Crystal structure of the CRISPR-Cas RNA silencing Cmr complex bound to a target analog.

Article

Crystal Structure of the CRISPR-Cas RNA Silencing Cmr Complex Bound to a Target Analog Graphical Abstract

Authors Takuo Osawa, Hideko Inanaga, Chikara Sato, Tomoyuki Numata

Correspondence [email protected]

In Brief Osawa et al. solved the crystal structure of the CRISPR-Cas Cmr complex bound to a target analog. The complex recognizes the crRNA 50 tag and deforms the guide-target duplex at 6 nt intervals. Together with biochemical experiments, this study revealed the periodic RNA cleavage mechanism by the type III CRISPR-Cas interference machinery.

Highlights d

d

Crystal structure of the Cmr complex bound to a target analog was determined The crRNA 50 tag is recognized and the duplex start site is defined in the complex

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The complex regularly deforms the duplex and degrades the target at 6 nt intervals

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The structure reveals the periodic RNA cleavage mechanism at atomic resolution

Osawa et al., 2015, Molecular Cell 58, 1–13 May 7, 2015 ª2015 Elsevier Inc. http://dx.doi.org/10.1016/j.molcel.2015.03.018

Accession Numbers 3X1L

Please cite this article in press as: Osawa et al., Crystal Structure of the CRISPR-Cas RNA Silencing Cmr Complex Bound to a Target Analog, Molecular Cell (2015), http://dx.doi.org/10.1016/j.molcel.2015.03.018

Molecular Cell

Article Crystal Structure of the CRISPR-Cas RNA Silencing Cmr Complex Bound to a Target Analog Takuo Osawa,1 Hideko Inanaga,1 Chikara Sato,1 and Tomoyuki Numata1,* 1Biomedical Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), 1-1-1 Higashi, Tsukuba-shi, Ibaraki 305-8566, Japan *Correspondence: [email protected] http://dx.doi.org/10.1016/j.molcel.2015.03.018

SUMMARY

In prokaryotes, Clustered regularly interspaced short palindromic repeat (CRISPR)-derived RNAs (crRNAs), together with CRISPR-associated (Cas) proteins, capture and degrade invading genetic materials. In the type III-B CRISPR-Cas system, six Cas proteins (Cmr1–Cmr6) and a crRNA form an RNA silencing Cmr complex. Here we report the 2.1 A˚ crystal structure of the Cmr1-deficient, functional Cmr complex bound to single-stranded DNA, a substrate analog complementary to the crRNA guide. Cmr3 recognizes the crRNA 50 tag and defines the start position of the guide-target duplex, using its idiosyncratic loops. The b-hairpins of three Cmr4 subunits intercalate within the duplex, causing nucleotide displacements with 6 nt intervals, and thus periodically placing the scissile bonds near the crucial aspartate of Cmr4. The structure reveals the mechanism for specifying the periodic target cleavage sites from the crRNA 50 tag and provides insights into the assembly of the type III interference machineries and the evolution of the Cmr and Cascade complexes.

INTRODUCTION Clustered regularly interspaced short palindromic repeat (CRISPR)-Cas (CRISPR-associated) constitutes a prokaryotic adaptive immune system, which is an RNA-based defense system against invading genetic elements (Sorek et al., 2013; Terns and Terns, 2014; van der Oost et al., 2014; Wiedenheft et al., 2012). CRISPR loci contain arrays of short invariant repeats interspaced by variable spacers derived from previously encountered foreign genetic materials (Bolotin et al., 2005; Mojica et al., 2005; Pourcel et al., 2005). The CRISPR arrays are transcribed, and the primary transcripts are processed within each repeat to generate crRNAs (Brouns et al., 2008; Carte et al., 2008; Hale et al., 2008). The crRNA is then loaded into Cas protein(s) to form ribonucleoprotein interference complexes that target and degrade invading nucleic acids complementary to the guide region of the crRNA (Brouns et al., 2008; Hale et al., 2009; Jinek et al., 2012). Based on the signature Cas proteins Cas3, Cas9,

