EMBO reports - Peer Review Process File - EMBO-2016-43521
Manuscript EMBO-2016-43521
Structural basis for ligand capture and release by the
endocytic receptor ApoER2
Hidenori Hirai, Norihisa Yasui, Keitaro Yamashita, Sanae Tabata, Masaki Yamamoto, Junichi
Takagi and Terukazu Nogi
Corresponding author: Terukazu Nogi, Graduate School of Nanobioscience, Yokohama City
University
Review timeline:
Submission date:
Editorial Decision:
Revision received:
Editorial Decision:
Revision received:
Accepted:
14 October 2016
18 November 2016
17 February 2017
02 March 2017
09 March 2017
15 March 2017
Editor: Martina Rembold
Transaction Report:
(Note: With the exception of the correction of typographical or spelling errors that could be a source of ambiguity,
letters and reports are not edited. The original formatting of letters and referee reports may not be reflected in this
compilation.)
1st Editorial Decision
18 November 2016
Thank you for the submission of your research manuscript to our journal. We have now received the
full set of referee reports that is copied below.
As you will see, the referees acknowledge the potential interest of the findings. However, referee 1
has a number of suggestions for how the study should be strengthened. As these are listed below I
prefer not to repeat them here but I think that all of them should be addressed. Importantly, referee 1
points out that caution should be applied when translating the findings obtained with ApoER2 to
LDLR as the splice form of ApoER2 used lacks the LA4 and LA5 repeats present in LDLR. Please
consider this and the referee's other comments on the R2193A/K2194A mutation and the role of the
beta-propeller in the release of reelin in the revision and make sure that you discuss your results in
the most appropriate and careful manner.
Given these constructive comments, we would like to invite you to revise your manuscript with the
understanding that the referee concerns (as detailed above and in their reports) must be fully
addressed and their suggestions taken on board. Please address all referee concerns in a complete
point-by-point response. Acceptance of the manuscript will depend on a positive outcome of a
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Revised manuscripts should be submitted within three months of a request for revision; they will
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otherwise be treated as new submissions. Please contact us if a 3-months time frame is not sufficient
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REFEREE REPORTS
Referee #1:
This manuscript is a follow-up study from the author's 2010 paper showing the co-structure of the
first LA repeat of apoER2 bound to the R56 region of reelin. The current paper now provides a
structure encompassing much of the ectodomain of apoER2 in complex with the R56 region and a
structure of the LA1-2 region alone. The co-structure shows new contacts of the R56 fragement with
both LA2 and the beta propeller of apoER2. A key observation is that the reelin-bound apoER2 is in
a conformation consistent with the "closed" conformation of LDLR family members. This is a
surprise because the LDLR is thought to bind LDL in an "open" conformation and only adopt a
"closed" conformation in the acidic lumens of endosomes as part of the lipoprotein dissociation
process. The authors' observations suggest that other LDLR family members may also bind their
ligands in a "closed" conformation and thus may place these associations in a context that is poised
for acid-dependent acceleration of ligand release via the beta propeller region.
Specific comments:
Caution should be exercised when translating the results of the apoER2-reelin data to the LDLRLDL system. First, the apoER2 used in the structure determination lacks three LA repeats (LA4-6).
This is based upon an alternatively spliced product of apoER2, which does not have a correlate in
the LDLR. The low resolution cryoEM structure of LDL in complex with the LDLR does suggest
association of the beta propeller with LDL; however, deletion of the beta propeller has no effect on
the binding affinity of the LDLR for LDL. The cryoEM structure was not at sufficient resolution to
discern the LA repeats on the surface of LDL and it is not clear how the beta propeller is positioned
relative to the LA repeats ("open" vs "closed") when LDL is bound.
Fig 1 indicates that SPR data presented is mean of triplicates, but no error bars are provided. Please
provide error bars and N values for all SPR data in Fig 1, 2, and 5.
