Architecture of the synaptotagmin–SNARE machinery for neuronal exocytosis

May combine two claims.

Syt1 binds in a Ca2+-dependent manner to anionic membranes, with anionic phospholipids and synaptotagmin C2 domains together coordinating calcium.

“Syt1 binds in a Ca2+-dependent manner to anionic membranes; during binding, anionic phospholipids and synaptotagmin C2 domains together coordinate calcium14–17.”

• cites [14] Davletov, B. A. & Südhof, T. C. A single C2 domain from synaptotagmin I is sufficient for high affinity Ca2+/phospholipid binding. J. Biol. Chem. 268, 26386–26390 (1993) sourceNot checked — no full text of the source
• cites [15] Brose, N., Petrenko, A. G., Südhof, T. C. & Jahn, R. Synaptotagmin: a calcium sensor on the synaptic vesicle surface. Science 256, 1021–1025 (1992) sourceNot checked — no full text of the source
• cites [16] Fernández-Chacón, R. et al. Synaptotagmin I functions as a calcium regulator of release probability. Nature 410, 41–49 (2001) sourceNot checked — no full text of the source
• cites [17] Vrljic, M. et al. Post-translational modifications and lipid binding profile of insect cell-expressed full-length mammalian synaptotagmin 1. Biochemistry 50, 9998–10012 (2011) sourceSupported

May combine two claims.

Spontaneous release in the reconstituted system reflects fusion probability at exactly zero Ca2+ concentration, whereas spontaneous release in neurons is driven by resting Ca2+ concentrations and is largely blocked by removing all calcium.

“We note that spontaneous release observed in our reconstituted system38 reflects the fusion probability at exactly zero Ca2+ concentration (that is, in absence of a Ca2+-sensor for mini-release) whereas spontaneous release in neurons is driven by resting Ca2+-concentrations and is largely blocked by removing all calcium41.”

• cites [38] Lai, Y. et al. Complexin inhibits spontaneous release and synchronizes Ca2+-triggered synaptic vesicle fusion by distinct mechanisms. Elife 3, 1–14 (2014) sourceSupported
• cites [41] Xu, J., Pang, Z. P., Shin, O.-H. & Südhof, T. C. Synaptotagmin-1 functions as a Ca2+ sensor for spontaneous release. Nature Neurosci. 12, 759–766 (2009) sourcePartly supported

  The Results section (“Spontaneous release is Ca2+-dependent”) reports that intracellular Ca2+ chelation blocked more than 95% of spontaneous mIPSCs, supporting the claim about removing calcium. But the Discussion says release is likely triggered by Ca2+ sparks rather than resting presynaptic Ca2+ concentrations. The citation is attached to the neuronal-release claim, not the description of the reconstituted system. (LLM, conf=0.88)

  Experimental — accuracy not yet measured

May combine two claims.

A reasonably complete data set was obtained from a few hundred images using 148 crystals, in contrast to the tens of thousands to millions of crystals typically used in XFEL-based crystallography.

“Moreover, in contrast to the tens of thousands to millions of crystals typically used in XFEL-based crystallography experiments34, we obtained a reasonably complete data set from a few hundred images using 148 crystals (Methods).”

• cites [34] Redecke, L. et al. Natively inhibited Trypanosoma brucei cathepsin B structure determined by using an X-ray laser. Science 339, 227–230 (2013) sourceSupported

May combine two claims.

Region I mutants retained some rescue ability, whereas region II mutants were nearly non-functional, including the R398Q, R399Q mutant.

“Interestingly, the region I mutants retained some rescue ability, in contrast to region II mutants that were nearly non-functional (Figs. 4a, b), including the R398Q, R399Q mutant as previously reported36.”

• cites [36] Xue, M., Ma, C., Craig, T. K., Rosenmund, C. & Rizo, J. The Janus-faced nature of the C2B domain is fundamental for synaptotagmin-1 function. Nature Struct. Mol. Biol. 15, 1160–1168 (2008) sourceSupported

May combine two claims.

Upon Ca2+ binding to the Syt1 C2B domain, the Ca2+ binding loops partially insert into the membrane with a preference for membrane curvature, possibly supported by ionic interactions via the polybasic region and with continuing maintenance of the Syt1-SNARE interface.

“Upon Ca2+ binding to the Syt1 C2B domain, the Ca2+ binding loops partially insert into the membrane23 with a preference for membrane curvature20,21, possibly supported by ionic interactions via the polybasic region and with continuing maintenance of the Syt1-SNARE interface.”

