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Structural basis of meiotic chromosome synapsis through SYCP1 self-assembly

Abstract

Meiotic chromosomes adopt unique structures in which linear arrays of chromatin loops are bound together in homologous chromosome pairs by a supramolecular protein assembly, the synaptonemal complex. This three-dimensional scaffold provides the essential structural framework for genetic exchange by crossing over and subsequent homolog segregation. The core architecture of the synaptonemal complex is provided by SYCP1. Here we report the structure and self-assembly mechanism of human SYCP1 through X-ray crystallographic and biophysical studies. SYCP1 has an obligate tetrameric structure in which an N-terminal four-helical bundle bifurcates into two elongated C-terminal dimeric coiled-coils. This building block assembles into a zipper-like lattice through two self-assembly sites. N-terminal sites undergo cooperative head-to-head assembly in the midline, while C-terminal sites interact back to back on the chromosome axis. Our work reveals the underlying molecular structure of the synaptonemal complex in which SYCP1 self-assembly generates a supramolecular lattice that mediates meiotic chromosome synapsis.

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Acknowledgements

O.R.D. thanks D. W. Sinclair for scientific inspiration and support. We thank Diamond Light Source and the staff of beamlines I02, I04-1 and B21 (proposals mx9948, mx13587, sm14435, sm15580, sm15897 and sm15836). We thank A. Baslé and H. Waller for assistance with X-ray crystallographic and circular dichroism data collection, and L. J. Salmon and V. A. Jatikusumo for work in the early stages of this project. I.U. is funded by grants BIO2015-64216-P and MDM2014-0435-01 (MINECO, Spanish Ministry of Economy and Competitiveness). C.M. is supported by a MINECO BES-2015-071397 scholarship associated to the Structural Biology Maria de Maeztu Unit of Excellence. S.M. is supported by a Wellcome Trust Career Re-entry Fellowship (062376). O.R.D. is a Sir Henry Dale Fellow jointly funded by the Wellcome Trust and Royal Society (grant number 104158/Z/14/Z).

Author information

J.M.D. performed the majority of biochemical, biophysical and crystallographic experiments. O.M.D. performed SAXS experiments and analyses. M.R. crystallized truncated αN-end and analyzed αN constructs. C.M. and I.U. solved the αC-end crystal structures. O.R.D. solved the αN-end crystal structures and built and refined all structures. S.M. assisted with initial experiments. J.M.D. and O.R.D. designed experiments; O.R.D. wrote the manuscript.

Correspondence to Owen R. Davies.

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The authors declare no competing interests.

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Integrated supplementary information

Supplementary Figure 1 Summary of SYCP1 constructs.

(a) Schematic of the SYCP1 sequence showing the α-helical core, consisting of αN-tetramer and αC-dimer, with αN-tip (magenta), along with αN-end and αC-end self-assembly sites (striped). The schematic is aligned with a secondary structure prediction, amino acid conservation amongst vertebrates and all constructs that contributed towards the identification of principal structural regions of SYCP1. Amino acid boundaries are provided along with oligomer states for stable constructs and annotation of constructs that were insoluble, unstable, degraded or unfolded. The key constructs included in the manuscript are highlighted. (b) SDS-PAGE analysis with Coomassie staining of the purified recombinant SYCP1 protein samples used in this study.

Supplementary Figure 2 SYCP1 is a parallel tetramer that bifurcates into two C-terminal coiled-coil dimers.