The relatively small difference in affinity (12 nM (wt) vs 38 nM (E108Q)) and the poor electron
density for the LA2 module in the structure suggest that the contribution of LA2 to reelin binding is
minimal. Please indicate the p-value for the affinity difference in affinity in Fig 2 or in Table 2.
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It is not clear to me whether use of the LA1-2 structure to place LA2 in the co-structure is
informative, given the flexibility between LA repeats. How did the rigid-body placement of LA2
change/improve the R-factor for the overall structure?
Affinity of the R2193A/K2194 (160 nM) for R56 is weaker than that for LA1 alone in complex with
R56 (73 nM), suggesting that the R2193A/K2194A mutations have an effect beyond that of an
interaction with LA2. A contact-independent effect may be responsible for the entire affinity
difference. Work with other tandemly repeated structures have shown that the folded state of a
repeat can be affected by the folded state of adjacent repeat even when each repeat is capable of
folding in isolation. Could the effect of the two mutations be simply a free energy consequence of a
difference in domain folding or orientation?
The authors identify a contact between R5A of reelin and the beta propeller of apoER2 (contact 3),
but do not show a function for this interaction. Authors show that the affinity at neutral pH (7.5) of
R56 for apoER2 ECD is the same whether or not the R5A loop is present or not. Of interest is
whether the affinity of the interaction is also the same at acidic pH (5.5). The beta propeller may be
positioned as it is in the structure, not to influence the reelin interaction at neutral pH, but rather to
influence the interaction at acidic pH.
The proposal that H96 and H99 accelerate R56 release at acidic pH should be tested using either
alanine or asparagine substitutions. Binding and release at pH 7.5 and 5.5 of LA1-2 with R56 should
also be done and compared to the apoER2 ECD interaction. If H96 and H99 mediate acid-dependent
release then the LA1-2 region should exhibit a similar off-rate at pH 5.5 as seen with the apoER2
ECD. This experiment is important because it tests whether reelin release requires the beta propeller.
A finding that the beta propeller is not required would be consistent with binding and release of
reelin from the full-length apoER2 and the apoER2 ECD, which lacks LA4-6. Assuming domain
orientation is similar in full-length apoER2 and the apoER2 ECD, the beta propeller would interact
with LA1-2 of apoER2 ECD, but LA5-6 in full-length apoER2.
The statement "Modulation of binding affinity using histidine residues as the pH-sensor has also
been proposed for regulation of the interactions between RAP and LDLR-related proteins" is
misleading with respect to the author's proposed role for H96 and H99 in R56 release. Release of
RAP from LDLR family members is thought to mostly involve an acid-dependent destabilization of
RAP mediated by protonation of histidines of RAP, not the bound LDLR family member. RAP
interacts with LA repeat pairs of many LDLR family members, few of which have histidines
arranged as they are in LA2 of apoER2.
The authors propose that the beta propeller of apoER2 contacts the interaction surface on LA1-2 for
reelin in analogy to the interaction of the LDLR beta propeller with the LA4-5 region of the LDLR.
The authors also propose that the beta propeller interaction accelerates release of reelin from
apoER2. It is possible that the same surface on LA1-2 is used for both reelin and beta propeller
binding, but it is not clear how such an interaction would accelerate the off-rate. Use of the same
surface would imply a competitive mechanism and competitive inhibitors do not accelerate off-rates.
RAP for example acts as a competitive inhibitor for ligand binding to many LDLR family members,
but addition of RAP does not accelerate the rate of release of ligands already bound to these
receptors. A competitive interaction might however be important to prevent re-binding of reelin to
apoER2 in endosomes, which because of their small luminal volumes, may develop high
concentrations of free reelin as a consequence of acid-dependent release.
Referee #2:
This is a really beautiful piece of work that combines high quality SPR data with a lot of
mutagenesis and crystallography to elucidate a physiologically relevant structure between two
highly flexible molecules, apoER2 and reelin. The authors go to heroic lengths to fit the
crystallography data, because these proteins contain a lot of flexible regions. Ordinarily I would
worry about such approaches, but the approach in this case is rigorous and tested by follow-up
mutagenesis. The report adds a lot to our understanding of how LDL receptors bind their ligands.