• cites [23] Rufener, E., Frazier, A. A., Wieser, C. M., Hinderliter, A. & Cafiso, D. S. Membrane-bound orientation and position of the synaptotagmin C2B domain determined by site-directed spin labeling. Biochemistry 44, 18–28 (2005) sourceNot checked — no full text of the source
• cites [20] Hui, E., Johnson, C. P., Yao, J., Dunning, F. M. & Chapman, E. R. Synaptotagmin-mediated bending of the target membrane is a critical step in Ca2+-regulated fusion. Cell 138, 709–721 (2009) sourcePartly supported

  Split into 2 atoms — 1 confirmed, 1 partially supported, 0 disputed, 0 misattributed, 0 unverifiable. Expand the claim to see per-atom verdicts.

  Experimental — accuracy not yet measured
• cites [21] Martens, S., Kozlov, M. M. & McMahon, H. T. How synaptotagmin promotes membrane fusion. Science 316, 1205–1208 (2007) sourceNot checked — no full text of the source

May combine two claims.

Covalent lanthanide labels attached to SNAP-25 residues 41 and 166 would likely disrupt the primary interface, so the crystal structures cannot be compared with that NMR study.

“In a recent study, lanthanide labels were covalently attached to SNAP-25 residues 41 and 166 for pseudocontact chemical shift NMR measurements in the presence of 125 thiocyanate30; these covalent labels would likely disrupt the primary interface (Fig. 2c), so it is not possible to compare our crystal structures with this NMR study.”

• cites [30] Brewer, K. D. et al. Dynamic binding mode of a Synaptotagmin-1–SNARE complex in solution. Nature Struct. Mol. Biol. 22, 555–564 (2015) sourceSupported

Probably not a substantial claim: may not pass S3 (load-bearing).

The observed smFRET histograms suggested several possible interfaces between Syt1 and the SNARE complex.

“The observed smFRET histograms28 suggested several possible interfaces between Syt1 and the SNARE complex.”

• cites [28] Choi, U. B. et al. Single-molecule FRET-derived model of the synaptotagmin 1-SNARE fusion complex. Nature Struct. Mol. Biol. 17, 318–324 (2010) sourcePartly supported

  The Discussion (page not provided) says that broad, multi-peaked intermolecular smFRET distributions suggest multiple conformations of the Syt1–SNARE complex. It does not say that the histograms suggest several distinct binding interfaces; the Results instead describe a dominant modeled interface. (LLM, conf=0.82)

  Experimental — accuracy not yet measured

Probably not a substantial claim: may not pass S3 (load-bearing).

A gain-of-function mutation in the Ca2+-binding region of the C2A domain suggested that the Syt1-SNARE interaction may be functionally important.

“A gain-of-function mutation in the Ca2+-binding region of the C2A domain suggested that the Syt1-SNARE interaction may be functionally important26, but the molecular basis and the significance of the interaction between Syt1 and the SNARE complex remain unknown.”

• cites [26] Pang, Z. P., Shin, O.-H., Meyer, A. C., Rosenmund, C. & Südhof, T. C. A gain-of-function mutation in synaptotagmin-1 reveals a critical role of Ca2+-dependent soluble N-ethylmaleimide-sensitive factor attachment protein receptor complex binding in synaptic exocytosis. J. Neurosci. 26, 12556–12565 (2006) sourceSupported

May combine two claims.

The gain-of-function mutation D232N in the Ca2+-binding region of the C2A domain is close to the tertiary interface and is also part of the C2A-C2B interface.

“Interestingly, the gain-of-function mutation D232N in the Ca2+-binding region of the C2A domain26 is close to the tertiary interface and it is also part of the C2A-C2B interface (Fig. 1b).”

• cites [26] Pang, Z. P., Shin, O.-H., Meyer, A. C., Rosenmund, C. & Südhof, T. C. A gain-of-function mutation in synaptotagmin-1 reveals a critical role of Ca2+-dependent soluble N-ethylmaleimide-sensitive factor attachment protein receptor complex binding in synaptic exocytosis. J. Neurosci. 26, 12556–12565 (2006) sourceSupported

May combine two claims.

The palmitoylated cysteine residues of SNAP-25 would be able to interact with the plasma membrane, and the membrane-proximal side of syntaxin is closely juxtaposed with the membrane, as inferred by the requirement of tight membrane-coupling of syntaxin for evoked release.

“The palmitoylated cysteine residues of SNAP-25 would be able to interact with the plasma membrane, and the membrane-proximal side of syntaxin is closely juxtaposed with the membrane, as inferred by the requirement of tight membrane-coupling of syntaxin for evoked release47.”

