Structural insights into arginine symmetric dimethylation by PRMT5

Litao Suna,b,1,MingzhuWanga,1, Zongyang Lva,b, Na Yanga,YingfangLiua, Shilai Baoc,WeiminGonga,2, and Rui-Ming Xua,2

aNational Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China; bGraduate University of Chinese Academy of Sciences, Beijing 100049, China; and cState Key Laboratory of Molecular and Developmental Biology, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101, China

Edited by* Dinshaw J. Patel, Memorial Sloan-Kettering Cancer Center, New York, NY, and approved October 19, 2011 (received for review May 2, 2011)

Symmetric and asymmetric dimethylation of arginine are isomeric shares high with its human counter- protein posttranslational modifications with distinct biological part, and the recombinant protein displays robust and specific effects, evidenced by the methylation of arginine 3 of histone H4 symmetric arginine dimethylase activity in vitro (Fig. 1). The (H4R3): symmetric dimethylation of H4R3 leads to repression of structure shows the PRMT5 is composed of four clearly defined expression, while asymmetric dimethylation of H4R3 is asso- domains, a previously unsuspected TIM-barrel at the N-terminal ciated with gene activation. The catalyzing these modifi- end, a middle Rossmann-fold domain, a C-terminal β-barrel cations share identifiable sequence similarities, but the relationship domain, and a ∼60 residue dimerization domain inserted be- between their catalytic mechanisms is unknown. Here we analyzed tween β1 and β2 of the β-barrel domain (Fig. 2A). The first three the structure of a prototypic symmetric arginine dimethylase, domains are packed in a triangular manner, with direct contacts PRMT5, and discovered that a conserved phenylalanine in the between sequential domains, and the oligomerization domain active site is critical for specifying symmetric addition of methyl bridges the TIM-barrel and β-barrel domains. The structure of groups. Changing it to a methionine significantly elevates the the SAH-bound PRMT5 differs from that of free protein in overall methylase activity, but also converts PRMT5 to an enzyme that a N-terminal loop (L0) and helix (αA) are ordered in the that catalyzes both symmetric and asymmetric dimethylation of SAH-bound structure. We will use the SAH-bound structure for arginine. Our results demonstrate a common catalytic mechanism analysis unless explicitly noted. BIOCHEMISTRY intrinsic to both symmetric and asymmetric arginine dimethylases, PRMT5 exists as a homodimer, shown both in the crystal struc- and show that steric constrains in the active sites play an essential ture and in solution, as determined by analytic ultracentrifugation role in determining the product specificity of arginine methylases. (Fig. 2B, Fig. S1). The dimeric interface buries a total pair wise 2 This discovery also implies a potentially regulatable outcome of surface area of 2;305 Å , and the intermolecular interactions arginine dimethylation that may provide versatile control of eukar- occur between the dimerization domains and that between the yotic gene expression. TIM-barrel and β-barrel domains. Human PRMT5 has a shorter oligomerization domain, but extensive conservation of amino histone methylation ∣ transtriptional regulation ∣ RNA splicing ∣ acids involved in intermolecular interactions implies that it also crystal structure forms a dimer, consistent with the report of dimeric and higher oligomeric forms of human PRMT5 (5). In fact, all arginine methy- rotein arginine methyltransferase 5 (PRMT5) catalyzes the lases with known structures dimerize via a homologous dimeriza- Pevenly addition of two methyl groups to the two ω-guanidino tion domain, also known as the dimerization “arm” (Fig. 3A), G 0G nitrogen atoms of arginine, resulting in ω-N , N symmetric (22–26). Thus, protein dimerization appears to be an evolutionarily dimethylation of arginine (sDMA) of the target protein (1–5). conserved property of arginine methylases, although the functional PRMT5 functions in the nucleus as well as in the cytoplasm, significance remains poorly understood. and its substrates include histones, spliceosomal , tran- scription factors, and proteins involved in piRNA biogenesis Comparison with Arginine Asymmetric Dimethylases. The overall (6). Symmetric dimethylation of these proteins profoundly impact fold and spatial positioning of the Rossmann-fold and β-barrel many biological