordinates stay the same as long as the scattering factor remains radially symmetric in the reciprocal space. Nonetheless, refined B factors and occupancies might be biased. Local changes in bonding are more problematic: (2) atoms actually move as show in your simulation (Fig 7a). Even when atoms are not moving significantly, (3) re-distribution of bonding electrons (Fig 7b) can give difference peaks in the map (Fig 5a/b) and shift refined atomic coordinates, because we see electron densities, not nuclei themselves, in the X-ray diffraction. These points are of practical importance and should be emphasized and mentioned earlier. > 2.4 > "it will impact the electron density recovered" > 4.1 > "a damage-free result can be considered one in which the XFEL damage does not cause errors in the interpretation or the atomic structure" > 4.2 > "The movement of the nuclei is ultimately what is referred to as radiation damage, since it is their positions that are interpreted as the molecular structure." For example, these sentences should go to the Introduction section. * Minor points > 1. > "permit experiments on membrane proteins at room temperature" This is not limited to membrane proteins. Please remove "membrane". > Introduction > "These correction factors differ from the Debye-Waller (temperature) factors, since they describe system that are far from thermal equilibrium" Protein crystallographers are familiar with global (non-specific) and local (specific) damages in the context of non-XFEL, lower flux crystallography. Global damage in this context can be modeled by B factors because it is caused by gradual loss of crystalline order. Please emphasize the difference between XFEL and synchrotron regimes. > Figure 1 > "Average ionization" What does "Average ionization" mean? Average number of electrons lost? > "the bulk samples trap unbound electrons" Does "bulk samples" mean solvent? Then "bulk solvent" is a more familiar term. > Figure 3 > Ionic scattering factors What do "two s-electrons", "one s-electron", "no s-electron" mean? > 2.1 > "where x-ray energy is deposited at much higher rates 1." Probably "1" is a typo of some citation number? > 2.2 > "which results in a second electron (the Auger electron) being emitted." > 2.3 > "a cascade of low energy electrons (called secondary electrons)" Are "second electron" and "secondary electrons" the same concept or not? > 3.3 > "was conducted by by Hau-Riege et al." Typo. Duplicated "by". > 4.1 > "sulphur dimers" "disulfide bonds" is a more familiar term. > "The carbonyl groups show a loss of electron density in the ring and an asymmetric increase of density near the exterior" What do "the ring" and "the exterior" of carbonyl groups mean? Finally, I recommend the original data of Fig 7 (input and output of QM and MD calculation programs) to be deposited in a public data bank (e.g. Zenodo).

Author Response

(NOTE: I am an export of SFX data processing but not an expert on physics; so I reviewed this manuscript from a perspective of a structural biologist) For a protein crystallographer, a practical concern is whether obtained atomic coordinates are affected by radiation damage or not. In early days of SFX, people had believed that SFX structures were completely damage free. This has turned out to be false but many people still believe the damage free myth. This manuscript is a good opportunity to debunk this problem. I enjoyed reading this manuscript and can recommend publication with minor changes to improve readability for structure biologists. 

 

In the current order of narrative, protein crystallographers might lose interests in reading further, because it is unclear until later in the manuscript whether damages actually change the refined structure or not. Note that many people wrongly believe atoms cannot move significantly within few tens of femtoseconds of an XFEL pulse. If I understand your manuscript correctly, many things happen: changes in electronic states (1, 3) and movement of atoms (2). (1) The global change in scattering factors is of smaller concern, because the refined atomic coordinates stay the same as long as the scattering factor remains radially symmetric in the reciprocal space. Nonetheless, refined B factors and occupancies might be biased. Local changes in bonding are more problematic: (2) atoms actually move as show in your simulation (Fig 7a). Even when atoms are not moving significantly, (3) re-distribution of bonding electrons (Fig 7b) can give difference peaks in the map (Fig 5a/b) and shift refined atomic coordinates, because we see electron densities, not nuclei themselves, in the X-ray diffraction. These points are of practical importance and should be emphasized and mentioned earlier. > 2.4 > "it will impact the electron density recovered" > 4.1 > "a damage-free result can be considered one in which the XFEL damage does not cause errors in the interpretation or the atomic structure" > 4.2 > "The movement of the nuclei is ultimately what is referred to as radiation damage, since it is their positions that are interpreted as the molecular structure." For example, these sentences should go to the Introduction section. 