• cites [47] Zhou, P., Bacaj, T., Yang, X., Pang, Z. P. & Südhof, T. C. Lipid-anchored SNAREs lacking transmembrane regions fully support membrane fusion during neurotransmitter release. Neuron 80, 470–483 (2013) sourcePartly supported

  Split into 2 atoms — 1 confirmed, 1 partially supported, 0 disputed, 0 misattributed, 0 unverifiable. Expand the claim to see per-atom verdicts.

  Experimental — accuracy not yet measured

May combine two claims.

Assays based on lipid mixing alone can produce misleading results since lipid mixing can occur without content mixing.

“More importantly, assays based on lipid mixing alone can produce misleading results since lipid mixing can occur without content mixing86.”

• cites [86] Zick, M. & Wickner, W. T. A distinct tethering step is vital for vacuole membrane fusion. Elife 3, e03251 (2014) sourceSupported

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

Ramachandran analysis with MolProbity indicated that 98% of residues are in favored regions and none are in disallowed regions for the quintuple mutant of the Syt1 C2B domain.

“Ramachandran analysis with MolProbity75 indicated that 98% of the residues are in the favored regions and none are in disallowed regions.”

• cites [75] Chen, V. B. et al. MolProbity: All-atom structure validation for macromolecular crystallography. Acta Crystallogr. D 66, 12–21 (2010) sourceSupported

May combine two claims.

Addition of complexin-1 greatly enhanced Ca2+-triggered amplitude and synchronization, and suppressed spontaneous release in this system.

“Moreover, addition of complexin-1 greatly enhanced Ca2+-triggered amplitude and synchronization, and suppressed spontaneous release in this system38.”

• cites [38] Lai, Y. et al. Complexin inhibits spontaneous release and synchronizes Ca2+-triggered synaptic vesicle fusion by distinct mechanisms. Elife 3, 1–14 (2014) sourceSupported

Probably not a substantial claim: may not pass S3 (load-bearing).

The size distributions of the SV and PM vesicles were analyzed by cryo-EM.

“The size distributions of the SV and PM vesicles were analyzed by cryo-EM, as described previously37.”

• cites [37] Diao, J. et al. Synaptic proteins promote calcium-triggered fast transition from point contact to full fusion. Elife 1, e00109 (2012) sourceSupported

May combine two claims.

The single vesicle-vesicle assay uses full-length Syt1 and monitors content mixing, a correlate for neurotransmitter release.

“Our single vesicle-vesicle assay thus uses full-length Syt1 and it monitors content mixing, a correlate for neurotransmitter release37,38,81.”

• cites [37] Diao, J. et al. Synaptic proteins promote calcium-triggered fast transition from point contact to full fusion. Elife 1, e00109 (2012) sourceSupported
• cites [38] Lai, Y. et al. Complexin inhibits spontaneous release and synchronizes Ca2+-triggered synaptic vesicle fusion by distinct mechanisms. Elife 3, 1–14 (2014) sourceSupported
• cites [81] Kyoung, M. et al. In vitro system capable of differentiating fast Ca2+-triggered content mixing from lipid exchange for mechanistic studies of neurotransmitter release. Proc. Natl Acad. Sci. USA 108, E304–E313 (2011) sourceNot checked — no full text of the source

May combine two claims.

A variety of complexin-1 truncations and mutations qualitatively reproduced effects observed in neuronal cultures for both spontaneous and Ca2+-triggered release, lending credence to this reconstituted system to investigate mechanistic questions.

“A variety of complexin-1 truncations and mutations qualitatively reproduced effects observed in neuronal cultures for both spontaneous and Ca2+-triggered release38, lending credence to this reconstituted system to investigate mechanistic questions.”

• cites [38] Lai, Y. et al. Complexin inhibits spontaneous release and synchronizes Ca2+-triggered synaptic vesicle fusion by distinct mechanisms. Elife 3, 1–14 (2014) sourceSupported

Probably not a substantial claim: may not pass S3 (load-bearing).

Ramachandran analysis with MolProbity indicated that 98% of residues are in favored regions and none are in disallowed regions for both the Ca2+- and Mg2+-bound Syt1-SNARE complexes in the short unit cell crystal form.