processes; e.g., epigenetic control of gene expres- domains are similar to that of type-I enzymes, represented by sion (7), splicing regulation (2, 3, 8, 9), circadian rhythms (9, 10), PRMT1 and CARM1 (Fig. 3A) (24–26). The root-mean-squared DNA damage response (11, 12), and germ development and deviation of Cα positions between PRMT5 and PRMT1 is pluripotency (13–16). Interestingly, both PRMT5 and a group of approximately 2.1 Å when both the Rossmann-fold and β-barrel asymmetric (type-I) arginine dimethylases, which add two methyl domains are compared, whereas it is 1.4 Å for the Rossmann-fold groups to the same ω-guanidino nitrogen atom (aDMA), share domain alone. In particular, a segment including a N-terminal common recognition sequences, and the target arginine can often loop (L0) and a following helix (αA) (a.a. 359–380) became be symmetrically or asymmetrically dimethylated. Yet, these iso- ordered upon SAH binding. Helix αA is positioned similar to that meric modifications have distinct biological effects. One such ex- ample occurs at arginine-3 of histone H4 (H4R3). Symmetric dimethylation of H4R3 has been linked to repression of gene Author contributions: L.S., Y.L., S.B., W.G., and R.-M.X. designed research; L.S., M.W., Z.L., – N.Y., and R.-M.X. performed research; S.B. contributed new reagents/analytic tools; L.S., expression (17 19), while asymmetric dimethylation of H4R3 is M.W., Z.L., N.Y., Y.L., S.B., W.G., and R.-M.X. analyzed data; and L.S., M.W., and R.-M.X. associated with gene activation (20, 21). The startling difference wrote the paper. in biological effects of sDMA and aDMA modifications necessi- The authors declare no conflict of interest. tates the understanding of the enzymatic mechanisms differentiat- *This Direct Submission article had a prearranged editor. ing the two chemically isomeric but functionally antagonistic Data deposition: The atomic coordinates have been deposited in the , posttranslational modifications. www.pdb.org (PDB ID codes 3UA3 and 3UA4). 1L.S. and M.W. contributed equally to this work. Results 2To whom correspondence may be addressed. E-mail: [email protected] or wgong@ Overall Structure. We have determined the crystal structures of sun5.ibp.ac.cn. full-length PRMT5 from Caenorhabditis elegans, alone and in This article contains supporting information online at www.pnas.org/lookup/suppl/ complex with S-Adenosyl-L-homocysteine (SAH). The nematode doi:10.1073/pnas.1106946108/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1106946108 PNAS Early Edition ∣ 1of6 Downloaded by guest on September 29, 2021 Fig. 1. Structural and functional conservation of PRMT5. (A) A schematic representation of domain structures of C. elegans and human PRMT5, and a re- presentative type-I arginine methylase, PRMT1 of rat. The lengths of the boxes are approximately drawn in scale with the protein lengths, and the residue numbers at domain boundaries are labeled. Areas filled in tan, cyan, green, and yellow represent TIM-barrel, Rossmann-fold, β-barrel, and oligomerization domains, respectively. Levels of identity and similarity of the individual domains between C. elegans and human PRMT5s, and that between human PRMT5 and rat PRMT1 are shown. (B) Sequence alignment. The full-length sequences of C. elegans and human PRMT5s, and the regions of the solved structures of rat PRMT1 and mouse CARM1 are aligned. Residues conserved in all four proteins are shown in white letters over purple background, and similar residues are indicated with red letters. Residues conserved in PRMT5 proteins and type-I arginine methylases are highlighted tan and yellow, respectively. Blue stars mark the CePRMT5 residues subjected to mutagenesis. At the top of the sequences, a schematic representation of the secondary structure elements of CePRMT5 is shown. Every ten residues are indicated with a “·” sign. (C) Enzymatic activity assay. Top box, coomassie-stained gel of enzymes and substrate (histone H4) used. GST-tagged rat PRMT1 and poly(His)-tagged C. elegans PRMT5 were expressed in E. coli, and flag-tagged human PRMT5 was purified from HEK293 cells. Approximately 5 μg of enzymes and histone H4 each were used in the assay. Top 2nd box, autoradiograph generated with the use of 0.25 mCi of SAM with tritiated methyl group. Top 3rd box, Western blot detection of asymmetrically dimethylated histone H4R3. Bottom box, Western blot detection of symme- trically dimethylated histone H4R3.

