Monitoring the Production of High Diffraction-Quality Crystals of Two Enzymes in Real Time Using In Situ Dynamic Light Scattering

Round 1

Reviewer 1 Report

- Line 35: "Databank" should be written "Data Bank".

- Line 38: put the F of "Free" in lower case.

- Line 39: past tense the verb enable.

- Lines 56-59: "The concept of such a system emerged in the nineties in the frame of a European research consortium on crystal growth (European Initiative for Biocrystallogenesis) and was developed in the context of the OptiCryst European consortium [10]."

This sentence should be clarified, i.e. were the two European consortia following each other?

- Line 63: the authors should only refer to panels A and A' of Figure 1 whereas panels B and C should be referred to in other places in the text.

- Lines 64-66: assuming that water is the only volatile chemical species? This should be clarified.

- Lines 66-67: "The instrument also provides diagnostic means to track the drop content along the experiment"

This statement should also be clarified.

- Lines 69-70: "as well as of nanocrystals by dynamic light diffusion (DLS)". Either suppress "of" or complete by "that", etc.

- Lines 71-72: "Experimental conditions can be varied and monitored in real time to stabilize a specific phase or drive the system in the phase diagram towards another phase."

This sentence is a repetition of what is said two sentences above.

- Line 77: replace "physical-chemical" by "physicochemical".

- Line 144: what is meant by "in water (Roche)"? Are the authors talking about milliQ water?

- Lines 145-146: "Stock solutions were filtered on a 0.22 μm Ultrafree-MC membrane (Millipore) prior to concentration measurement." is more or less a repetition of the sentence at line 139.

- Line 162: "for storage and/or incubation". Is there any difference between storage and incubation? To be clarified.

- Line 163: "Standard DLS measurements". It is not clear whether the same DLS equipment is used in the XtalController setup. This should be made clear.

- Paragraph "2.4 Crystal analysis" starting at line 171: the resolution limit of PhaCCA crystals is not given whereas that of lysosyme crystals is mentioned. It should be indicated too.

- Lines 216-219: "After 48h, the monitoring is stopped and the cover slip is transferred from the instrument to a classical 24-well Linbro plate to be stored in equilibrium with a reservoir containing a crystallant solution at the same concentration as that reached at the completion of the protocol (i.e. 1 M diammonium phosphate, 0.1 M ammonium acetate pH 4.5)."

The time scale for the experiment in the XtalController setup as indicated in Figure 2A and B (total time 40,000 sec, ~11 h) is less than the 48 h (172,800 sec) mentioned in the text. What does occur in the meantime?

- Lines 232-234: "If the process is too fast and evolves towards precipitation, the whole system can be driven back to lower concentrations favoring the dissolution of precipitates and the solubilization of monomers."

In essence, folded monomers should be considered soluble. Is "re-solubilization" meant here? This should be further explained.

- Lines 251-252: "The close-up view on the left hand side shows typical PhaCCA crystals used for data collection (Table 1)." It rather seems that this is the right hand side that shows these crystals.

- Lines 255-256: for sake of clarity, the four cycles of drop concentration/dilution should be indicated on Figure 3A.

- Line 265: Replace "S. Engilberge observed" by "we observed", which is more adapted to the style of a scientific article.

- Lines 277-278: "Figure 4 shows that the addition of Tb-Xo4 triggers the instantaneous formation of particles of larger size and this phenomenon of association, which is not observed upon water addition, leads to the growth of large crystals after weeks of incubation."

Two verbs (i.e. trigger, lead) should be given in the past tense.

- Line 281: is Figure 5 meant here? Besides, there is no reference to panel A in the text.

- Lines 294-295: 20,000 sec is not 8 h but 5.6 h. There is some inconsistency between numbers given in figure 4 and its legend.

- Lines 298-308: the whole paragraph should be rephrased to take into account the above remark and replacing "left" and "middle" by "A" and "B". Reference to panel C should also be introduced in the main text.

- Table 1: true PDB IDs should be given rather than "6xxx" and "6yyy".

- Lines 372-373: "Two alternate positions are observed but their ligand is not visible due to low occupancy."

Do the authors mean two other sites or are there real alternate positions of the same site. With that respect, the following sentence (lines 373-374) should also be clarified. The corresponding section in the main text is clearly in favor of three different binding sites (one major site and two minor sites).

- Line 388: "- ensure the reproducibility of crystals in the context of structural biology investigations,"

Why not in other contexts, e.g. rational drug design, fragment-based screening, etc where many crystals are required?

- Line 390: "diffraction analyses using X-ray free electron lasers and CryoEM, or, conversely, to promote the"

"μED" rather than "CryoEM"?