(a) Far UV CD spectra and (b) CD thermal denaturation of SYCP1 αCore-ΔNtip (112-783) (black), αN-ΔNtip (112-362) (grey), αN-tetramer (206-362) (narrow dashes) and αC-dimer (358-783) (wide dashes). Helical content was estimated (as indicated) through deconvolution of spectra with data fitted at normalised rmsd values of 0.005, 0.009, 0.003 and 0.003 respectively. Thermal denaturation was recorded as % unfolded based on the helical signal at 222 nm; melting temperatures were estimated at 52 °C, 38 °C, 38 °C and 37 °C respectively. (c-f) SEC-SAXS analysis of SYCP1 αCore-ΔNtip, αN-tetramer and αC-dimer, and their N-terminal MBP fusions. (c-d) Scattering intensity plots with fits used for P(r) distributions shown as black lines. (e) Guinier analysis to determine the radius of gyration (Rg) with linear fits shown in black. Q.Rg values were < 1.3. The Guinier regions of αCore-ΔNtip and αC-dimer were too narrow for analysis; their Rg values were calculated as 254 Å and 196 Å from real space P(r) distributions. (f) Guinier analysis to determine the radius of gyration of the cross-section (Rc), with the linear fits highlighted in black. Q.Rc values were < 1.3. (g-j) SEC-MALS analysis of SYCP1 fusion proteins; light scattering (LS) and differential refractive index (dRI) are shown as solid and dashed lines respectively, with fitted molecular weights (Mw) plotted as diamonds across elution peaks. (g) MBP-αCore-ΔNtip (black) is a 477 kDa tetramer (theoretical tetramer - 499 kDa) whilst MBP-αC-dimer (grey) is a 185 kDa dimer (theoretical dimer - 191 kDa). (h) MBP-αN-tetramer (black) is a 232 kDa tetramer (theoretical tetramer - 255 kDa) whilst MBP (grey) is a 43 kDa monomer (theoretical monomer - 45 kDa). (i) RecE-αN-tetramer (black) and RecE (grey) are tetramers of 208 kDa (theoretical tetramer - 214 kDa) and 132 kDa (theoretical tetramer - 136 kDa) respectively. (j) GST-αN-tetramer (black, right) is a 194 kDa tetramer (theoretical tetramer - 195 kDa), whilst GST-αC-dimer (black, left) and GST (grey) are dimers of 157 kDa (theoretical dimer - 160 kDa) and 58 kDa (theoretical dimer - 59 kDa) respectively. (k-n) SEC-SAXS analysis of GST fusions of αN-tetramer and αC-dimer. (k) Scattering intensity plots. (l-m) Guinier analysis to determine the radius of gyration (Rg). (n) P(r) distributions of GST-αN-tetramer (black), GST-αC-dimer (wide dashes) and GST (grey) showing maximum dimensions of 282 Å, 677 Å and 99 Å respectively. In GST-αN-tetramer, the presence of short inter-atomic distance peaks, and the absence of peaks close to Dmax values, is consistent with the relative orientation of GST molecules within parallel coiled-coil structures. Peaks corresponding to intra-GST and inter-GST distances are indicated.

Supplementary Figure 3 Crystal structures of SYCP1 αN-end (101–206) and αN-end truncated (101–175).

(a-b) Sample 2Fo-Fc electron density maps contoured at 1.2σ and superimposed on the refined crystallographic models for (a) SYCP1 αN-end (101-206) and (b) SYCP1 αN-end truncated (101-175). (c-e) Superposition of the SYCP1 αN-end (101-206) (purple) and αN-end truncated (101-175) (green) crystal structures with an rmsd of 2.28 Å. (c) Overall structural superposition (in a series of 45° rotations) reveals a similar global conformation but with a key distinction that chain B copies of αN-end splay from the midline to create an open rather than closed head-to-head assembly. (d) Close-up of the head-to-head assembly demonstrating how chain A copies of the αN-end structure adopt a similar conformation to the αN-end truncated structure, whilst chain B copies splay apart to create the open interface. (e) Cross-sections through the αN-end head-to-head open and closed interfaces. The open interface contains no hydrophobic core and is asymmetrical in nature, whereas the closed interface is formed of symmetry-related chains and contains a hydrophobic core of residues L102, L109 and I116. (f) Superposition of the two unique chains of the αN-end (101-206) structure (cyan and red) with the sole chain of the αN-end truncated (101-175) structure (blue); rmsd values between all chains are shown. Whilst αN-end chain A and αN-end truncated adopt similar conformations at the N-terminus, αN-end chain B deviates to form the open conformation. Chain A and B of αN-end further differ in angulation along the length of the dimeric coiled-coil.

Supplementary Figure 4 SYCP1 N-terminal self-assembly requires both αN-end and αN-tetramer sequences.