Importantly, it reveals new interfaces that were suspected but structurally unproven by previous
work. The observation that some of the LAs adopt still different orientations relative to one another
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to achieve this binding interface with several different sub-interfaces is really interesting. The results
help explain some un-explained observations in the field and will be very useful for future work in
designing inhibitors of LDLR interactions. A beautiful study and well-worthy of publication in
EMBO reports!
1st Revision - authors' response
17 February 2017
Point-by-point response to the comments by reviewer #1:
We would like to express our gratitude to this reviewer for the important comments on our
manuscript. We performed several additional experiments to validate our model according to the
reviewer’s suggestions. We obtained some data supporting our model in those experiments and
revised the manuscript as follows.
Reviewer’s comment:
Caution should be exercised when translating the results of the apoER2-reelin data to the LDLRLDL system. First, the apoER2 used in the structure determination lacks three LA repeats (LA4-6).
This is based upon an alternatively spliced product of apoER2, which does not have a correlate in
the LDLR. The low resolution cryoEM structure of LDL in complex with the LDLR does suggest
association of the beta propeller with LDL; however, deletion of the beta propeller has no effect on
the binding affinity of the LDLR for LDL. The cryoEM structure was not at sufficient resolution to
discern the LA repeats on the surface of LDL and it is not clear how the beta propeller is positioned
relative to the LA repeats ("open" vs "closed") when LDL is bound.
Response:
We also think that we should not simply translate our results to the LDLR system. As the reviewer
pointed out, major splicing variants of ApoER2 have different numbers of LA modules compared
with LDLR. Therefore, we modified the manuscript as follows:
(Page 4, line 17) The ectodomain of ApoER2 consists of the same set of structural modules in the
same order as LDLR although splicing variants containing different numbers of the LA repeats in
the ligand-binding domain are identified for ApoER2
(Page 18, line 8) Although the number of LA repeats is different between LDLR and the splicing
variant of ApoER2 studied in the current work, our ApoER2 structure in complex with reelin R56 is
expected to serve as a structural basis for analyzing the pH-dependent ligand uptake mechanism
conserved among the LDLR family members.
In addition, we also added a citation of the above-mentioned EM study of LDLR in the
Introduction. As the reviewer stated above, it was reported in that study that the YWTD repeat
assuming a β-propeller fold interacts with LDL, but the resolution was not sufficient and the precise
conformation of the entire ectodomain remains to be resolved. It is this very problem that we have
attempted to address in the current work by using ApoER2 as a surrogate. With this approach, we
elucidated for the first time a conformation of the ectodomain in the ligand-binding state that is
representative for LDLR and its close homologues.
Reviewer’s comment:
Fig 1 indicates that SPR data presented is mean of triplicates, but no error bars are provided.
Please provide error bars and N values for all SPR data in Fig 1, 2, and 5.
Response:
In the SPR analysis, we processed the sensorgrams independently from triplicate experiments for
each combination of ApoER2 and reelin constructs. For each sample, we estimated three KD values
by steady-state affinity analysis, and then calculated the mean and standard deviation of the KD
values from the three results. For each set of triplicates, one representative set of sensorgram
readings and the fitted curve is shown in Figs 1 and 4. The curves shown in the figure were not
plotted using the mean Req values of the triplicates, because this alternative method does not allow
us to calculate the error in KD from independent fits of each replicate, even though it allows us to
calculate the errors for each individual Req value. In the revised manuscript, we again stated, “A
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representative result from triplicate SPR measurements is plotted for each condition.” in the Figure
captions. The statistics of the triplicates, such as mean, standard deviation, and p-value, are
summarized in Tables 1 and 2.
Reviewer’s comment:
The relatively small difference in affinity (12 nM (wt) vs 38 nM (E108Q)) and the poor electron
density for the LA2 module in the structure suggest that the contribution of LA2 to reelin binding is
minimal. Please indicate the p-value for the affinity difference in affinity in Fig 2 or in Table 2.