 

Answer: We thank the reviewer for this suggestion to increase the interest of our manuscript to the structural biology community. The sentences referred to throughout the manuscript are unchanged, but a new paragraph has been added near the end of the introduction to emphasise these important points early in the manuscript. The new paragraph reads:

 

“Radiation damage becomes problematic for structural biology when it impacts the structural interpretation of the electron density. Most of the x-rays scattered to high angles interact with bound electrons and, hence, the recovered electron density is primarily determined by the location of the ions. The evidence so far for ``damage free" experiments with XFELs~\cite{Boutet2012,Chapman2014} does not rule out local reproducible ion motion in the vicinity of a heavier element, so called ``damage hot spots", at least on sub-{\AA}ngstr{\"o}m scales. An ongoing concern is, thus, whether local damage could impact the interpretation of structure and dynamics of metal clusters in time-resolved studies of metaloproteins.”

 

* Minor points > 1. > "permit experiments on membrane proteins at room temperature" This is not limited to membrane proteins. Please remove "membrane". 

 

Answer: The word “membrane” has been removed.

 

> Introduction > "These correction factors differ from the Debye-Waller (temperature) factors, since they describe system that are far from thermal equilibrium" Protein crystallographers are familiar with global (non-specific) and local (specific) damages in the context of non-XFEL, lower flux crystallography. Global damage in this context can be modeled by B factors because it is caused by gradual loss of crystalline order. Please emphasize the difference between XFEL and synchrotron regimes. 

 

Answer: We thank the reviewer for this suggested improvement. We have added the following text to the end of the 2nd paragraph of the introduction:

“Essentially, the Debye-Waller (temperature) factors from synchrotron-based protein crystallography model the thermal motion of atoms at a fixed temperature, whereas XFEL correction factors model the rapid heating of the sample by the XFEL pulse. The significance of x-ray induced heating effects depends on pulse duration, pulse energy and the wavelength~\cite{Caleman2015-jd}. Early XFEL protein crystallography experiments identified beam conditions for which the heating effects were highly significant~\cite{Barty2012} and others for which they could be effectively ignored~\cite{Boutet2012}.”

 

> Figure 1 > "Average ionization" What does "Average ionization" mean? Average number of electrons lost? 

 

Answer: Yes it does mean the average number of electrons lost. The bold subtitle for the figure has been reworded to read:

“Average number of electrons lost in a Photosystem I (PSI) crystal vs time.”

 

> "the bulk samples trap unbound electrons" Does "bulk samples" mean solvent? Then "bulk solvent" is a more familiar term. 

 

Answer: This sentence in the 2nd last paragraph of the introduction has been reworded to improve clarity. The term “bulk samples” has been replaced by “protein crystals”.

 

> Figure 3 > Ionic scattering factors What do "two s-electrons", "one s-electron", "no s-electron" mean?

 

Answer: A sentence has been added to the caption of Figure 3 to clarify this term:

“The term "s-electrons" in the legend refers to the number of electrons occupying the 1s shell of the ion.”

 

 > 2.1 > "where x-ray energy is deposited at much higher rates 1." Probably "1" is a typo of some citation number? 

 

Answer: This was intended to be a reference to Figure 1. The text “(see Figure 1)” has been appended to the sentence.

 

> 2.2 > "which results in a second electron (the Auger electron) being emitted." 

> 2.3 > "a cascade of low energy electrons (called secondary electrons)" Are "second electron" and "secondary electrons" the same concept or not? 

 

Answer: “Secondary electrons” is a technical term as defined in the manuscript. The term “second electron” only appears once in the manuscript in the context of Auger decay and is meant literally in common language to mean a second electron in addition to the photoelectron was emitted by the ionized atom during the decay process. 

 

Our  view is these terms are correctly used throughout the manuscript.

 

> 3.3 > "was conducted by by Hau-Riege et al." Typo. Duplicated "by". 

 

Answer: This has been corrected.

 

> 4.1 > "sulphur dimers" "disulfide bonds" is a more familiar term. 

 

Answer: The term suggested by the reviewer is now used throughout the manuscript.

 

> "The carbonyl groups show a loss of electron density in the ring and an asymmetric increase of density near the exterior" What do "the ring" and "the exterior" of carbonyl groups mean? 

 

Answer: The term “carbonyl group” was a typo in this sentence, and should have stated “aromatic side chains”. The sentence has been rewritten as follows:

“Elsewhere in the structure, the aromatic side chains show a loss of electron density in the phenyl ring and an asymmetric increase of density near the exterior of the ring.”

 

Finally, I recommend the original data of Fig 7 (input and output of QM and MD calculation programs) to be deposited in a public data bank (e.g. Zenodo).

 

Answer: We have created a Supplementary Material where we have put the input and output files for both the QM/MM and the NLTE simulation.