“Ramachandran analysis with MolProbity75 indicated that 98% of the residues are in the favored regions and none are in disallowed regions for both the Ca2+- and Mg2+-bound Syt1-SNARE complexes in the short unit cell crystal form”

• cites [75] Chen, V. B. et al. MolProbity: All-atom structure validation for macromolecular crystallography. Acta Crystallogr. D 66, 12–21 (2010) sourceSupported

Probably not a substantial claim: may not pass S3 (load-bearing).

Similar improvements in limiting resolution of XFEL versus synchrotron diffraction images have been observed for GPCRs.

“We note that similar improvements in limiting resolution of XFEL versus synchrotron diffraction images have been observed for GPCRs60.”

• cites [60] Cohen, A. E. et al. Goniometer-based femtosecond crystallography with X-ray free electron lasers. Proc. Natl Acad. Sci. USA 111, 17122–17127 (2014) sourceSupported

Probably not a substantial claim: may not pass S3 (load-bearing).

Ramachandran analysis with MolProbity indicated that 97% of residues are in favored regions and none are in disallowed regions for the Ca2+-bound Syt1-SNARE complex in the long unit cell crystal form.

“and that 97% of the residues are in the favored regions and none are in disallowed regions for the Ca2+-bound Syt1-SNARE complex in the long unit cell crystal form.”

• cites [75] Chen, V. B. et al. MolProbity: All-atom structure validation for macromolecular crystallography. Acta Crystallogr. D 66, 12–21 (2010) sourcePartly supported

  The 'Torsion-angle combinations: updated Ramachandran and rotamer analyses' section (page not specified) confirms that MolProbity evaluates Ramachandran conformations and reports percentages of favored residues and outliers. It does not report an analysis of the Ca2+-bound Syt1-SNARE complex or substantiate the claimed 97% favored and zero disallowed residues. (LLM, conf=0.87)

  Experimental — accuracy not yet measured

May combine two claims.

The C2A domain forming the tertiary interface is in the same orientation in both crystal forms and in Ca2+- and Mg2+-bound structures, and the interacting side chains are in similar positions.

“The C2A domain that forms the tertiary interface is in the same orientation in both crystal forms and in the Ca2+- and Mg2+-bound crystal structures (Extended Data Figs. 5c, d) and the interacting side chains are in similar positions.”

May combine two claims.

The residues involved in the primary interface have relatively low temperature factors and well-defined electron densities, suggesting a specific interaction.

“The residues involved in the primary interface have relatively low temperature factors, among the lowest in these structures (Extended Data Fig. 5e), and the electron densities of the sidechains that form this interface are well-defined (Figs. 2c, d), suggesting a specific interaction.”

May combine two claims.

The secondary interface involves another Syt1 C2B domain and the SNARE complex, while the tertiary interface involves a Syt1 C2A domain and the SNARE complex.

“The secondary interface involves another Syt1 C2B domain and the SNARE complex, while the tertiary interface involves a Syt1 C2A domain and the SNARE complex (Fig. 1b).”

May combine two claims. The quote only partly states this claim.

The primary interface between the Syt1 C2B domain and the SNARE complex is large, specific, Ca2+-independent, and conserved.

“The structures revealed several interfaces, including a large, specific, Ca2+-independent, and conserved interface.”

May combine two claims.

The primary interface is very similar in both complexes and both crystal forms, suggesting it is not affected by crystal packing.

“The largest, primary interface between the Syt1 C2B domain and the SNARE complex is very similar in both complexes (Fig. 1b) and in both crystal forms (Extended Data Fig. 5a), suggesting that it is not affected by crystal packing.”

May combine two claims.

Removal of Syt1 abolished synchronous release monitored by eIPSCs, a phenotype rescued by re-introduction of WT but not mutant Syt1 cDNA.

“As expected7, removal of Syt1 abolished synchronous release monitored by recording evoked inhibitory postsynaptic currents (eIPSCs), a phenotype that can be rescued by re-introduction of WT but not mutant Syt1 cDNA (Figs. 4a, b).”

• cites [7] Geppert, M. et al. Synaptotagmin I: a major Ca2+ sensor for transmitter release at a central synapse. Cell 79, 717–727 (1994) sourceNot checked — no full text of the source

May combine two claims.

The electron density maps from the XFEL data were notably superior to those of the synchrotron datasets and of sufficient quality to obtain accurate rotamers for most sidechains, including at interfaces.

“The electron density maps obtained from the XFEL diffraction data were notably superior to those of the synchrotron datasets (Methods). In particular, the electron density maps calculated from the XFEL data set were of sufficient quality to obtain accurate rotamers for most sidechains, including those at the interfaces between molecules (Extended Data Figs. 2d–f).”

May combine two claims.

