statistics.md

General numbers

SNAP

ID	Mutation	Vesicles per Tomogram	Tethers per AZ	AZ surface area [µm²]	Connectors per synapse (0-250 nm)
73	4E	459	23	0.23	269
80	4E	105	6	0.21	84
84	4E	109	10	0.12	107
88	4E	154	10	0.13	159
102	4E	103	0	0.29	94
114	4K	123	2	0.09	68
115	4K	137	1	0.26	70
116	4K	278	1	0.13	154
123	4K	55	3	0.09	52
128	WT-KO	243	12	0.18	683
132	WT-KO	126	2	0.24	110
133	WT-KO	505	27	0.17	229
134	WT-KO	600	7	0.34	144

Time Res

ID	Timepoint [ms]	Vesicles per Tomogram	Tethers per AZ	AZ surface area [µm²]	Connectors per synapse (0-250 nm)
Control 1	0	220	15	0.11	331
Control 2	0	104	8	0.04	264
Control 3	0	127	4	0.05	230
Control 4	0	143	3	0.03	482
Control 5	0	213	12	0.11	361
Control 6	0	104	7	0.06	199
Control 7	0	184	9	0.03	360
Control 8	0	132	19	0.09	226
Control 9	0	134	6	0.08	326
Spray 1	late	697	5	0.03	272
Spray 2	late	115	3	0.08	88
Spray 3	late	429	21	0.19	882
Spray 5	early	534	57	0.07	1412
Spray 5_2 (second AZ, same synaptosome)	early		32	0.15
Spray 6	late	371	1	0.03	397
Spray 7	early	107	5	0.02	156
Spray 8	late	99	4	0.02	202
Spray 10	late	76	4	0.02	96

significance for vesicle occupancy

SNAP (Fig. 3A)

	occupancy	count	std	sem	p_distance	p_treatment	fdr_bh_corr_p_distance	fdr_bh_corr_p_treatment
('0-45', 'WT')	0.133124	4	0.00860717	0.00430359	0.00467712	nan	0.0397555	nan
('0-45', '4E')	0.107469	5	0.039373	0.0176081	nan	0.247191	nan	0.466917
('0-45', '4K')	0.0863832	4	0.0144041	0.00720205	nan	0.00141816	nan	0.0241088
('45-75', 'WT')	0.0940015	4	0.0156622	0.00783111	nan	nan	nan	nan
('45-75', '4E')	0.103423	5	0.0350639	0.015681	nan	0.636297	nan	0.832081
('45-75', '4K')	0.106511	4	0.0309522	0.0154761	nan	0.497903	nan	0.769486
('150-250', 'WT')	0.162134	4	0.0826631	0.0413315	0.15644	nan	0.466917	nan
('150-250', '4E')	0.117767	5	0.0489696	0.0218999	nan	0.346687	nan	0.589368
('150-250', '4K')	0.0974842	4	0.0475772	0.0237886	nan	0.224007	nan	0.466917
('250-450', 'WT')	0.141476	4	0.0694036	0.0347018	0.230445	nan	0.466917	nan
('250-450', '4E')	0.0770829	5	0.0631539	0.0282433	nan	0.188593	nan	0.466917
('250-450', '4K')	0.109623	4	0.0801068	0.0400534	nan	0.569798	nan	0.807214
('450-900', 'WT')	0.106749	4	0.0679294	0.0339647	0.727122	nan	0.882934	nan
('450-900', '4E')	0.0367801	5	0.0706431	0.0315926	nan	0.177067	nan	0.466917
('450-900', '4K')	0.106304	4	0.0613511	0.0306755	nan	0.992553	nan	0.992553

time-resolved (Fig. 3B)