(a) Far UV CD spectra and (b) CD thermal denaturation of SYCP1 αN (101-362) (black) and Ntail~αN (1-362) (grey). Helical content was estimated (as indicated) through deconvolution of spectra with data fitted at normalised rmsd values of 0.007 and 0.012 respectively. Thermal denaturation was recorded as % unfolded based on the helical signal at 222 nm; melting temperatures were estimated at 41 °C for both αN and Ntail~αN. (c) Far UV CD spectra of SYCP1 αN-end (101-206) (black), αN-end truncated (101-175) (grey) and Ntail~αN-end (1-175) (wide dashes). Helical content was estimated (as indicated) through deconvolution of spectra with data fitted at normalised rmsd values of 0.005, 0.010 and 0.021 respectively. (d) CD thermal denaturation of SYCP1 αN-end (101–206) (black) and αN-end truncated (101-175) (grey). Thermal denaturation was recorded as % unfolded based on the helical signal at 222 nm; melting temperatures were estimated at 24 °C and 23 °C for αN-end and αN-end truncated respectively. (e) SEC-MALS analysis revealing that SYCP1 αN and Ntail~αN undergo assembly in solution into large molecular weight species. SYCP1 αN forms species of 3-25 MDa; whilst SYCP1 Ntail~αN forms similar assemblies of 2-12 MDa, it is also present in smaller species of 0.15-1 MDa.

Supplementary Figure 5 Crystal structure of SYCP1 αC-end (676–770).

(a-b) Sample 2Fo-Fc electron density maps contoured at 1.2σ and superimposed on the refined crystallographic models for (a) SYCP1 αC-end (676-770) crystal form 1 and (b) SYCP1 αC-end (676-770) crystal form 2. (c) Crystal structure of SYCP1 αC-end (676–770) in I4122 spacegroup (crystal form 2) demonstrating an anti-parallel tetrameric assembly of length 145 Å. The structure includes a central tetrameric interface flanked by C703 pinch points that lead to lateral four-helical bundles. (d) Superposition of SYCP1 αC-end (676–770) structures in C2 spacegroup (crystal form 1) (red) and I4122 spacegroup (crystal form 2) (blue) with an rmsd of 1.39 Å. Unique chains of crystal form 1 are indicated. Crystal form 2 is symmetrical; both ends contain C703 pinch points consisting of partial disulphide interactions (occupancies of 38% cysteine, 62% disulphide), with flanking chains undergoing a smooth angulation of 17° at residue E731. This is most similar to the disulphide end of crystal form 1 in which flanking chains A and C have smooth angulations at E731 of 16 and 18°, and is distinct from the non-disulphide end in which flanking chains B and D have sharp angulations at E731 of 28 and 31°. (e) Superposition of the SYCP1 αC-end (676-770) C2 spacegroup structure (crystal form 1) (blue) with itself following 180° rotations in both x- and y-axes (red), such that chains ABCD are mapped to DCBA, with an rmsd of 3.44 Å. This highlights the differences between the smoother disulphide end and the sharply angled non-disulphide end in which flanking chains are formed by chains A-C and B-D respectively. (f) Superposition of the four unique chains of the SYCP1 αC-end (676-770) structure in C2 spacegroup (crystal form 1) (cyan, red, yellow and green) with the sole chain of the structure in I4122 spacegroup (crystal form 2) (blue); rmsd values between all chains are shown. (g) The SYCP1 αC-end (676-770) I4122 structure (crystal form 2) coloured according to amino acid conservation amongst vertebrate sequences (red = highly conserved; blue = poorly conserved). (h) Theoretical model of the central tetrameric interface of the H717W Y721F mutant. The phenylalanine and tryptophan residues provide a hydrophobic core that eliminates the pH-sensitivity of histidine and removes the central hydrogen bonding network, but with the indole nitrogen of tryptophan retaining hydrogen bonding with flanking residue Q720. (i) Superposition of the SYCP1 αC-end (C2 spacegroup; red) and the tetrameric assembly present within the crystal lattice of a structure of a similar fragment of SYCP1 (PDB 4YTO; blue) that was misinterpreted as an anti-parallel dimer on the basis of asymmetric unit contents (Seo, E.K. et al., PLoS One. 11, e0161379, 2016). The structures have an rmsd of 2.46 Å.

Supplementary Figure 6 SYCP1 αC-end is a parallel dimer that undergoes pH-induced tetrameric self-assembly.