The primary interface is divided into two regions dominated by polar interactions: region I comprises SNAP-25 E37, K40, N159, M163, D166 and Syt1 E295, K297, N336 and Y338; region II comprises SNAP-25 D51, E52, E55, syntaxin-1A D231, E234, E238, and Syt1 R281, K288, R398, and R399.

“We divide the primary interface into two regions (Fig. 2) dominated by polar interactions: region I comprises SNAP-25 E37, K40, N159, M163, D166 and Syt1 E295, K297, N336 and Y338; region II comprises SNAP-25 D51, E52, E55, syntaxin-1A D231, E234, E238, and Syt1 R281, K288, R398, and R399.”

May combine two claims.

The E295A, Y338W mutant could rescue the unclamping of spontaneous release phenotype while region II mutants (or combinations thereof) could not.

“Interestingly, the E295A, Y338W mutant could rescue this phenotype while the region II mutants (or combinations thereof) could not (Figs. 4f, g).”

May combine two claims.

The paper reports atomic-resolution crystal structures of a Syt1-SNARE complex in two different crystal forms and in the presence of either Ca2+ or Mg2+.

“Here we report atomic-resolution crystal structures of Ca2+- and Mg2+-bound complexes between synaptotagmin-1 and the neuronal SNARE complex, one of which was determined with diffraction data from an X-ray free electron laser, leading to an atomic-resolution structure with accurate rotamer assignments for many sidechains.”

• Also stated in: § Introduction · plain

  “Here we report atomic-resolution crystal structures of a Syt1-SNARE complex in two different crystal forms and in the presence of either Ca2+ or Mg2+.”

May combine two claims.

The primary interface is very similar in Ca2+-bound and Mg2+-bound crystal structures, implying a Ca2+-independent interface.

“The primary interface is also very similar in the Ca2+-bound as well as in the Mg2+-bound crystal structures (Extended Data Fig. 5d), implying a Ca2+-independent interface.”

May combine two claims.

Mutation of either region I or II of the primary interface reduced Syt1 binding to syntaxin-1A/SNARE complex by ~50%, and simultaneous mutation of both regions reduced binding to ~33% compared to WT Syt1.

“Mutation of either regions I and II of the primary interface reduced Syt1 binding to syntaxin-1A/SNARE complex by ~50%, and simultaneous mutation of both regions (“Syt1 quintuple”) reduced binding to ~33% as compared to a Syt1 WT construct introduced in the same manner (Figs. 3b, c).”

May combine two claims.

The SNARE complex is positively charged and Syt1 is negatively charged in region I, whereas the opposite is observed for region II.

“The SNARE complex is positively charged and Syt1 is negatively charged in region I, whereas the opposite is observed for region II.”

May combine two claims.

All Syt1 mutants underwent facilitation and the severity of the phenotype for each mutant correlated well with the results for single evoked release.

“All Syt1 mutants underwent facilitation and the severity of the phenotype for each mutant correlated well with the results for single evoked release.”

May combine two claims.

ATP has no effect on spontaneous and Ca2+-triggered fusion and only a mild effect on vesicle association, suggesting that functionally important Syt1-SNARE interactions are not affected by ionic shielding.

“We observed no effect on both spontaneous and Ca2+-triggered fusion, and only a mild effect on vesicle association (Figs. 3d–g, right group of mutants), suggesting that the functionally important interactions between Syt1 and the SNARE complex are not affected by ionic shielding.”

May combine two claims.

During high frequency stimulation, WT cultured hippocampal neurons displayed depression while Syt1 cKO neurons showed asynchronous release with robust facilitation.

“During high frequency stimulation, WT cultured hippocampal neurons displayed depression while Syt1 cKO neurons showed asynchronous release with robust facilitation (Figs. 4c–e).”

May combine two claims.

Both groups of mutants reduce the amplitude and decrease the synchronization of Ca2+-triggered fusion, but do not affect spontaneous fusion.

“Likewise, both groups of mutants reduce the amplitude and decrease the synchronization of Ca2+-triggered fusion, but they do not affect spontaneous fusion (Figs. 3e–g).”

Probably not a substantial claim: may not pass S3 (load-bearing).

There were consistently two crystal forms in the same drop with identical morphology, referred to as short unit cell and long unit cell crystal forms.

“There were consistently two crystal forms in the same drop with identical morphology, referred to as “short unit cell” crystal form and “long unit cell” crystal form, respectively.”

Probably not a substantial claim: may not pass S3 (load-bearing).