Response:
We confirmed that the difference is statistically significant with p < 0.005 and added the respective
p-values to the tables.
Reviewer’s comment:
It is not clear to me whether use of the LA1-2 structure to place LA2 in the co-structure is
informative, given the flexibility between LA repeats. How did the rigid-body placement of LA2
change/improve the R-factor for the overall structure?
Response:
As we discussed, the electron density for LA2 was very weak. Hence, we were not able to build a
reliable model for LA2 without an independently determined high-resolution structure of LA12.
Actually, the assignment of the LA2 module in the complex did not significantly improve the Rfactor. This is partly because the size of the LA2 module (about 40 a.a.) is quite small compared
with that of the entire complex for ApoER2 ECD and reelin R56 (over 1,000 a.a.). We also analyzed
the lower ranking solutions from constrained real-space search, and confirmed that most of the highscoring solutions placed the LA2 model in similar positions and orientations to those in the top
solution, as shown in Fig EV2. In addition, we have performed binding analyses targeted to
Interface-2 to validate the reliability of the model assignment. To emphasize this point, we revised
the manuscript as follows:
(Page 9, line 22) To examine the reliability of the model assignment and the contribution of
Interface-2 to the complex stabilization, we introduced mutations to the binding site identified on
reelin R5B.
Reviewer’s comment:
Affinity of the R2193A/K2194 (160 nM) for R56 is weaker than that for LA1 alone in complex with
R56 (73 nM), suggesting that the R2193A/K2194A mutations have an effect beyond that of an
interaction with LA2. A contact-independent effect may be responsible for the entire affinity
difference. Work with other tandemly repeated structures have shown that the folded state of a
repeat can be affected by the folded state of adjacent repeat even when each repeat is capable of
folding in isolation. Could the effect of the two mutations be simply a free energy consequence of a
difference in domain folding or orientation?
Response:
To examine the structural integrity of the R56 fragment, we have performed additional experiments
such as analytical gel filtration and thermal shift assays. Consequently, the R2193A/K2194A mutant
showed similar hydrodynamic properties and thermal stability to the wild type, indicating that the
domain folding and orientation are consistent between the wild type and the mutant. We also
performed the same experiment and confirmed the structural integrity for a reelin R56 GS3 linker
mutant, in which the appendage-like loop involved in Interface-3 was replaced to three repeats of
Gly-Ser.
Reviewer’s comment:
The authors identify a contact between R5A of reelin and the beta propeller of apoER2 (contact 3),
but do not show a function for this interaction. Authors show that the affinity at neutral pH (7.5) of
R56 for apoER2 ECD is the same whether or not the R5A loop is present or not. Of interest is
whether the affinity of the interaction is also the same at acidic pH (5.5). The beta propeller may be
positioned as it is in the structure, not to influence the reelin interaction at neutral pH, but rather to
influence the interaction at acidic pH.
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Response:
Actually, we hypothesize that Interface-3 will be affected by calcium depletion rather than pH shift
during endocytosis, because the concaved surface of EGF-AB is maintained by a calcium ion bound
between the two EGF modules. It is known that the corresponding calcium ion in LDLR dissociates
in the calcium-depleted endosomal compartment. We therefore examined the stability of the
complex under low calcium conditions. Specifically, we performed the SPR measurements using a
running buffer containing 3 µM CaCl2. The contribution of Interface-3 to the complex formation is
expected to be marginal, as the KD value increased only slightly.
Reviewer’s comment:
The proposal that H96 and H99 accelerate R56 release at acidic pH should be tested using either
alanine or asparagine substitutions. Binding and release at pH 7.5 and 5.5 of LA1-2 with R56
should also be done and compared to the apoER2 ECD interaction. If H96 and H99 mediate aciddependent release then the LA1-2 region should exhibit a similar off-rate at pH 5.5 as seen with the
apoER2 ECD. This experiment is important because it tests whether reelin release requires the beta
propeller. A finding that the beta propeller is not required would be consistent with binding and
release of reelin from the full-length apoER2 and the apoER2 ECD, which lacks LA4-6. Assuming
domain orientation is similar in full-length apoER2 and the apoER2 ECD, the beta propeller would
interact with LA1-2 of apoER2 ECD, but LA5-6 in full-length apoER2.