(a) Far UV CD spectra and (b) CD thermal denaturation of SYCP1 αC-end (676-770) at pH 7.5 or 8.0 (solid) and pH 5.5 (dashed). Helical content at pH 7.5 was estimated (as indicated) through deconvolution of spectra with data fitted at a normalised rmsd values of 0.004. Thermal denaturation was recorded as % unfolded based on the helical signal at 222 nm; melting temperatures were estimated at 35 °C and 38 °C for pH 8.0 and pH 5.5 respectively. (c-e) SEC-SAXS analysis of SYCP1 αC-end (676-770) and αC-end tethered dimer at pH 5.5 and pH 8.0. (c) Scattering intensity plots with the fits used for P(r) distributions shown as black lines. (d) Guinier analysis to determine the radius of gyration (Rg) with linear fits shown in black. Q.Rg values were < 1.3. (e) Guinier analysis to determine the radius of gyration of the cross-section (Rc). Q.Rc values were < 1.3. (f-h) SEC-MALS analysis of SYCP1 αC-end fusion proteins; light scattering (LS) and differential refractive index (dRI) are shown as solid and dashed lines respectively, with fitted molecular weights (Mw) plotted as diamonds across elution peaks. (f) MBP-αC-end (black, right), αC-end-MBP (grey) and MBP-αC-end-MBP (black, left) are dimeric species of 94 kDa, 87 kDa and 161 kDa respectively (theoretical dimers – 112 KDa, 104 kDa and 194 kDa). (g) At pH 5.5, MBP-αC-end is a 214 kDa tetramer (theoretical tetramer – 224 kDa). (h) GST-αC-end (black) is an 80 kDa dimer (theoretical dimer – 82 kDa); the GST dimer of 58 kDa (theoretical dimer – 59 kDa) is shown for comparison (grey). (i-k) SEC-SAXS analysis of MBP and GST fusions of αC-end at pH 8.0, and MBP-αC-end at pH 5.5. (i) Scattering intensity plots. (j) Guinier analysis to determine the radius of gyration (Rg). The Guinier region of MBP-αC-end-MBP was too narrow for analysis; its Rg value was calculated as 86 Å from its real space P(r) distribution. (k) P(r) distributions of GST-αC-end (black) and GST (grey) reveal maximum dimensions of 194 Å and 99 Å respectively.

Supplementary Figure 7 SYCP1 αC-end extended undergoes pH-induced tetrameric self-assembly.

(a) Far UV CD spectra and (b) CD thermal denaturation of SYCP1 αC-end extended (676-783) at pH 7.5 or 8.0 (solid) and pH 4.6 (dashed). Helical content at pH 7.5 was estimated (as indicated) through deconvolution of spectra with data fitted at a normalised rmsd value of 0.004. Thermal denaturation was recorded as % unfolded based on the helical signal at 222 nm; melting temperatures were estimated at 46 °C and 60 °C for pH 8.0 and pH 4.6 respectively. (c) SEC-MALS analysis reveals that SYCP1 αC-end extended undergoes assembly from a 25 kDa dimer (theoretical dimer – 26 kDa) at pH 8.0 (grey) to a 48 kDa tetramer (theoretical tetramer – 52 kDa) at pH 4.6 (black). Light scattering (LS) and differential refractive index (dRI) are shown as solid and dashed lines respectively, with fitted molecular weights (Mw) plotted as diamonds across elution peaks. (d-i) SEC-SAXS analysis of SYCP1 αC-end extended at pH 4.6 and pH 8.0. (d) Scattering intensity plots, (e) Guinier analysis to determine the radius of gyration (Rg) with linear fits shown in black (Q.Rg values were < 1.3) and (f) Guinier analysis to determine the radius of gyration of the cross-section (Rc) (Q.Rc values were < 1.3). (g) P(r) distributions of SYCP1 αC-end extended reveal maximum dimensions of 170 Å at pH 4.6 (black) and pH 8.0 (grey), matching its theoretical coiled-coil length of 167 Å. Cross-sectional radius of gyration (Rc) values of 10.7 Å and 7.3 Å correspond to the known dimensions of four-helical and dimeric coiled-coils respectively. (h-i) SAXS ab initio models of the tetrameric and dimeric conformations of SYCP1 αC-end extended at (h) pH 4.6 and (i) pH 8.0. The models resulting from 10 independent DAMMIF runs were averaged, with mean normalised spatial discrepancy (NSD) values of 0.654 (±0.042) and 0.690 (±0.059) respectively. The χ2 values of reference DAMMIF models for averaging were 1.99 and 1.98 respectively. The αC-end tetrameric crystal structure and a theoretical dimeric coiled-coil of 108 amino acids were docked into the respective envelopes. (j-l) SEC-MALS analysis of SYCP1 αC-end extended mutants. (j) H717W Y721F (black) forms 40 kDa tetrameric assemblies at pH 8.0 (theoretical tetramer – 52 kDa); wild type is shown in grey for comparison. (k) H717E (black) fails to undergo pH-induced assembly and remains as a 27 kDa dimer (theoretical dimer – 26 kDa) at pH 4.6; wild type is shown in grey for comparison. (l) L679A I688A (black) fails to undergo pH-induced assembly and remains mostly as a 26 kDa dimer (theoretical dimer – 26 kDa) at pH 4.6; wild type is shown in grey for comparison.