	occupancy	count	std	sem	p_distance	p_treatment	fdr_bh_corr_p_distance	fdr_bh_corr_p_treatment
('0-45', 'spray_ctrl')	0.109982	9	0.024676	0.00822534	0.0282501	nan	0.0776878	nan
('0-45', 'spray_early')	0.123332	3	0.0396709	0.022904	nan	0.495593	nan	0.77879
('0-45', 'spray_late')	0.0686147	6	0.022716	0.00927378	nan	0.00599179	nan	0.0219699
('45-75', 'spray_ctrl')	0.0817971	9	0.0249044	0.00830145	nan	nan	nan	nan
('45-75', 'spray_early')	0.0682967	3	0.00449321	0.00259415	nan	0.386528	nan	0.708634
('45-75', 'spray_late')	0.0808933	6	0.0419075	0.0171087	nan	0.958736	nan	0.98064
('75-150', 'spray_ctrl')	0.12884	9	0.0176375	0.00587916	0.000281204	nan	0.00309324	nan
('75-150', 'spray_early')	0.1158	3	0.00819847	0.00473339	nan	0.254934	nan	0.560854
('75-150', 'spray_late')	0.128561	6	0.0263766	0.0107682	nan	0.98064	nan	0.98064
('150-250', 'spray_ctrl')	0.139951	9	0.0332375	0.0110792	0.00067761	nan	0.00372686	nan
('150-250', 'spray_early')	0.134966	3	0.014355	0.00828787	nan	0.810751	nan	0.98064
('150-250', 'spray_late')	0.136525	6	0.0336375	0.0137325	nan	0.848646	nan	0.98064

significances for min distance to the AZ for near SVs (Figure 3 A first 100nm)

SNAP

	10	20	30	40	50	60	70	80	90	100
WT-4E t-test	-0.87	1.3	5.18	7.43	5.78	0.36	-5.73	-5.45	-3.86	-2.64
WT-4E p-value	0.38	0.2	2.8e-6	4.6e-10	2.8e-7	0.72	3.4e-7	1.0e-6	2.7e-4	0.01
WT-4K t-test	1.15	5.56	16.53	10.86	3.64	-0.9	-5.51	-3.67	-1.83	-1.26
WT-4K p-value	0.25	7.9e-7	3.3e-23	2.1e-15	6.0e-4	0.37	9.3e-7	5.5e-4	0.07	0.21

significances for min distance to the AZ for near SVs (Figure 3 C/D)

SNAP

asdf	mean	std	sem	n	t-confidence	fdr_bh corrected
WT	5.85	3.45	0.54	41	1
4E	8.16	5.96	0.93	51	0.0302	0.0371
4K	8.54	6.08	1.47	17	0.0371	0.0371

Time Res

	mean	std	sem	n	t-confidence
ctrl	8.93	5.49	0.58	90	1
early	7.65	4.06	0.61	45	0.168
late	9.7	6.01	0.97	38	0.484

Fraction of Proximal SVs in RRP(Figure 4 A/B)

SNAP

	Fraction	n	chi2	confidence
WT	0.171	41	0	1
4E	0.146	51	0.0914	0.7624
4K	0	17	3.3008	0.0692

Time Res

	Fraction	n	chi2	confidence	fdr_bh corrected
ctrl	0.078	90	0	1
early	0.289	45	10.5946	0.0011	0.0022
late	0.132	38	0.9103	0.34	0.34

Number of tethers (Figure 4 C/D)

SNAP

	mean	std	n	t-test p-value	frd_bh corrected
WT	1.171	1.482	41	1
4E	0.961	1.264	51	0.473	0.473
4K	0.412	0.618	17	0.008	0.016

Time Res

	mean	std	n	t-test p-value	frd_bh corrected
ctrl	0.889	1.116	90	1
early	2.089	2.244	45	0.0001	0.0002
late	1	1.208	38	0.617	0.617

Number of connectors per SV (Figure 5 A and B)

SNAP-25 (panel A)