- Lines 392-394: "More generally, this type of versatile instruments provides a more rational approach to crystallization and a great alternative to extensive blind screening. We do believe that this technology has a bright future."

Thus, it would be very helpful to the reader (i.e. crystallographers) to know a bit more of technical details about the instrument and a rough estimate of its cost.

Author Response

Reviewer 1

**********

Comments and Suggestions for Authors

We thank Reviewer 1 for her/his constructive comments, which have all been taken into account. Our modifications appear in red in the revised manuscript and in our answers below.

- Line 35: "Databank" should be written "Data Bank".

Done

- Line 38: put the F of "Free" in lower case.

Done

- Line 39: past tense the verb enable.

We changed it to “has enabled” since it remains true

- Lines 56-59: "The concept of such a system emerged in the nineties in the frame of a European research consortium on crystal growth (European Initiative for Biocrystallogenesis) and was developed in the context of the OptiCryst European consortium [10]."

This sentence should be clarified, i.e. were the two European consortia following each other?

We added corresponding periods: “The concept of such a system emerged in the nineties in the frame of a European research consortium on crystal growth (European Bio-crystallogenesis Initiative, 1998-2000) and was developed in the context of the OptiCryst European consortium (2006-2010)“

- Line 63: the authors should only refer to panels A and A' of Figure 1 whereas panels B and C should be referred to in other places in the text.

Done

- Lines 64-66: assuming that water is the only volatile chemical species? This should be clarified.

We precised: “by evaporation of volatile chemicals (generally water)”

- Lines 66-67: "The instrument also provides diagnostic means to track the drop content along the experiment"

This statement should also be clarified.

We modified the paragraph as follows: “The instrument provides diagnostic means to track the drop content along the experiment: the early occurrence of association events leading to nucleation, as well as of nanocrystals can be detected in real time by dynamic light scattering (DLS) and the growth of crystals by video microscopy as soon as they reach a size exceeding a few microns. The XtalController also provides means to navigate in the phase diagram, from an undersaturated solution to a supersaturated state leading to crystal growth or precipitation. Hence, a specific phase may be stabilized or the system may be driven in the phase diagram towards another phase by varying experimental conditions in real time using the piezo injectors.”

- Lines 69-70: "as well as of nanocrystals by dynamic light diffusion (DLS)". Either suppress "of" or complete by "that", etc.

done

- Lines 71-72: "Experimental conditions can be varied and monitored in real time to stabilize a specific phase or drive the system in the phase diagram towards another phase."

This sentence is a repetition of what is said two sentences above.

This has been changed, see above.

- Line 77: replace "physical-chemical" by "physicochemical".

Done

- Line 144: what is meant by "in water (Roche)"? Are the authors talking about milliQ water?

Correct

- Lines 145-146: "Stock solutions were filtered on a 0.22 μm Ultrafree-MC membrane (Millipore) prior to concentration measurement." is more or less a repetition of the sentence at line 139.

We changed the text to avoid the repetition but still precised that Lysozyme stock solutions were filtered.

- Line 162: "for storage and/or incubation". Is there any difference between storage and incubation? To be clarified.

We kept “incubation”, which, in our mind, means a long term storage with potential changes in the outcome of the experiment.

- Line 163: "Standard DLS measurements". It is not clear whether the same DLS equipment is used in the XtalController setup. This should be made clear.

We changed the sentence as follows: “In parallel to DLS measurements performed inside the XtalController, a benchtop Nanostar light scattering instrument (Wyatt Technology, Inc.) was used to record the effect of Tb-Xo4 on lysozyme.”

- Paragraph "2.4 Crystal analysis" starting at line 171: the resolution limit of PhaCCA crystals is not given whereas that of lysosyme crystals is mentioned. It should be indicated too.

Done: “yielding a dataset at a resolution of 2.28 Å”

- Lines 216-219: "After 48h, the monitoring is stopped and the cover slip is transferred from the instrument to a classical 24-well Linbro plate to be stored in equilibrium with a reservoir containing a crystallant solution at the same concentration as that reached at the completion of the protocol (i.e. 1 M diammonium phosphate, 0.1 M ammonium acetate pH 4.5)."

The time scale for the experiment in the XtalController setup as indicated in Figure 2A and B (total time 40,000 sec, ~11 h) is less than the 48 h (172,800 sec) mentioned in the text. What does occur in the meantime?

In Figure 2, we zoomed on a time window corresponding the first 11h because the situation did not evolve between 11h and 48h. This is now precised in the legend:

“Panels A) and B) show the experimental records over the first 40000 sec (~11 h). No visible change occurred in the plots between t = 11 and 48 h when the experiment was transferred to a Linbro plate.”