found in type-I enzymes, sheltering SAH from exposing to the PRMT5 proteins (Pro366, Leu367, and Leu371) and forms a solvent and creating a secluded catalytic active site. However, solvent inaccessible area for catalysis; (ii) the N-terminal end key residues responsible for the disordered-to-ordered conforma- of L0 makes a U-turn and contacts the dimerization domain, tional transition upon SAH/SAM binding are separately con- which stabilizes the loop in a conformation endowed with the served among PRMT5 family members (Fig. 1B). Among which, ability to influence substrate binding (Figs. 3 A and B). Hence, Tyr376 and Phe379 on αA interact with the ribose and homocys- the highly conserved PRMT5 loop is important for SAH/SAM teine moieties via hydrogen bonds and van der Waals contacts, binding, as well as in a position to regulate substrate binding. respectively. Loop L0 contains several amino acids uniquely conserved in PRMT5s across species. The corresponding region The Active Site. The active site of PRMT5 is identified by the loca- in PRMT1 is disordered, and that of CARM1 adopts a helical tion of the sulfur atom of SAH and a pair of invariant glutamate conformation. This PRMT5 loop has two apparent functions, residues, Glu499 and Glu508, found in all protein arginine (i) contact the SAH/SAM molecule via residues conserved in methylases (Fig. 3B). These two residues are located on a hairpin

2of6 ∣ www.pnas.org/cgi/doi/10.1073/pnas.1106946108 Sun et al. Downloaded by guest on September 29, 2021 Fig. 2. Overall structure of PRMT5. (A) A ribbon representation of PRMT5 monomer. Domains are colored as in Fig. 1A. Helices and strands are labeled TA to TH and T1 to T8, respectively, for the TIM-barrel domain; αAtoαFandβ1toβ5, respectively, for the Rossmann-fold domain; and OA-OB and O1-O2, respectively, for the oligomerization domain. Strands in the β-barrel domain are labeled b1 to b10. L0 indicates the N-terminal loop of the Rossmann-fold domain, and the SAH molecule is shown in a stick model. (B) A PRMT5 dimer is shown as a ribbon model superimposed onto a surface representation. The surface for one monomer is colored light green and the other in light blue. The red line approximately traces the dimeric interface.

loop connecting β4 and αF, also known as the “double-E” lases are Met, Arg, Tyr, and His, respectively, and they are also loop (24), and are absolutely required for enzymatic activities conserved among type-I enzymes. We reasoned that some of (Fig. S2). An examination of the active site reveals that there these differences might be important for PRMT5’s catalytic ac- are four residues separately conserved among PRMT5 proteins. tivity, and carried out analyses of these residues by mutagenesis. These four residues are Phe379, Lys385, Ser503, and Ser669 Interestingly, changing Phe379 to a methionine (F379M) resulted

(Fig. 1B). The corresponding residues in type-I arginine methy- in a more active enzyme, while an F279Y mutant is inactive, and BIOCHEMISTRY

Fig. 3. Structural features of the PRMT5 active site. (A) Structural comparison with type-I arginine methylases PRMT1 (Pdb id: 1OR8; magenta) and CARM1 (Pdb id: 2V74; light blue). For visual clarity, only three regions of major differences, enclosed in red circles and labeled I, II and III, are superimposed onto the structure of PRMT5. (B) An up-close view of the active site. Key residues of PRMT5 (carbon: yellow; oxygen: red; nitrogen: blue) and the SAH molecule (carbon: orange; sulfur: gold) are shown in a stick model superimposed with a ribbon representation of PRMT5. The double-E loop of PRMT1 (while ribbon), Glu153 on it, and the bound substrate arginine (stick model; carbon: white) are also shown. (C) Methylase activities of Phe379 mutants. Top box: coomassie-stained gel of enzymes and substrate used. Bottom box: autoradiography detection. (D) Circular dichroism spectra of the wild-type and Phe379 mutants of PRMT5.