	n_connection	count	std	sem	p_distance	p_treatment	corr_p_distance	corr_p_treatment	fdr_bh_corr_p_distance	fdr_bh_corr_p_treatment
(45, '4E')	1.54902	51	1.54031	0.215686	nan	0.333858	nan	0.418108	nan	0.418108
(45, '4K')	1.82353	17	2.21459	0.537117	nan	0.851538	nan	0.851538	nan	0.851538
(45, 'WT')	1.95122	41	2.40781	0.376037	nan	nan	nan	nan	nan	nan
(75, '4E')	1.11429	35	1.20712	0.204041	nan	0.535421	nan	0.568885	nan	0.568885
(75, '4K')	0.444444	18	0.783823	0.184749	nan	0.0350148	nan	0.0744065	nan	0.0744065
(75, 'WT')	1.4	10	1.50555	0.476095	0.494222	nan	0.560118	nan	0.560118	nan
(150, '4E')	1.75776	161	1.9293	0.15205	nan	0.0195284	nan	0.0474262	nan	0.0474262
(150, '4K')	1.62222	90	1.73256	0.182627	nan	0.0164181	nan	0.0474262	nan	0.0474262
(150, 'WT')	2.392	125	2.63627	0.235795	0.344324	nan	0.418108	nan	0.418108	nan
(250, '4E')	1.53695	203	1.78341	0.125171	nan	1.66988e-10	nan	9.46263e-10	nan	9.46263e-10
(250, '4K')	1.5	106	1.74711	0.169694	nan	5.9964e-07	nan	2.54847e-06	nan	2.54847e-06
(250, 'WT')	3.23431	239	3.30957	0.214078	0.0182112	nan	0.0474262	nan	0.0474262	nan
(450, '4E')	1.27451	255	1.67753	0.105051	nan	6.54299e-15	nan	1.11231e-13	nan	1.11231e-13
(450, '4K')	2.57542	179	2.7598	0.206277	nan	0.32203	nan	0.418108	nan	0.418108
(450, 'WT')	2.81958	521	2.87196	0.125823	0.0600592	nan	0.102101	nan	0.102101	nan
(900, '4E')	1.42081	221	1.79122	0.120491	nan	7.57199e-11	nan	6.43619e-10	nan	6.43619e-10
(900, '4K')	2.35519	183	2.05409	0.151843	nan	0.0540546	nan	0.102101	nan	0.102101
(900, 'WT')	2.80112	538	2.88698	0.124467	0.0667945	nan	0.103228	nan	0.103228	nan

Time resolved (panel B)

	n_connection	count	std	sem	p_distance	p_treatment	fdr_bh_corr_p_distance	fdr_bh_corr_p_treatment
(45, 'spray_ctrl')	1.62222	90	1.57635	0.166162	nan	nan	nan	nan
(45, 'spray_early')	2.68889	45	2.87483	0.428554	nan	0.00611329	nan	0.0134492
(45, 'spray_late')	0.947368	38	1.08919	0.176691	nan	0.0176205	nan	0.0276894
(75, 'spray_ctrl')	1.47059	68	1.56902	0.190271	0.549462	nan	0.755511	nan
(75, 'spray_early')	1.40909	22	1.53248	0.326725	nan	0.872711	nan	0.981532
(75, 'spray_late')	1.45652	46	1.48633	0.219147	nan	0.961833	nan	0.981532
(150, 'spray_ctrl')	2.38802	384	1.88936	0.0964159	0.000401512	nan	0.00220831	nan
(150, 'spray_early')	2.98026	152	2.29156	0.18587	nan	0.00222876	nan	0.00612908
(150, 'spray_late')	2.82041	245	2.23065	0.142511	nan	0.00937208	nan	0.0171822
(250, 'spray_ctrl')	2.91697	554	2.2885	0.0972289	3.13053e-07	nan	3.44358e-06	nan
(250, 'spray_early')	3.42705	281	2.19476	0.130929	nan	0.00210109	nan	0.00612908
(250, 'spray_late')	2.92051	390	2.35562	0.119281	nan	0.981532	nan	0.981532

significances for fractions of connected vesicles per distance group (Figure 5 C/D)

SNAP

	WT				4E				4K
	number of connected SVs	number of non connected SVs	p-value	fdr_bh corrected	number of connected SVs	number of non connected SVs	p-value	fdr_bh corrected	number of connected SVs	number of non connected SVs	p-value	fdr_bh corrected
0-45	23	18	1		28	13	1		9	8	1
45-75	6	4	0.89	0.96	19	13	(0.59)		6	12	(0.4)
75-150	87	38	0.16	0.272	89	38	(0.98)		58	32	(0.53)
150-250	184	55	0.008	0.0272	88	49	(0.77)		69	37	(0.49)
250-450	399	122	0.0063	0.026775	126	89	(0.32)		124	55	(0.27)
450-900	369	169	0.14	0.2644	124	96	(0.21)		146	37	(0.026)