Response:
According to the reviewer’s suggestions, we have performed detailed mutational analysis targeted
to H96/H99. We constructed not only H96A/H99A, but also H96K/H99K double mutant of ApoER2
ECD to test if these His residues act as pH sensors. H96A/H99A is expected to abolish electrostatic
repulsion due to protonation while H96K/H99K mimics the constantly protonated state. We
performed SPR analysis using these two double-mutants at both neutral and acidic pH, and obtained
results consist with a role as a pH sensor. Specifically, H96K/H99K destabilized the complex even
under neutral pH conditions, while an increase of affinity was observed for H96A/H99A under both
the neutral and acidic conditions.
We think that the YWTD β-propeller domain is somehow involved in the release of reelin R56.
The increase of KD value derived from the mutation H96K/H99K (4.5 x 10-8 M) is much smaller
than that derived from the pH shift (4.1 x 10-7 M). It is therefore likely that regions other than
Interface-2 are sensitive to pH shift and modulate the binding affinity between ApoER2 and reelin
R56. However, identifying where and how affinity is lost at interfaces in the complex at acidic pH
demands careful design and execution of several new experiments. We would like to address this
problem in our future study.
Reviewer’s comment:
The statement "Modulation of binding affinity using histidine residues as the pH-sensor has also
been proposed for regulation of the interactions between RAP and LDLR-related proteins" is
misleading with respect to the author's proposed role for H96 and H99 in R56 release. Release of
RAP from LDLR family members is thought to mostly involve an acid-dependent destabilization of
RAP mediated by protonation of histidines of RAP, not the bound LDLR family member. RAP
interacts with LA repeat pairs of many LDLR family members, few of which have histidines
arranged as they are in LA2 of apoER2.
Response:
We agree to the reviewer’s comment and think that our statement was confusing. We therefore
deleted the sentence “Modulation of binding affinity using histidine residues as the pH-sensor has
also been proposed for regulation of the interactions between RAP and LDLR-related proteins”
from the Discussion section.
Reviewer’s comment:
The authors propose that the beta propeller of apoER2 contacts the interaction surface on LA1-2 for
reelin in analogy to the interaction of the LDLR beta propeller with the LA4-5 region of the LDLR.
The authors also propose that the beta propeller interaction accelerates release of reelin from
apoER2. It is possible that the same surface on LA1-2 is used for both reelin and beta propeller
binding, but it is not clear how such an interaction would accelerate the off-rate. Use of the same
surface would imply a competitive mechanism and competitive inhibitors do not accelerate off-rates.
RAP for example acts as a competitive inhibitor for ligand binding to many LDLR family members,
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but addition of RAP does not accelerate the rate of release of ligands already bound to these
receptors. A competitive interaction might however be important to prevent re-binding of reelin to
apoER2 in endosomes, which because of their small luminal volumes, may develop high
concentrations of free reelin as a consequence of acid-dependent release.
Response:
We agree with the reviewer’s comment that the intramolecular interactions between LA modules
and YWTD do not accelerate the dissociation of reelin R56. Alternatively, it should reduce the
affinity for reelin R56 as the competitive inhibitor. We therefore modified the discussion as follows:
(Page 16, line 1) In fact, it is also known that histidine mutations, such as H562Y, cause FH, likely
because it reduces the release activity of LDL, suggesting the importance of the closed conformation
for ligand release through a competitive inhibition mechanism.