Sun et al. PNAS Early Edition ∣ 3of6 Downloaded by guest on September 29, 2021 to an alanine or a glycine pronouncedly reduced the (Fig. 4C). Furthermore, we probed whether the F379M mutant enzymatic activity (Fig. 3C). Circular dichroism spectra show that exclusively carries out symmetric dimethylation of arginine. Sur- the observed differences in enzymatic activities are not due to prisingly, both symmetric and asymmetric dimethylation of H4R3 gross structural alterations caused by mutations (Fig. 3D). were detected (Fig. 4B). This phenomenon appears to be specific of other conserved residues near the active site, such to the change to a methionine, as no such activity was detected as changing Ser503 to a tyrosine (S503Y) or a double substitution for the F379A and F379G mutants (Fig. S3). These observation of Val668 and Ser669 to a threonine and a histidine (V668T/ imply that: (i) symmetric and asymmetric dimethylation of argi- S669H), greatly diminished the enzymatic activity (Fig. S2). nine shares a common catalytic mechanism, as the same active ’ These residues are unlikely to be directly involved in catalysis, as site is involved; (ii) Phe379 occupies a key position for PRMT5 s suggested by their distances from the SAH molecule. Val668 and sDMA product specificity. The above observation should hold Ser669 are situated on a β-barrel domain loop between strands b5 for all type-II PRMTs, as the phenylalanine is absolutely con- and b6 that interacts with critical elements of the Rossmann-fold served among them. The corresponding mutation (F327M) in hu- domain: the αA-αB junction and the double-E loop. The confor- man PRMT5 also resulted in the gaining of asymmetric arginine mation of this loop is considerably different from those in type-I dimethylase activity (Fig. 4D). arginine methylases (Fig. 3A, region II). Ser503 is located on the Discussion tip of the double-E loop and makes hydrogen bonds with the car- A surprising finding of this study is the role of Phe379 in specify- bonyl and amide groups of Phe671 of this β-barrel domain loop. ing sDMA specificity of PRMT5. Phe379’s conformation is tightly Thus, the mutagenesis data of Ser503 and Val88/Ser669 indicate fixed in the structure, and its closest distance to the sulfur atom of that interdomain contacts mediated by the b5–b6 loop is critical SAH is ∼4 Å. The structure suggests that Phe379 will be juxta- for precise positioning of key residues for catalysis. posed with the methyl group of SAM and the substrate arginine, thus, serving as a steric factor restricting the reaction product to Determinants for Arginine Symmetric Dimethylation. An in-depth sDMA. A methionine in this position has more conformational evaluation of the enzymatic property of the F379M mutant shows flexibility, as seen from the structures of PRMT1 and CARM1, that it is more active than the wild-type enzyme over a broad which may allow the production of both sDMA and aDMA. range of enzyme concentrations (Fig. 4A). Enzyme kinetic mea- We also mutated the corresponding methionine in PRMT1 to surements indicate that a drop of the Km value is largely respon- a phenylalanine to see if the reverse is true, but the result is in- sible for the elevated enzymatic activity of the mutant enzyme conclusive due to a much lowered level of the mutants’s overall

Fig. 4. Enzymatic properties of the F379M mutant. (A) Top box: coomassie-stained gel showing varying amounts of the wide-type and the F379M mutant of PRMT5, and a constant amount of histone H4 (5.0 μg) used for enzymatic assay. “C” indicates no enzyme added. Bottom box: autoradiography detection with 0.25 mCi of [3H]-SAM used in each reaction. (B) Western blot detection of asymmetric (middle box) and symmetric (bottom box) dimethylation of histone H4R3. Top box is a coomassie-blue stained gel showing proteins used in the activity assays. Please note, for a comparable level of Werstern blot signal, the amount of the F379M protein is adjusted to ∼1∕10 of the wild-type protein, and the amount of PRMT1 is even smaller. (C) Double reciprocal plot analysis of the wild-type and F379 mutant of PRMT5. Derived kinetic parameters are tabulated. (D) Conserved property of the Phe-to-Met mutants of human and nematode PRMT5s. Top two boxes: coomassie staining of the wild-type and mutant enzymes and the substrate used. The right pointing arrow indicates the position of human PRMT5 proteins; an asterisk indicates the position of rat PRMT1; and the left pointing arrow marks the position of C. elegans PRMT5. Third box: Western blot detection of the flag-tagged wild-type and mutant human PRMT5. Fourth box: Western blot detection of asymmetrically dimethylated H4R3. Bottom box: Western blot detection of symmetrically dimethylated H4R3.