	WT			4E				4K
	number of connected SVs	number of non connected SVs	p-value	number of connected SVs	number of non connected SVs	p-value	fdr_bh corrected	number of connected SVs	number of non connected SVs	p-value	fdr_bh corrected
0-45	23	18		28	13	0.36	0.51	9	8	0.94	0.96
45-75	6	4		19	13	0.74	0.8986	6	12	0.33	0.51
75-150	87	38		89	38	0.96	0.96	58	32	0.52	0.68
150-250	184	55		88	49	0.011	0.031167	69	37	0.03	0.07286
250-450	399	122		126	89	0.0000015	0.0000255	124	55	0.06	0.1275
450-900	369	169		124	96	0.0018	0.0153	146	37	0.005	0.026775

TimeRes

	spay control				spray early				spray late
	number of connected SVs	number of non connected SVs	p-value	fdr_bh corrected	number of connected SVs	number of non connected SVs	p-value	fdr_bh corrected	number of connected SVs	number of non connected SVs	p-value	frd_bh corrected
0-45	63	27			32	13	(1)		20	18
45-75	46	22	0.89	0.99	16	6	(0.88)		32	14	(0.172 )
75-150	323	61	0.0032	0.0176	129	23	(0.06)		213	32	(0.0000008)
150-250	483	71	0.00005	0.00055	258	23	(0.00011)		333	57	(0.0000013)

	spay control			spray early				spray late
	number of connected SVs	number of non connected SVs	p-value	number of connected SVs	number of non connected SVs	p-value	fdr_bh corrected	number of connected SVs	number of non connected SVs	p-value	fdr_bh corrected
0-45	63	27		32	13	0.95	0.99	20	18	0.09	0.2475
45-75	46	22		16	6	0.86	0.99	32	14	0.99	0.99
75-150	323	61		129	23	0.93	0.99	213	32	0.39	0.858
150-250	483	71		258	23	0.06	0.22	333	57	0.48	0.88

Number of connectors per RRP / non-RRP proximal SV (Figure 5 E/F)

SNAP (Figure 5E)

RRP

	mean	std	sem	n	t-confidence
WT	1.571	1.272	0.481	7	1
4E	1.333	1.211	0.494	6	0.738
4K	0	0	0	0	-

non RRP

	mean	std	sem	n	t-confidence
WT	2.029	2.588	0.444	34	1
4E	1.578	1.588	0.237	45	0.342
4K	1.824	2.215	0.537	17	0.781

Time Res (Figure 5F)

RRP

	mean	std	sem	n	t-confidence	fdr_bh corrected
ctrl	1.429	1.813	0.685	7	1
early	2.692	2.496	0.692	13	0.25	0.5
late	1.200	1.304	0.583	5	0.81	0.81

non RRP

	mean	std	sem	n	t-confidence	fdr_bh corrected
ctrl	1.639	1.566	0.172	83	1
early	2.688	3.053	0.54	32	0.017	0.034
late	0.909	1.071	0.186	33	0.016	0.034

number of connector per tethered / non tethered proximal SVs (Figure 5 G/H)

SNAP

tethered

	mean	std	sem	n	t-confidence
WT	2.609	2.572	0.536	23	1
4E	2.037	1.675	0.322	27	0.35
4K	3	2.28	0.931	6	0.74

non tethered

	mean	std	sem	n	t-confidence
WT	1.111	1.937	0.457	18	1
4E	1	1.18	0.241	24	0.82
4K	1.182	1.99	0.6	11	0.93

Time Res

tethered

	mean	std	sem	n	t-confidence	fdr_bh corrected
ctrl	1.653	1.601	0.229	49	1
early	2.583	2.687	0.448	36	0.05	0.0693
late	0.85	1.137	0.254	20	0.045	0.0693

non tethered

	mean	std	sem	n	t-confidence	fdr_bh corrected
ctrl	1.585	1.565	0.244	41	1
early	3.111	3.689	1.23	9	0.052	0.0693
late	1.056	1.056	0.249	18	0.197	0.197

(Supplement 1 A/B chi2)

SNAP

		Fraction (0 tethers)	n	p-value
WT	tethered/non-tethered	0.439	23/18	1
4E	tethered/non-tethered	0.471	27/24	0.927
4K	tethered/non-tethered	0.647	6/11	0.249