Improved diffraction quality could be achieved by placing consecutive shots far apart across the length of the crystal from one another, then later filling between the first and second shots in the same shuttling manner.

“During data collection we observed that improved diffraction quality could be achieved by placing consecutive shots far apart (i.e., across the length of the crystal from one another), then later “filling” between the first and second shots in the same “shuttling” manner.”

Probably not a substantial claim: may not pass S3 (load-bearing).

Only one out of 85 screened crystals in the long unit cell crystal form diffracted to 4.1 Å resolution at the APS NE-CAT microfocus synchrotron beamline.

“Only one out of 85 screened crystals in the long unit cell crystal form diffracted to 4.1 Å resolution at the APS NE-CAT microfocus synchrotron beamline”

May combine two claims.

Exposure to Cre-recombinase results in the total absence of synchronous release, which is typical for Syt1 KO neurons.

“Exposure to Cre-recombinase results in the total absence of synchronous release which is typical for Syt1 KO neurons (Fig. 4a, b).”

May combine two claims.

Only 1 out of 85 screened crystals in the long unit cell form diffracted to 4.1 Å at APS, while 61 out of about 72 long unit cell crystals diffracted to at least 3.5 Å at LCLS.

“It is notable that only 1 out of 85 screened crystals in the long unit cell form diffracted to 4.1 Å at APS, while 61 out of ~ 72 long unit cell crystals diffracted to at least 3.5 Å at LCLS.”

Probably not a substantial claim: may not pass S3 (load-bearing).

The long unit cell form occurred much more frequently, at about 80%.

“The long unit cell form occurred much more frequently (about 80%).”

May combine two claims.

The primary interfaces have relatively low B-factors, similar to the majority of the structure, while parts of the C2A and C2B domains involved in the secondary and tertiary interfaces have higher B-factors.

“Note that the primary interfaces have relatively low B-factors, similar to the majority of the structure, while parts of the C2A and C2B domains involved in the secondary and tertiary interfaces have higher B-factors”

May combine two claims.

The short unit cell crystal form produced a limiting resolution of 3.6 Å at APS NE-CAT, but the density maps are substantially less well defined for sidechains compared to the LCLS data set.

“Interestingly, while the short unit cell crystal form produced a limiting resolution of 3.6 Å at APS NE-CAT, the density maps are substantially less well defined for sidechains compared to the LCLS data set (compare Extended Data Figs. 2c–f and Figs. 3b, c).”

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

Of the 148 crystals used in the experiment, 113 produced 578 images that could be processed; the other crystals did not diffract or showed multiple lattices.

“Of the 148 crystals used in the experiment, 113 crystals produced 578 images that could be processed; the other crystals did not diffract or showed multiple lattices.”

May combine two claims.

The primary interface is very similar in both crystal forms: the RMSD for the primary interface between both crystal forms is 0.38 Å (bright-orange) and 0.42 Å (white) for complex I and complex II, respectively.

“The primary interface is very similar in both crystal forms: the RMSD for the primary interface between both crystal forms is 0.38 Å (bright-orange) and 0.42 Å (white) for complex I and complex II, respectively”

May combine two claims.

Using a reverse vapor-diffusion method led to thicker crystal plates and eventually produced Bragg peaks past 4 Å resolution.

“Using a reverse vapor-diffusion method led to thicker crystal plates (Extended Data Fig. 1e), and eventually produced Bragg peaks past 4 Å resolution.”

May combine two claims.

The entire crystal was damaged when exposed at room temperature, requiring many more crystals and much more time to collect a complete diffraction dataset.

“Furthermore, the entire crystal was damaged when exposed at room temperature, requiring many more crystals and much more time to collect a complete diffraction dataset.”

Probably not a substantial claim: may not pass S3 (load-bearing).

The final model of the quintuple mutant of the Syt1 C2B domain consists of four Syt1 C2B molecules in the asymmetric unit.

“The final model consists of four Syt1 C2B molecules in the asymmetric unit.”

May combine two claims.

This morphological change of the plasma membrane juxtaposes the membranes closer than the critical distance (0.9 nm) to promote stalk formation, and subsequently leads to fusion pore opening.

“This morphological change of the plasma membrane juxtaposes the membranes closer than the critical distance (0.9 nm) to promote stalk formation48 (Fig. 5d), and subsequently leads to fusion pore opening (Fig. 5e).”