- Lines 232-234: "If the process is too fast and evolves towards precipitation, the whole system can be driven back to lower concentrations favoring the dissolution of precipitates and the solubilization of monomers."

In essence, folded monomers should be considered soluble. Is "re-solubilization" meant here? This should be further explained.

Correct

- Lines 251-252: "The close-up view on the left hand side shows typical PhaCCA crystals used for data collection (Table 1)." It rather seems that this is the right hand side that shows these crystals.

Correct

- Lines 255-256: for sake of clarity, the four cycles of drop concentration/dilution should be indicated on Figure 3A.

done

- Line 265: Replace "S. Engilberge observed" by "we observed", which is more adapted to the style of a scientific article.

done

- Lines 277-278: "Figure 4 shows that the addition of Tb-Xo4 triggers the instantaneous formation of particles of larger size and this phenomenon of association, which is not observed upon water addition, leads to the growth of large crystals after weeks of incubation."

Two verbs (i.e. trigger, lead) should be given in the past tense.

Done

- Line 281: is Figure 5 meant here? Besides, there is no reference to panel A in the text.

The referee is right, two figures should be mentioned here to illustrate two distinct ideas: “the monomer seems to be converted in a slightly larger entity (Figure 5), possibly bridged by the nucleant (Figure 6B,C)”

- Lines 294-295: 20,000 sec is not 8 h but 5.6 h. There is some inconsistency between numbers given in figure 4 and its legend.

Same problem as in Figure 2. We changed the legend and added the following sentence: “No visible change was observed afterwards and, as the drop remained clear (C), it was transferred after 8 h to a Linbro plate for incubation.”

- Lines 298-308: the whole paragraph should be rephrased to take into account the above remark and replacing "left" and "middle" by "A" and "B". Reference to panel C should also be introduced in the main text.

Done

- Table 1: true PDB IDs should be given rather than "6xxx" and "6yyy".

Done

- Lines 372-373: "Two alternate positions are observed but their ligand is not visible due to low occupancy."

Do the authors mean two other sites or are there real alternate positions of the same site. With that respect, the following sentence (lines 373-374) should also be clarified. The corresponding section in the main text is clearly in favor of three different binding sites (one major site and two minor sites).

The referee is right, the description was misleading: we actually observe a major site with a Tb3+ ion and its ligand, and a minor site with two alternate positions showing lower occupancy. We changed the text to clarify this point.

“The dataset collected at 1.51 Å resolution (Table 1) allowed the identification of three anomalous peaks corresponding to one major Tb³⁺ site for which the ligand could be built, and one minor site with two alternate positions (Figure 6C). These sites are consistent with those described in PDBid 6F2I determined with an HEWL crystal grown in 0.8 M NaCl and 100 mM Tb-Xo4 [20]. The low occupancy of Tb³⁺ positions corresponding to the minor site and the absence of visible ligand are also consistent with the low concentration of Tb-Xo4 (10 mM) which provides a ratio of two Tb-Xo4 complexes for one lysozyme molecule.”

- Line 388: "- ensure the reproducibility of crystals in the context of structural biology investigations,"

Why not in other contexts, e.g. rational drug design, fragment-based screening, etc where many crystals are required?

excellent suggestion!

- Line 390: "diffraction analyses using X-ray free electron lasers and CryoEM, or, conversely, to promote the"

"μED" rather than "CryoEM"?

Done

- Lines 392-394: "More generally, this type of versatile instruments provides a more rational approach to crystallization and a great alternative to extensive blind screening. We do believe that this technology has a bright future."

Thus, it would be very helpful to the reader (i.e. crystallographers) to know a bit more of technical details about the instrument and a rough estimate of its cost.

References giving a detailed description are already indicated in the introduction [11, 16, 17, 18].

Submission Date

25 December 2019

Date of this review

05 Jan 2020 15:41:32

Reviewer 2 Report

The paper “Monitoring the production of high diffraction-quality crystals of two enzymes in real time using in situ dynamic light scattering” clearly describes controlled crystallisation experiment and informs readers on the abilities of novel instrument XtalController, which allow to detect early nucleation and produce high quality crystals, which makes crystallisation experiment more reproducible.

The authors used known crystallisation conditions for Planococcus halocryophilus CCA-adding enzyme  as the starting point and successfully improved crystal  size and increased the diffraction properties of the crystals. They follow it up with another type of experiment, HEWL crystals nucleation by Tb-Xo4 chemical. It will be interesting to see other applications for this instument, for example, growing very bid crystals for neutron diffraction. The paper is very clear and easy to follow up and can be published after minor changes.