(Page 16, line 16) The intramolecular interaction between LA1 and YWTD under acidic conditions,
if it occurs, should further decrease the apparent binding affinity for reelin R56 by preventing R56
from rebinding to LA1. Further studies are required to address the issue whether or not the closed
conformation is formed through LA1, or any other LA modules, and promotes the ligand release
through the competitive inhibition mechanism in this splicing variant of ApoER2.
Response to the comments by reviewer #2:
We would like to express our sincere gratitude to this reviewer for the positive review of our
manuscript. It is truly our pleasure that the reviewer accepted the significance of our study in this
research field.
We would like to again thank both of the reviewers for their constructive suggestions, and we
hope that these revisions will satisfy all the reviewers’ concerns as well as the editorial needs for
publication in EMBO reports. We look forward to your reply.
2nd Editorial Decision
02 March 2017
Thank you for your patience while we have reviewed your revised manuscript. As you will see from
the reports below, referee 1 is now also all positive about its publication in EMBO reports. I am
therefore writing with an 'accept in principle' decision, which means that I will be happy to accept
your manuscript for publication once a few minor issues/corrections have been addressed, as
follows.
- Please provide a running title (max. 40 characters incl. spaces) and up to five keywords.
- EMBO reports papers are accompanied online by A) a short (1-2 sentences) summary of the
findings and their significance, B) 2-3 bullet points highlighting key results and C) a synopsis image
that is 550x200-400 pixels large (width x height). You can either show a model or key data in the
synopsis image. Please send us this information along with the revised manuscript.
- Panel C seems to miss a header in Figure 1
- Please indicate the method used to calculate the p-value in the legends of the table 1 and 2.
- The abstract should be written in present tense. Please find my suggestion attached to this mail.
REFEREE REPORTS
Referee #1:
This manuscript provides direct structural and functional data that are paradigm shifting for the
mechanisms by which LDLR family members bind and release their ligands. The original
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manuscript had several minor flaws all of which have been fully addressed in the revision. In my
opinion, this work has the potential to be a landmark publication.
2nd Revision - authors' response
09 March 2017
We are now submitting a revised manuscript including tables and figures and some additional
information such as a short summary and synopsis according to your suggestions.
Comment:
- Please provide a running title (max. 40 characters incl. spaces) and up to five keywords.
Response:
The running title “3D structure of ligand-bound ApoER2” and keywords were added in the revised
manuscript.
Comment:
- EMBO reports papers are accompanied online by A) a short (1-2 sentences) summary of the
findings and their significance, B) 2-3 bullet points highlighting key results and C) a synopsis image
that is 550x200-400 pixels large (width x height). You can either show a model or key data in the
synopsis image. Please send us this information along with the revised manuscript.
Response:
We also added the short summary and 3 bullet points in the revised manuscript. In addition, we are
submitting a TIF image file for the synopsis.
Comment:
- Panel C seems to miss a header in Figure 1
Response:
We corrected Figure 1 according to the comment.
Comment:
- Please indicate the method used to calculate the p-value in the legends of the table 1 and 2.
Response:
As you can find in the revised tables, we added the following description to the legends: “…with the
indicated p-value, which is calculated by the unpaired two-tailed t-test”.
Comment:
- The abstract should be written in present tense. Please find my suggestion attached to this mail.
Response:
We revised the abstract according to the suggestion. In addition, we changed some capitalized terms
such as “Extended” and “Contracted-closed” to those in lower letters.
Additional minor revision:
We found a mistake in Figure 7 of the previous manuscript. In the previous Fig. 7, PCSK9 already
binds with ApoER2 in a conformational equilibrium at the cell surface. We therefore deleted PCSK9
to correct this figure.
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We would like to again thank both of the reviewers for their review of our manuscript. We hope that
the above revisions will satisfy the editorial needs for publication in EMBO reports. We look
forward to your reply.
© European Molecular Biology Organization
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Corresponding Author Name: Terukazu Nogi, Ph.D.
Journal Submitted to: EMBO reports
Manuscript Number: EMBOR-­‐2016-­‐43521V2
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è the exact sample size (n) for each experimental group/condition, given as a number, not a range;
è a description of the sample collection allowing the reader to understand whether the samples represent technical or biological replicates (including how many animals, litters, cultures, etc.).