• cites [48] Aeffner, S., Reusch, T., Weinhausen, B. & Salditt, T. Energetics of stalk intermediates in membrane fusion are controlled by lipid composition. Proc. Natl Acad. Sci. USA 109, E1609–E1618 (2012) sourceNot checked — no full text of the source

• AssumptionThe authors state this as an assumption: Prior to Ca2+ influx, there is a membrane juxtaposed, hemifusion-free state, the likely starting state for fast Ca2+ triggered fusion, and a partially folded trans-SNARE complex.
• AssumptionThe authors state this as an assumption: The Syt1 C2B-SNARE unit can assemble in the pre-Ca2+ state.

May combine two claims.

The primary Syt1-SNARE interface is critical for the role of Syt1 as a Ca2+ sensor of evoked release, but is not required for spontaneous release in neurons.

“Together, these observations support the notion that the primary Syt1-SNARE interface is critical for the role of Syt1 as a Ca2+ sensor of evoked release, but that this Syt1-SNARE interface is not required for spontaneous release in neurons, consistent with the differential effect of Syt1 mutations on spontaneous and evoked release11–13.”

• cites [11] Yoshihara, M. & Littleton, J. T. Synaptotagmin I functions as a calcium sensor to synchronize neurotransmitter release. Neuron 36, 897–908 (2002) sourceNot checked — no full text of the source
• cites [12] Maximov, A. & Südhof, T. C. Autonomous function of synaptotagmin 1 in triggering synchronous release independent of asynchronous release. Neuron 48, 547–554 (2005) sourceNot checked — no full text of the source
• cites [13] Kochubey, O. & Schneggenburger, R. Synaptotagmin increases the dynamic range of synapses by driving Ca2+-evoked release and by clamping a near-linear remaining Ca2+ sensor. Neuron 69, 736–748 (2011) sourceNot checked — no full text of the source

Probably not a substantial claim: may not pass S3 (load-bearing).

All of these interactions may be anchored by the primary interface, which is the most stable and extensive contact site.

“All of these interactions may be anchored by the primary interface, which is the most stable and extensive contact site.”

May combine two claims.

Tests of the primary interface by mutagenesis suggest that it is essential for Ca2+-triggered neurotransmitter release in neuronal synapses and for Ca2+-triggered vesicle fusion in a reconstituted system.

“Tests of this interface by mutagenesis suggest that it is essential for Ca2+-triggered neurotransmitter release in neuronal synapses and for Ca2+-triggered vesicle fusion in a reconstituted system.”

May combine two claims.

Other synaptotagmin isoforms not involved in fast synchronous release exhibit sequence variations and differences in electrostatic potential maps in both regions, suggesting that the primary interface may be specific for fast synchronous release.

“In contrast, other synaptotagmin isoforms not involved in fast synchronous release exhibit sequence variations and differences in the electrostatic potential maps in both regions (Extended Data Figs. 6a, b), suggesting that the primary interface may be an interaction that is specific for fast synchronous release.”

Classified as probably not a substantial claim.

The entire Syt1-SNARE assembly would be poised to confer cooperativity upon Ca2+-triggering on a sub-millisecond timescale.

“This entire Syt1-SNARE assembly would thus be poised to confer cooperativity upon Ca2+-triggering on a sub-millisecond timescale.”

• AssumptionThe authors state this as an assumption: Prior to Ca2+ influx, there is a membrane juxtaposed, hemifusion-free state, the likely starting state for fast Ca2+ triggered fusion, and a partially folded trans-SNARE complex.
• AssumptionThe authors state this as an assumption: The Syt1 C2B-SNARE unit can assemble in the pre-Ca2+ state.

Probably not a substantial claim: may not pass S3 (load-bearing).

Increased ensemble lipid mixing was observed upon addition of the soluble Syt1 C2AB fragment and Ca2+.

“For example, increased ensemble lipid mixing was observed upon addition of the soluble Syt1 C2AB fragment and Ca2+ (ref. 21).”

• cites [21] Martens, S., Kozlov, M. M. & McMahon, H. T. How synaptotagmin promotes membrane fusion. Science 316, 1205–1208 (2007) sourceNot checked — no full text of the source

Probably not a substantial claim: may not pass S3 (load-bearing).

Only a lack of available XFEL beamtime prevented the authors from collecting a complete diffraction dataset beyond 3.5 Å.

“In fact, only a lack of available XFEL beamtime prevented us from collecting a complete diffraction dataset beyond 3.5 Å.”

Classified as probably not a substantial claim. May combine two claims.

The presence and purity of reconstituted proteins was confirmed by SDS-PAGE, and the directionality of the membrane proteins facing outward was assessed by chymotrypsin digestion followed by SDS-PAGE.