Minor corrections

Page 4 line 69-70 ‘dynamic light diffusion’ (DLS) is dynamic light scattering (DLS)

Table 1 , Please change the resolution limits to conventional from low resolution to high resolution

Resolution range (.) 47 – 2.28(2.42 – 2.28) 35 – 1.51 (1.60 – 1.51)

You can also include Rp.i.m in addition to Rmeas, which may show improved merging statistics in the last shell, as it will take redundancy into account.

Author Response

Reviewer 2

**********

Comments and Suggestions for Authors

We thank Reviewer 2 for her/his constructive comments, which have all been taken into account. Our modifications appear in red in the revised manuscript and in our answers below.

The paper “Monitoring the production of high diffraction-quality crystals of two enzymes in real time using in situ dynamic light scattering” clearly describes controlled crystallisation experiment and informs readers on the abilities of novel instrument XtalController, which allow to detect early nucleation and produce high quality crystals, which makes crystallisation experiment more reproducible.

The authors used known crystallisation conditions for Planococcus halocryophilus CCA-adding enzyme as the starting point and successfully improved crystal size and increased the diffraction properties of the crystals. They follow it up with another type of experiment, HEWL crystals nucleation by Tb-Xo4 chemical. It will be interesting to see other applications for this instument, for example, growing very bid crystals for neutron diffraction. The paper is very clear and easy to follow up and can be published after minor changes.

Minor corrections

Page 4 line 69-70 ‘dynamic light diffusion’ (DLS) is dynamic light scattering (DLS)

Done

Table 1 , Please change the resolution limits to conventional from low resolution to high resolution

Resolution range (.) 47 – 2.28(2.42 – 2.28) 35 – 1.51 (1.60 – 1.51)

Done

You can also include Rp.i.m in addition to Rmeas, which may show improved merging statistics in the last shell, as it will take redundancy into account.

Done

Submission Date

25 December 2019

Date of this review

04 Jan 2020 00:35:28

Reviewer 3 Report

Very interesting paper

Acceptable after minor revision.

My proposition of changes:

Line 2

Title: In my opinion "in real time" and "in situ" are synonimus

Line 63 Please specify 70 pl droplets

Line 70 Please change "dynamic ligt scattering" is more correct

Line 145 Please explain for potential readers why concentrations rangin from 25-71 mg/ml. It may be useful for them.

Line 162 Linbro plate. Please refine (specify).

Line 165 To this line sybol μl was used, now μL (please standarize throughout).

Line 194 2† (should be as an index)

Line 221

should be ...see chapter 3.3

Line 287 should be ...dot at the end of sentence

Line 292 As the first should be A panel described not B (in sequence, in turn)

Line 295 20000s not equal 8h (this is confusing)

Line 338 Tb 2† (should be as an index)

Line 312 shold be opposite ..50 and 71...not 71 and 50

Line 438 double "in"...

 

Comments for author File: Comments.pdf

Author Response

Reviewer 3

**********

We thank Reviewer 3 for her/his constructive comments, which have all been taken into account. Our modifications appear in red in the revised manuscript and in our answers below.

My proposition of changes:

Line 2

Title: In my opinion "in real time" and "in situ" are synonimus

We would like to keep the original title because “in situ” measurements (DLS here) do not necessarily mean that they are performed “in real time”. It is indeed the advantage of this instrument to provide “real time” (i.e. continuous) DLS monitoring thanks to an integrated DLS system (i.e. in situ).

Line 63 Please specify 70 pl droplets

done

Line 70 Please change "dynamic ligt scattering" is more correct

done

Line 145 Please explain for potential readers why concentrations rangin from 25-71 mg/ml. It may be useful for them.

This is explained in section 3.2, lines 305-308

Line 162 Linbro plate. Please refine (specify).

Done

Line 165 To this line sybol μl was used, now μL (please standarize throughout).

Done

Line 194 2† (should be as an index)

Done

Line 221

should be ...see chapter 3.3

Done

Line 287 should be ...dot at the end of sentence

Done

Line 292 As the first should be A panel described not B (in sequence, in turn)

the legend was modified accordingly.

Line 295 20000s not equal 8h (this is confusing)

Done. See answers to Referee 1.

Line 338 Tb 2† (should be as an index)

Done

Line 312 shold be opposite ..50 and 71...not 71 and 50

Done

Line 438 double "in"...

Done

peer-review-6056534.v1.pdf

Submission Date

25 December 2019

Date of this review

03 Jan 2020 15:35:15