4of6 ∣ www.pnas.org/cgi/doi/10.1073/pnas.1106946108 Sun et al. Downloaded by guest on September 29, 2021 enzymatic activity. It should be noted that changing Phe379 of beamline BL17U of Shanghai Synchrotron Radiation Facility (SSRF) using a PRMT5 to a methionine appears to relax PRMT5's sDMA con- Mar CCD-225 detector (Mar Research). Diffraction data were processed with P2 2 2 straint rather than a complete switch to aDMA. It is conceivable the HKL2000 software (30). The crystal belongs to the 1 1 1 space group, that other factors are needed in conjunction with Phe379 to and there are two PRMT5 molecules per asymmetric unit. Se sites were found achieve a complete switch. For example, Lys385 and Tyr386 are by SHELXD (31) and PHENIX (32) was used for phasing and generation of an initial model. Iterative cycles of model building and refinement were carried in the vicinity of Phe379 and SAH, and they are exclusively con- out using COOT (33) and PHENIX. The data used for refinement were sub- served in PRMT5 proteins (Fig, 3B). The two amino acids make jected to anisotropic scaling performed using the UCLA Anisotropy Server. hydrogen bonds with the carboxylate group of SAH and the cat- Noncrystallography symmetry restrains, secondary structure restrains, and alytic Glu499. In type-I enzymes, a conserved arginine serves the TLS refinement (four TLS groups: A46-330, A359-734, B46-330, B359-734) functions of the pair of residues in PRMT5. Further structure and were applied to improve the electron density map. The coordinates for function studies are needed to fully delineate requirements for the refined models have been deposited in PDB under the accession codes symmetric vs. asymmetric arginine dimethylation. Another inter- 3UA3 and 3UA4. Detailed statistics of data collection and refinement are esting point is that Phe379 is located on αA, which undergoes shown in Table S1. large conformational changes upon SAH binding. It is conceiva- ble that the conformation of αA may be regulated, such as by Analytical Ultracentrifugation. Protein sample prepared after gel-filtration was diluted to OD280 ¼ 0.8 in a buffer of 20 mM Tris-HCl (pH 7.4) and protein-protein interactions, and the conformational dynamics of α 150 mM NaCl. Sedimentation velocity experiment was carried out with the A is likely to impact the enzyme activity and product specificity ProteomeLab XL-I (Beckman Coulter). Experiment was performed at of the arginine dimethylases. 4,800 rpm at 20 °C for 5 h. Velocity data were collected in a continuous scan This first structure of a symmetric arginine dimethylase also mode at 280 nm, and sedimentation coefficients were calculated with the reveals that the N-terminal domain of PRMT5 has a TIM-barrel program Sedfit (34). fold, and this domain is important for homodimerization. As noted previously, human PRMT5 lacking the N-terminal region Circular Dichroism (CD) Measurements. CD spectra were recorded using an encompassing the TIM-barrel domain is still capable of forming a Applied Photophysics Pi-Star 180 spectropolarimeter at 20 °C, with the pro- dimer at high protein concentrations, but the catalytic activity is tein samples at 0.1 mg∕mL in PBS buffer. Measurements were made under severely compromised (27). This observation suggests that the nitrogen with a 0.1 cm path length. Data were collected at 1 nm wave length TIM-barrel domain has other essential functions in addition to interval from 200 to 260 nm. Mean residue molar ellipticity was calculated using the formula ½θ¼θ × 103∕ðc × l × NÞ, where c is the mean protein con- being important for PRMT5 dimerization. Human PRMT5 was centration in millimolar, θ is the ellipticity in millidegrees (mdeg), l is the path purified as a large protein complex, known as the methylosome, length in millimeters, and N is the number of amino acids. BIOCHEMISTRY that contains pICLn and MEP50 (2, 3, 28). pICLn interacts with the PRMT5 region now known to have a TIM-barrel structure, In Vitro Methylation Assay. Wild-type and various mutants of His-tagged C. and it stimulates PRTM5’s activity towards Sm proteins (27). An- elegans PRMT5, and GST-tagged rat PRMT1 were expressed in E. coli and pur- other protein, RioK1 competes with pICLn for binding to ified using a standard protocol. Flag-tagged human PRMT5 (hPRMT5) or its PRMT5 and directs PRMT5’s activity