Supplementary Figure 8 The unstructured C-terminal tail of SYCP1 contains obligate DNA-binding sequences.

(a) Far UV CD spectra and (b) CD thermal denaturation of SYCP1 αC-end extended (640-783) (black) and αC-end~Ctail (640-976) (narrow dashes). Helical content was estimated (as indicated) through deconvolution of spectra with data fitted at a normalised rmsd values of 0.003 and 0.006 respectively. Thermal denaturation was recorded as % unfolded based on the helical signal at 222 nm; melting temperatures were estimated at 56 °C and 47 °C respectively. (c) SEC-MALS analysis reveals that MBP-αC-end~Ctail is a 143 kDa dimer (theoretical dimer – 168 kDa). Light scattering (LS) and differential refractive index (dRI) are shown as solid and dashed lines respectively, with fitted molecular weights (Mw) plotted as diamonds across elution peaks. (d) Electrophoretic mobility shift assay showing that αC-end extended (640–783) fails to bind a linear double-stranded DNA substrate at pH 8.0 over the concentration range at which αC-end~Ctail readily interacts. (e-g) SEC-MALS analysis. (e) MBP-αCore~Ctail (101–976) forms large molecular weight assemblies of 0.3–4 MDa. (f) MBP-αC-dimer~Ctail (358–976) is a 206 kDa dimer (theoretical dimer - 235 kDa). (g) MBP-Ctail (784-976) is a 68 kDa monomer (theoretical monomer - 67 kDa). (h) Electrophoretic mobility shift assays showing that αCore (101–783) and αC-dimer (358–783) fail to bind linear double-stranded DNA substrates at pH 8.0 over the concentration range at which αCore~Ctail (101–976) and αC-dimer~Ctail (358–976) readily interact.

Supplementary Figure 9 Purification and characterization of refolded full-length SYCP1.

(a-b) Full length SYCP1 was purified following insoluble expression in bacteria through denaturation in 8 M urea (buffer 2) followed by refolding through dialysis into 500 mM L-arginine (buffer 3) and subsequent removal of L-arginine by dialysis (buffer 1). This resulted in the formation of a hydrogel of SYCP1 and sheared bacterial genomic DNA. The removal of DNA from denatured material through ion exchange chromatography completely abrogated hydrogel formation following the refolding steps, liberating soluble refolded full length SYCP1. (c) Far UV CD spectra and (d) CD thermal denaturation of SYCP1 full length (1–976) and ΔCtail (1–783). Helical content was estimated (as indicated) through deconvolution of spectra with data fitted at a normalised rmsd values of 0.005 and 0.006 respectively. Thermal denaturation was recorded as % unfolded based on the helical signal at 222 nm; melting temperatures were estimated at 38 °C and 41 °C respectively. (e-f) SEC-MALS analysis demonstrating that refolded SYCP1 full length and ΔCtail form large molecular weight assemblies. Light scattering (LS) and differential refractive index (dRI) are shown as solid and dashed lines respectively, with fitted molecular weights (Mw) plotted as diamonds across elution peaks.