è a statement of how many times the experiment shown was independently replicated in the laboratory.
è definitions of statistical methods and measures:
Ÿ common tests, such as t-­‐test (please specify whether paired vs. unpaired), simple χ2 tests, Wilcoxon and Mann-­‐Whitney tests, can be unambiguously identified by name only, but more complex techniques should be described in the methods section;
Ÿ are tests one-­‐sided or two-­‐sided?
Ÿ are there adjustments for multiple comparisons?
Ÿ exact statistical test results, e.g., P values = x but not P values < x;
Ÿ definition of ‘center values’ as median or average;
Ÿ definition of error bars as s.d. or s.e.m. Any descriptions too long for the figure legend should be included in the methods section and/or with the source data.
Please ensure that the answers to the following questions are reported in the manuscript itself. We encourage you to include a specific subsection in the methods section for statistics, reagents, animal models and human subjects. In the pink boxes below, provide the page number(s) of the manuscript draft or figure legend(s) where the information can be located. Every question should be answered. If the question is not relevant to your research, please write NA (non applicable).
B-­‐ Statistics and general methods
Please fill out these boxes ê (Do not worry if you cannot see all your text once you press return)
1.a. How was the sample size chosen to ensure adequate power to detect a pre-­‐specified effect size?
In SPR analysis, we carried out triplicate experiments for each condition to obtain a precise dissociation constant value.
1.b. For animal studies, include a statement about sample size estimate even if no statistical methods were used.
We did not perform animal studies.
2. Describe inclusion/exclusion criteria if samples or animals were excluded from the analysis. Were the criteria pre-­‐
established?
In SPR analysis, all results were included in the calculation for the binding affinity.
3. Were any steps taken to minimize the effects of subjective bias when allocating animals/samples to treatment (e.g. randomization procedure)? If yes, please describe. In the triplicates of SPR analysis, the serially diluted reelin fragments were injected in ascending, descending, and randome orders to avoid the bais.
For animal studies, include a statement about randomization even if no randomization was used.
4.a. Were any steps taken to minimize the effects of subjective bias during group allocation or/and when assessing results (e.g. blinding of the investigator)? If yes please describe.
4.b. For animal studies, include a statement about blinding even if no blinding was done
5. For every figure, are statistical tests justified as appropriate?
Do the data meet the assumptions of the tests (e.g., normal distribution)? Describe any methods used to assess it.
Is there an estimate of variation within each group of data?
Is the variance similar between the groups that are being statistically compared?
C-­‐ Reagents
In SPR analysis, we presented a representative result (sensorgram and curve fit of the steady-­‐state affinity analysis) for each condition in the figures while we summarized the statistical data in the tables. The two-­‐sided p-­‐value is used in tests of significance. 6. To show that antibodies were profiled for use in the system under study (assay and species), provide a citation, catalog number and/or clone number, supplementary information or reference to an antibody validation profile. e.g., Antibodypedia (see link list at top right), 1DegreeBio (see link list at top right).
7. Identify the source of cell lines and report if they were recently authenticated (e.g., by STR profiling) and tested for mycoplasma contamination.
We used the two mammalian cell lines, HEK293S GnT1(-­‐) and CHO-­‐lec 3.2.8.1. HEK293S GnT1(-­‐) was provided by Dr. H.G. Khorana at MIT and CHO-­‐lec3.2.8.1 was provided by Dr. P. Stanley at albert Einstein College of Medicine.
* for all hyperlinks, please see the table at the top right of the document
D-­‐ Animal Models
8. Report species, strain, gender, age of animals and genetic modification status where applicable. Please detail housing and husbandry conditions and the source of animals.
9. For experiments involving live vertebrates, include a statement of compliance with ethical regulations and identify the committee(s) approving the experiments.