Time Res

		Fraction (0 tethers)	n	p-value	fdr_bh corrected
ctrl	tethered/non-tethered	0.456	49/41	1
early	tethered/non-tethered	0.2	36/9	0.0067	0.0134
late	tethered/non-tethered	0.474	20/18	0.995	0.995

(Supplement 1 C/D chi2)

SNAP

	overall
WT	1
4E	0.968

	0	1	2	3
WT/4E	1	1	1	0.741

Time Res

	overall
ctrl	1
early	0.457
late	0.438

	0	1	2	3	4
ctrl/early/late	1	0.487	0.420	0.869	0.787

(Supplement 1 E/F chi2)

SNAP

	overall
WT	1
4E	0.129
4K	0.562

	0	1	2	3	4
WT/4E/4K	1	0.623	0.0036	0.523	0.588

Time Res

	overall
ctrl	1
early	0.526
late	0.323

	0	1	2	3	4
ctrl/early/late	1	0.359	0.435	0.071	0.319

(Supplement 1 G/H chi2)

SNAP

		Fraction	n	p-value	p-value
WT	tethered	0.739	23	1	1
4E	tethered	0.8	20	0.638	1
4K	tethered	0.833	6	0.631	1
WT	non-tethered	0.333	18	1	0.0094
4E	non-tethered	0.571	21	0.137	0.116
4K	non-tethered	0.364	11	0.868	0.064

Time Res

		Fraction	n	p-value	p-value
ctrl	tethered	0.694	49	1	1
early	tethered	0.694	36	0.996	1
late	tethered	0.5	20	0.129	1
ctrl	non-tethered	0.707	41	1	0.89
early	non-tethered	0.778	9	0.67	0.622
late	non-tethered	0.556	18	0.257	0.732

(Supplement 1 I/J chi2)

SNAP

		Fraction	n	p-value	p-value
WT	RRP	0.714	7	1	1
4E	RRP	0.667	6	0.853	1
4K	RRP	-	0	-	-
WT	non-RRP	0.529	34	1	0.369
4E	non-RRP	0.686	35	0.184	0.926
4K	non-RRP	0.529	17	1	-

Time Res

		Fraction	n	p-value	fdr_bh corrected
ctrl	RRP	0.429	7	1
early	RRP	0.692	13	0.251	0.502
late	RRP	0.6	5	0.558	0.744
ctrl	non-RRP	0.723	83	1
early	non-RRP	0.719	32	0.965	0.965
late	non-RRP	0.515	33	0.033	0.132

Tether length by length (t-test)

SNAP

	mean	count	std	p-value	fdr_bh corrected
WT	7.97	48	5.71
4E	6.01	49	3.63	0.0452	0.0904
4K	4.84	7	3.78	0.1667	0.1667

Time res

	mean	count	std	p-value	fdr_bh corrected
ctrl	5.96	83	4.77
early	4.61	94	2.82	0.021	0.042
late	5.39	38	4.59	0.538	0.538

Tether length by distance to AZ (t-test)

SNAP

	mean	count	std	p-value	fdr_bh corrected
WT	7.14	48	4.59
4E	5.17	49	3.21	0.0158	0.0316
4K	3.54	7	4.59	0.0487	0.0487

Time res

	mean	count	std	p-value
ctrl	6.91	83	3.85
early	6.12	94	4.22	0.20
late	7.53	38	5.19	0.47

Tether length binned (chi2)

SNAP

	WT	4E	4K	p-value ctrl-early	p-value ctrl-late	p-value early-late
<8 nm	19	27	4	0.18	0.64	0.76
8-10 nm	4	7	1	0.55	0.85	1
10-22 nm	21	15	2	0.26	0.73	0.74
>22 nm	4	0	0	0.12	0.99	-
p-value	-	0.066	0.66

Time res

	ctrl	early	late	p-value ctrl-early	p-value ctrl-late	p-value early-late
<8 nm	33	45	20	0.35	0.26	0.76
8-10 nm	9	9	2	0.98	0.52	0.64
10-22 nm	27	36	9	0.52	0.44	0.16
>22 nm	14	4	7	0.012	0.96	0.020
p-value	-	0.045	0.46