Supplementary Figure 10 Model for the three-dimensional assembly of SYCP1 within the synaptonemal complex.

(a) Speculative model of the three-dimensional organisation of SYCP1 within the synaptonemal complex. Two parallel layers of SYCP1 lattices, formed of midline horizontally orientated αN-end head-to-head interactions, are connected together through vertically orientated αC-end back-to-back assemblies within the lateral elements. This model is compatible with our findings regarding the in vitro structure and self-assembly of SYCP1, and with the known presence of two parallel chains of SYCP1 N-termini that are vertically separated by up to 100 nm and single chain of C-termini within the native synaptonemal complex (Schucker, K. et al., Proc Natl Acad Sci U S A. 112, 2029-33, 2015; Hernandez-Hernandez, A. et al., J Cell Sci. 129, 2016). In contrast, an alternative model in which vertically orientated αN-end head-to-head interactions connect SYCP1 planes in the midline would place N-termini in essentially the same horizontal plane, contradicting their observed vertical separation (Schucker, K. et al., Proc Natl Acad Sci U S A. 112, 2029-33, 2015). (b) Three-dimensional model of the mature SC in which the SYCP1 lattice is stabilised and elongated through central element assembly. Central element components may act as vertical and longitudinal supports between adjacent SYCP1 αN-tetramers to rigidify the SYCP1 hemi-lattice from each chromosome, orientating αN-ends in a manner conducive to long range cooperative head-to-head assembly. They may further act as transverse bridges that directly connect SYCP1 hemi-lattices across the midline to reinforce SYCP1 head-to-head interactions. (c) The SYCP1 lattice can accommodate slight narrowing and widening of the SC. Increased angulation of midline αN-end head-to-head interactions places opposing αN-tetramers closer together, thereby narrowing both the central region and central element. The geometry of the SYCP1 lattice means that this also increases the angulation of αC-dimer transverse filaments, reducing the density of C-terminal interactions with the chromosome axis. Thus, alterations in the structure of the chromosome axis may be transmitted through the SYCP1 lattice to result in narrowing or widening of the central region and central element. This may account for the approximately 10% narrowing of the central region and central element that has been observed in the SCs of female mice in comparison with males (Agostinho, A. et al., J Cell Sci. 131, 2018).

Supplementary information

Supplementary Figures 1–10 and Supplementary Table 1

Supplementary Figures 1–10 and Supplementary Table 1

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Supplementary Dataset 1

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Fig. 1: The obligate tetrameric structure of SYCP1.
Fig. 2: Crystal structures of the SYCP1 αN-end head-to-head assembly in open and closed conformations.
Fig. 3: Head-to-head assembly interfaces of SYCP1 αN-end.
Fig. 4: SYCP1 N-terminal self-assembly into higher order structures is mediated by αN-end head-to-head interactions.
Fig. 5: Crystal structure of the SYCP1 C-terminal tetrameric assembly.
Fig. 6: SYCP1 αC-end undergoes pH-induced assembly into an antiparallel tetramer.
Fig. 7: DNA binding by SYCP1.
Fig. 8: Meiotic chromosome synapsis through SYCP1 self-assembly.
Supplementary Figure 1: Summary of SYCP1 constructs.
Supplementary Figure 2: SYCP1 is a parallel tetramer that bifurcates into two C-terminal coiled-coil dimers.
Supplementary Figure 3: Crystal structures of SYCP1 αN-end (101–206) and αN-end truncated (101–175).
Supplementary Figure 4: SYCP1 N-terminal self-assembly requires both αN-end and αN-tetramer sequences.
Supplementary Figure 5: Crystal structure of SYCP1 αC-end (676–770).
Supplementary Figure 6: SYCP1 αC-end is a parallel dimer that undergoes pH-induced tetrameric self-assembly.
Supplementary Figure 7: SYCP1 αC-end extended undergoes pH-induced tetrameric self-assembly.
Supplementary Figure 8: The unstructured C-terminal tail of SYCP1 contains obligate DNA-binding sequences.
Supplementary Figure 9: Purification and characterization of refolded full-length SYCP1.
Supplementary Figure 10: Model for the three-dimensional assembly of SYCP1 within the synaptonemal complex.