10. We recommend consulting the ARRIVE guidelines (see link list at top right) (PLoS Biol. 8(6), e1000412, 2010) to ensure that other relevant aspects of animal studies are adequately reported. See author guidelines, under ‘Reporting Guidelines’. See also: NIH (see link list at top right) and MRC (see link list at top right) recommendations. Please confirm compliance.
E-­‐ Human Subjects
11. Identify the committee(s) approving the study protocol.
12. Include a statement confirming that informed consent was obtained from all subjects and that the experiments conformed to the principles set out in the WMA Declaration of Helsinki and the Department of Health and Human Services Belmont Report.
13. For publication of patient photos, include a statement confirming that consent to publish was obtained.
14. Report any restrictions on the availability (and/or on the use) of human data or samples.
15. Report the clinical trial registration number (at ClinicalTrials.gov or equivalent), where applicable.
16. For phase II and III randomized controlled trials, please refer to the CONSORT flow diagram (see link list at top right) and submit the CONSORT checklist (see link list at top right) with your submission. See author guidelines, under ‘Reporting Guidelines’. Please confirm you have submitted this list.
17. For tumor marker prognostic studies, we recommend that you follow the REMARK reporting guidelines (see link list at top right). See author guidelines, under ‘Reporting Guidelines’. Please confirm you have followed these guidelines.
F-­‐ Data Accessibility
18. Provide accession codes for deposited data. See author guidelines, under ‘Data Deposition’.
The atomic coordinates and structure factors of the ApoER2 ECD:reelin R56 complex and the ApoER2 LA12 fragment have been deposited in the Protein Data Bank with accession numbers of Data deposition in a public repository is mandatory for:
5B4X and 5B4Y, respectively.
a. Protein, DNA and RNA sequences
b. Macromolecular structures
c. Crystallographic data for small molecules
d. Functional genomics data e. Proteomics and molecular interactions
19. Deposition is strongly recommended for any datasets that are central and integral to the study; please consider the We would like to submit the raw data of SPR analysis in Figs. 1 & 4. journal’s data policy. If no structured public repository exists for a given data type, we encourage the provision of datasets in the manuscript as a Supplementary Document (see author guidelines under ‘Expanded View’ or in unstructured repositories such as Dryad (see link list at top right) or Figshare (see link list at top right).
20. Access to human clinical and genomic datasets should be provided with as few restrictions as possible while respecting ethical obligations to the patients and relevant medical and legal issues. If practically possible and compatible with the individual consent agreement used in the study, such data should be deposited in one of the major public access-­‐
controlled repositories such as dbGAP (see link list at top right) or EGA (see link list at top right).
21. As far as possible, primary and referenced data should be formally cited in a Data Availability section. Please state whether you have included this section.
Examples:
Primary Data
Wetmore KM, Deutschbauer AM, Price MN, Arkin AP (2012). Comparison of gene expression and mutant fitness in Shewanella oneidensis MR-­‐1. Gene Expression Omnibus GSE39462
Referenced Data
Huang J, Brown AF, Lei M (2012). Crystal structure of the TRBD domain of TERT and the CR4/5 of TR. Protein Data Bank 4O26
AP-­‐MS analysis of human histone deacetylase interactions in CEM-­‐T cells (2013). PRIDE PXD000208
22. Computational models that are central and integral to a study should be shared without restrictions and provided in a machine-­‐readable form. The relevant accession numbers or links should be provided. When possible, standardized format (SBML, CellML) should be used instead of scripts (e.g. MATLAB). Authors are strongly encouraged to follow the MIRIAM guidelines (see link list at top right) and deposit their model in a public database such as Biomodels (see link list at top right) or JWS Online (see link list at top right). If computer source code is provided with the paper, it should be deposited in a public repository or included in supplementary information.
G-­‐ Dual use research of concern
23. Could your study fall under dual use research restrictions? Please check biosecurity documents (see link list at top right) and list of select agents and toxins (APHIS/CDC) (see link list at top right). According to our biosecurity guidelines, provide a statement only if it could.