Architecture of the synaptotagmin–SNARE machinery for neuronal exocytosis

In the paper

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

What the sources say

• ?Not checked — no full text of the sourcecites [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) source
• ?Not checked — no full text of the sourcecites [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) source
• ?Not checked — no full text of the sourcecites [16] Fernández-Chacón, R. et al. Synaptotagmin I functions as a calcium regulator of release probability. Nature 410, 41–49 (2001) source
• Supported

  The Discussion states that Syt1 binds anionic phospholipids in a Ca2+-dependent manner, with calcium coordinated between its Ca2+-binding loops and anionic phospholipid headgroups. The Results section (“Full-Length Syt1 Is Folded”) likewise describes calcium coordination involving the C2A and C2B domains and anionic lipids. Page numbers were not supplied. (LLM, conf=0.95)

  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) source

In the paper

“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.”

What the sources say

• Supported

  The citation to this paper is attached to the reconstituted-system claim, not the subsequent claim about neurons. The Results and Materials and methods describe spontaneous fusion before Ca2+ injection, with Ca2+ removed from the preparation and sample chamber. This supports fusion under nominally calcium-free conditions, though the paper does not establish a mathematically exact zero concentration. (LLM, conf=0.87)

  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) source
• Partly 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)

  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) sourceExperimental — accuracy not yet measured

In the paper

“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).”

What the sources say

• Supported

  Citation 34 supports the comparison with large XFEL crystallography experiments, not the citing authors’ own result using 148 crystals. The source reports 178,875 indexed diffraction patterns from individual microcrystals, consistent with the cited scale of crystal use. (LLM, conf=0.88)

  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) source

In the paper

“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.”

What the sources say

• Supported

  The citation is attached specifically to the previously reported R398Q,R399Q result, not to the citing authors’ region I versus region II comparison. In Results, “The two C2B domain bottom arginines are crucial for release” (Fig. 2), the source reports that the double mutation practically abolished rescue activity. The Discussion notes weak residual activity under high-frequency stimulation, consistent with “nearly non-functional.” (LLM, conf=0.93)

  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) source

In the paper

“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.”

What the sources say

• ?Not checked — no full text of the sourcecites [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) source
• Partly supported

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

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

In the paper

“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.”

What the sources say

• Supported

  The citation is attached to the description of the NMR study, not to the citing authors’ subsequent inference about interface disruption and crystal-structure comparison. The Results section reports lanthanide-tagged SNAP-25 at residues 41 and 166 for pseudocontact-shift measurements; Online Methods specifies 125 mM KSCN and describes labeling the protein before SNARE-complex assembly. (LLM, conf=0.92)

  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) source

In the paper

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

What the sources say

• Partly 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)

  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) sourceExperimental — accuracy not yet measured

In the paper

“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.”

What the sources say

• Supported

  The Introduction identifies D232N as a mutation in the Ca2+-binding site of the C2A domain. The Results report that it selectively enhances Ca2+-dependent Syt1–SNARE binding, and the Discussion links that change to increased neurotransmitter release, supporting the claim of functional importance. Page numbers are not available in our copy. (LLM, conf=0.94)

  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) source

In the paper

“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).”

What the sources say

• Supported

  The Introduction identifies D232N as a mutation in the Ca²⁺-binding site of the C2A domain, and the results report that it enhances Ca²⁺-dependent SNARE binding. In the citing sentence, citation 26 follows that identification; the statements about the tertiary and C2A–C2B interfaces follow the citation and refer to the citing paper’s Fig. 1b, so they are not attributed to this source. (LLM, conf=0.90)

  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) source

In the paper

“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.”

What the sources say

• Partly supported

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

  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) sourceExperimental — accuracy not yet measured

In the paper

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

What the sources say

• Supported

  The Results explain that hemifusion or lysis followed by membrane re-annealing can produce lipid mixing detected by lipid-based assays, making a protected lumenal-content assay important. The Discussion also reports lipid mixing with substantially less content mixing under the same reaction condition. (LLM, conf=0.94)

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

In the paper

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

What the sources say

• Supported

  The citation identifies MolProbity as the tool used for Ramachandran analysis, not as the source of the quintuple mutant’s results. The Validation analyses section describes MolProbity’s Ramachandran evaluation and says its summary reports the percentage of residues in favored conformations and the percentage of outliers. (LLM, conf=0.92)

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

In the paper

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

What the sources say

• Supported

  The Results passage reports that adding complexin-1 increased the amplitude and decay rate of Ca2+-triggered fusion, indicating greater synchronization. It also reports a lower probability of spontaneous fusion with complexin-1 than without it in the paper’s vesicle-fusion assay. (LLM, conf=0.94)