“As described previously81, the presence and purity of reconstituted proteins was confirmed by SDS-PAGE of the vesicle preparations, and the directionality of the membrane proteins (facing outward) was assessed by chymotrypsin digestion followed by SDS-PAGE.”

• cites [81] Kyoung, M. et al. In vitro system capable of differentiating fast Ca2+-triggered content mixing from lipid exchange for mechanistic studies of neurotransmitter release. Proc. Natl Acad. Sci. USA 108, E304–E313 (2011) sourceNot checked — no full text of the source

Probably not a substantial claim: may not pass S3 (load-bearing).

As a consequence of the 16-degree rotation, the interactions between residues R281, K288, R398 of the Syt1 C2B (I′) domain and residues E224, E228 of syntaxin-1A are slightly changed.

“as a consequence, the interactions between residues R281, K288, R398 of the Syt1 C2B (I′) domain and residues E224, E228 of syntaxin-1A are slightly changed by this rotation.”

May combine two claims.

The best constructs resulted in a Syt1-SNARE complex that eluted in a mono-disperse peak and remained stable in SDS-PAGE without boiling, a hallmark for neuronal SNARE complex formation.

“The best constructs resulted in a Syt1-SNARE complex that eluted in a mono-disperse peak in the final size-exclusion chromatography step, and that remained stable in SDS-PAGE without boiling, a hallmark for neuronal SNARE complex formation (Extended Data Fig. 1b).”

May combine two claims.

The role of the region II interacting residues was unclear in the absence of structural information, which now reveals that they are part of a larger interface between Syt1 and the SNARE complex.

“However, the role of these residues was unclear in the absence of structural information, which now reveals that they are part of a larger interface between Syt1 and the SNARE complex.”

• AssumptionRests on an assumption: The role of the region II interacting residues was unclear in the absence of structural information.

May combine two claims.

In retrospect, the increased ensemble lipid mixing result was probably caused by an effect on vesicle association since multivalent binding of the soluble Syt1 C2AB fragment can induce vesicle clustering.

“However, in retrospect, this result was probably caused by an effect on vesicle association since multivalent binding of the soluble Syt1 C2AB fragment can induce vesicle clustering19.”

• cites [19] Araç, D. et al. Close membrane-membrane proximity induced by Ca2+-dependent multivalent binding of synaptotagmin-1 to phospholipids. Nature Struct. Mol. Biol. 13, 209–217 (2006) sourceNot checked — no full text of the source

May combine two claims.

The maps from XFEL data, superior to those of the short unit cell data crystal structure collected at APS NE-CAT at comparable limiting resolution, enabled better investigation of the binding interfaces between Syt1 and the SNARE complex, and were essential for the structural portion of the study.

“These maps, superior to those of the short unit cell data crystal structure collected at the APS NE-CAT microfocus synchrotron beamline at comparable limiting resolution (Extended Data Figs. 3b, c), enabled us to better investigate the binding interfaces between Syt1 and the SNARE complex, and was thus essential for the structural portion of our study.”

May combine two claims.

The LCLS diffraction data set proved notably superior to the particular synchrotron-derived datasets collected in terms of limiting resolution and quality of the electron density maps, and was thus essential for more accurately determining side chain positions.

“Taken together, the LCLS diffraction data set proved notably superior to the particular synchrotron-derived datasets that we collected in terms of limiting resolution and quality of the electron density maps, and was thus essential for more accurately determining side chain positions.”

May combine two claims.

Taking into account the smaller number of molecules in the asymmetric unit of the short unit cell crystal form, there are still fewer Ca2+ sites observed in maps derived from the APS data set, suggesting that Ca2+-binding sites have been affected by radiation damage.

“Taking into account the smaller number of molecules in the asymmetric unit of the short unit cell crystal form (2 Syt1 and 1 SNARE complex compared 3 Syt1 and 2 SNARE complexes), there are still fewer Ca2+ sites that were observed in maps derived from the data set collected at APS, suggesting that Ca2+-binding sites have been affected by radiation damage.”

Classified as probably not a substantial claim.

A subset of the interacting residues of region II were suggested to be functionally important in previous studies: Syt1 R398, R399, and SNAP-25 D51, E52, E55.

“A subset of the interacting residues of region II were suggested to be functionally important in previous studies: Syt1 R398, R399 (refs. 35,36,78), and SNAP-25 D51, E52, E55 (refs. 27,79,80).”

• cites refs. 35,36,78Not matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

• cites refs. 27,79,80Not matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.