towards the RNA-binding F327M mutant was prepared from HEK293 cells transfected with a pCMV2b protein (29). Thus, the TIM-barrel domain of PRMT5 vector carrying the wild-type or mutant cDNA, and the recombinant enzyme also serves as a scaffold for the binding of adaptor proteins, such was purified by resins conjugated with anti-FLAG antibody. FLAG-pull down of hPRMT5 samples from cells grown to ∼70% confluency in a 60 mm-die- as pICLn and RioK1. Hence, this domain is important for the μ assembly of PRMT5 complexes and their substrate selectivities. meter dish and transfected with 2 g of plasmid DNA were divided into two equal aliquots for enzymatic reactions. Approximately 5 μgofE. coli ex- In summary, the structural and biochemical properties of the pressed proteins, or an aliquot of hPRMT5 was individually incubated with PRMT5 uncovered here will serve as a guide for understanding 5 μg of histone H4, and 0.25 mCi of adenosyl-L-[methyl-3H] methionine the biochemical mechanisms of all type-II arginine methylases, (Amersham Biosciences, Inc.) at 30 °C for 2 h in a final volume of 20 μL. After and advance the understanding of symmetric arginine dimethyla- the reaction, the mixtures were boiled in the SDS sample buffer and sepa- tion in a wide-spectrum of biological processes. rated by SDS-PAGE. The gels were stained by Coomassie Blue and destained, then treated with Amplify (Amersham Biosciences, Inc.), dried, and exposed Materials and Methods to a film. Expression, Purification, and Crystallization of PRMT5. A bacterial expression plasmid of C. elegans PRMT5 was constructed by cloning the full-length cDNA Enzyme Kinetics Measurements. Enzyme kinetics parameters were determined into a pET21a vector (Novagen). The poly(His)-tagged recombinant protein using the Methyltransferase Colorimetric Assay kit (Cayman, 700140). was produced in the BL21(DE3) strain of Escherichia coli. Protein expression His-tagged PRMT5 and PRMT5-F379M proteins (0.5 μM), in the presence was induced with 0.4 mM IPTG when the cell density reached OD600 ¼ 0.8, of saturating SAM (≥100 μM), and varying concentrations of Histone H4 were after which point the temperature was shifted to 16 °C for 30 h. The poly incubated for 45 min at 37 °C in a 200 μL reaction mixture. During incubation, (His)-tagged PRMT5 was first purified using Ni-IDA resins, followed by anion color formation was assessed at 515 nm with Thermo Scientific Varioskan exchange and gel-filtration column chromatography. High purity fractions Flash (Thermo). Initial velocity data, measured as a function of substrate were pooled and concentrated to ∼8–10 mg∕mL for crystallization. PRMT5 concentration, were analyzed using the Michaelis-Menten equation, V ¼ V ½S∕ð½SþK Þ K ¼ V ∕½E ½E crystals were grown by vapor diffusion in hanging drops at 16 °C in a condi- max m ; and cat max , where is the total enzyme tion with 100 mM Tris (pH 7.0), 9% PEG-5000MME and 5% Tacsimate. concentration. All measurements were done in triplicate. Selenyl-methionine (SeMet) substituted PRMT5 was expressed using a defined medium supplemented with 25 mg∕L of SeMet. SeMet PRMT5 Western Blot Analysis. Western blot analysis was performed with antibodies was purified and crystallized in a manner identical to the wild-type protein. against histone H4 with symmetric dimethyl Arg 3 (ab5823/97454, Abcam), Single or multiple point mutations of PRMT5 were generated by PCR and H4 asymmetric dimethyl Arg 3 (39705, Active Motif), and an anti-FLAG anti- verified by DNA sequencing. Mutant proteins were expressed and purified body (F1804, Sigma). following the same protocol for the wild-type protein. ACKNOWLEDGMENTS. We thank SSRF beamline scientists for technical support Data Collection and Structure Solution. X-ray diffraction data were collected at during data collection. The work was supported by grants from the Ministry liquid nitrogen cryogenic temperature using a cryoprotectant with the well of Science and Technology of China (2009CB825501 and 2010CB944903), solution supplemented with 30% glycerol. A 3.0 Å single wave length anom- the Natural Science Foundation of China (90919029 and 3098801), Chinese alous dispersion (SAD) dataset was collected at a wave length of 0.9792 Å at Academy of Sciences (CAS), and the Novo Nordisk-CAS foundation.

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