  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) source

In the paper

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

What the sources say

• Supported

  The Results section identifies the donor and acceptor vesicles as liposomes designed to mimic synaptic vesicles and the plasma membrane. The Materials and methods section (‘Measurement of vesicle diameters’; page not available) describes calculating vesicle-size distribution histograms from cryo-EM images. The histograms pool vesicles rather than reporting separate distributions for the two types. (LLM, conf=0.88)

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

In the paper

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

What the sources say

• Supported

  The Results section, under “Ca2+-triggered fusion with SNAREs and synaptotagmin 1,” states that the single vesicle–vesicle assay reconstituted full-length synaptotagmin 1 and monitored content mixing. The Materials and methods section, under “Single vesicle–vesicle content/lipid mixing system,” explains that its content dye is only slightly larger than neurotransmitters such as glutamate or serotonin, supporting content mixing as a correlate of neurotransmitter release. (LLM, conf=0.92)

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

  The Results (Figure 2 caption; page not provided) explicitly describe the assay as using full-length synaptotagmin-1. Materials and methods (“Single vesicle–vesicle content-mixing assay”; page not provided) says it measures exchange of vesicle contents as a correlate of neurotransmitter release. (LLM, conf=0.94)

  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) source
• ?Not checked — no full text of the sourcecites [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) source

In the paper

“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.”

What the sources say

• Supported

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

  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) source

In the paper

“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”

What the sources say

• Supported

  The citation identifies MolProbity as the tool used for Ramachandran analysis. The paper’s “Torsion-angle combinations” section says MolProbity evaluates Ramachandran angles and reports percentages of favored conformations and outliers. The 98% result for the Syt1-SNARE complexes is presented as the citing authors’ analysis, not as a finding attributed to this source. (LLM, conf=0.93)

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

In the paper

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

What the sources say

• Supported

  The Raster Data Collection section reports improved diffraction resolution for β2-adrenoreceptor, a GPCR, at the XFEL: the best image reached 2.3 Å, versus 2.9 Å for crystals from the same batch at a synchrotron. This supports the cited comparison, although the Discussion notes that improvements were not observed for every crystal tested. (LLM, conf=0.94)

  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) source

In the paper

“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.”

What the sources say

• Partly 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)

  cites [75] Chen, V. B. et al. MolProbity: All-atom structure validation for macromolecular crystallography. Acta Crystallogr. D 66, 12–21 (2010) sourceExperimental — accuracy not yet measured

In the paper

“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.”

In the paper

“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.”

In the paper

“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).”

In the paper

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

In the paper

“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.”

In the paper

“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).”

What the sources say

• ?Not checked — no full text of the sourcecites [7] Geppert, M. et al. Synaptotagmin I: a major Ca2+ sensor for transmitter release at a central synapse. Cell 79, 717–727 (1994) source

In the paper

“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).”

In the paper

“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.”

In the paper

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

In the paper

“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+.”

In the paper

“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.”

In the paper

“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).”

In the paper

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

In the paper

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

In the paper

“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.”

In the paper

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

In the paper

“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).”

In the paper

“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.”

In the paper

“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.”

In the paper

“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”

In the paper

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

In the paper

“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.”

In the paper

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

In the paper

“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”

In the paper

“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).”

In the paper

“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.”

In the paper

“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”

In the paper

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

In the paper

“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.”

In the paper

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

In the paper

“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).”

What the sources say

• ?Not checked — no full text of the sourcecites [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) source

In the paper

“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.”

What the sources say

• ?Not checked — no full text of the sourcecites [11] Yoshihara, M. & Littleton, J. T. Synaptotagmin I functions as a calcium sensor to synchronize neurotransmitter release. Neuron 36, 897–908 (2002) source
• ?Not checked — no full text of the sourcecites [12] Maximov, A. & Südhof, T. C. Autonomous function of synaptotagmin 1 in triggering synchronous release independent of asynchronous release. Neuron 48, 547–554 (2005) source
• ?Not checked — no full text of the sourcecites [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) source

In the paper

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

In the paper

“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.”

In the paper

“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.”

In the paper

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

In the paper

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

What the sources say

• ?Not checked — no full text of the sourcecites [21] Martens, S., Kozlov, M. M. & McMahon, H. T. How synaptotagmin promotes membrane fusion. Science 316, 1205–1208 (2007) source

In the paper

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

In the paper

“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.”

What the sources say

• ?Not checked — no full text of the sourcecites [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) source

In the paper

“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.”

In the paper

“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).”

In the paper

“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.”

In the paper

“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.”

What the sources say

• ?Not checked — no full text of the sourcecites [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) source

In the paper

“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.”

In the paper

“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.”

In the paper

“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.”

In the paper

“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).”

What the sources say

• ?Not matched

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

  cites refs. 35,36,78
• ?Not matched

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

  cites refs. 27,79,80