and Cas10, the CRISPR-Cas systems are classified into three major types (I, II, and III, respectively), which are further divided into several subtypes (Makarova et al., 2011b). The interference complexes of the type I and II CRISPR-Cas systems target foreign DNA for degradation (Brouns et al., 2008; Gasiunas et al., 2012; Jinek et al., 2012; Westra et al., 2012). The type III CRISPR-Cas system was previously thought to be categorized into the DNA-targeting type III-A and the RNA-targeting type III-B interference complexes, which are known as the Csm and Cmr complexes, respectively (Hale et al., 2009; Marraffini and Sontheimer, 2008; Zhang et al., 2012). Very recently, however, the type III-A Csm complex was also revealed to target and degrade RNA in vitro (Staals et al., 2014; Tamulaitis et al., 2014). The type III-B Cmr complex in Pyrococcus furiosus comprises six Cas proteins (Cmr1–Cmr6) and a 39 or 45 nt crRNA (Hale et al., 2009, 2012). The crRNAs from the P. furiosus Cmr (PfCmr) complex contain the repeat-derived 8 nt 50 tag, followed by the spacer-derived 31 or 37 nt sequence that functions as a guide (Hale et al., 2009, 2012). A similar complex also exists in Thermus thermophilus, where two crRNA species of 40 and 46 nt were observed (Staals et al., 2013). Cryoelectron microscopy (cryoEM) reconstructions of the PfCmr and T. thermophilus Cmr (TtCmr) complexes revealed the general shapes of these particles at 12 and 26 A˚ resolutions, respectively, and suggested that Cmr4 and Cmr5 form the helical filaments of the complex (Spilman et al., 2013; Staals et al., 2013). The overall shape of the Cmr complex resembles the cryo-EM structures of the type III-A Csm complex of five Cas proteins (Csm1–Csm5) and a crRNA (Rouillon et al., 2013; Staals et al., 2014). Studies of the type III complexes also suggested their structural similarities to the type I-E interference complex known as Cascade (CRISPR-associated complex for antiviral defense), which adopts a seahorse-shaped architecture (Jackson et al., 2014; Jore et al., 2011; Mulepati et al., 2014; Wiedenheft et al., 2011; Zhao et al., 2014). The structural resemblance between the type I and III interference complexes may be attributable to the presence of several orthologs in each system (Makarova et al., 2011a). Although the type I and III interference complexes share a similar helical architecture, they employ distinct mechanisms to degrade their targets. Cascade recruits the helicase-nuclease Cas3 to destroy the target (Brouns et al., 2008; Sinkunas et al., 2013; Westra et al., 2012), while the Cmr and Csm complexes cleave it by their intrinsic nuclease activity (Hale et al., 2009; Staals et al., 2013, 2014; Tamulaitis et al., 2014; Zhang et al., 2012). Molecular Cell 58, 1–13, May 7, 2015 ª2015 Elsevier Inc. 1


Intriguingly, the Cmr complex reportedly degrades the target RNA at multiple positions with 6 nt intervals via a 50 ruler mechanism (Hale et al., 2014; Staals et al., 2013), suggesting the presence of multiple active sites that coordinate the specifically recognized 50 tag in the complex. Recent biochemical experiments also revealed that the Csm complex degrades RNA via a similar mechanism to the Cmr complex (Staals et al., 2014; Tamulaitis et al., 2014), showing their evolutionary conservation in the type III interference process. The crystal structures of the individual Cmr proteins (Cmr1, Cmr2, Cmr4, Cmr5, and Cmr6) and the Cmr2-Cmr3 subcomplex have been determined (Benda et al., 2014; Cocozaki et al., 2012; Osawa et al., 2013; Park et al., 2013; Shao et al., 2013; Sun et al., 2014). These individual structures were fitted into the cryo-EM map of the Cmr complex to construct the structural model, which, together with the mutational analysis, revealed that Cmr4 is the catalytic subunit of the Cmr complex (Benda et al., 2014; Ramia et al., 2014; Zhu and Ye, 2015). However, the precise mechanisms of the Cmr complex assembly, the target recognition by the complex, and the periodic target cleavages from the 50 end of the crRNA remain elusive, because of the lack of structural information at atomic resolution. In this study, to clarify the mechanism of crRNA-guided RNA silencing in the type III system, we determined the crystal structure of the Cmr complex bound to a target analog complementary to the crRNA guide. This is the first crystal structure of a type III CRISPR-Cas system RNAsilencing complex. The structure reveals a number of functional features of the Cmr complex and provides insight into the evolutionary relationship between the type I and III interference machineries. RESULTS AND DISCUSSION Structure Determination To reconstitute the Cmr complex for crystallization, we first prepared the P. furiosus Cmr2dHD-Cmr3 (PfCmr2dHD-Cmr3) and Cmr4-Cmr5-Cmr6 (PfCmr4-Cmr5-Cmr6) subcomplexes, separately. We also purified P. furiosus Cmr1 (PfCmr1). The PfCmr2dHD-Cmr3 complex interacted with the PfCmr4-Cmr5Cmr6 complex, while PfCmr1 easily dissociated from the other protein subunits (data not shown). Consistent with this, previous experiments also suggested the weak interaction between Cmr1 and the other Cmr protein subunits (Benda et al., 2014). We confirmed that the reconstituted PfCmr complex cleaved the 37-mer target RNA in a sequence-specific manner, using the 39-mer Pf7.01-crRNA (see Figure S1 and Table S1 available online). Intriguingly, the PfCmr1-deficient particle (PfCmrD1) also exhibited the same activity as that of the PfCmr complex (Figure S1). Consistently, Cmr1 has been demonstrated to be dispensable for the reaction (Zhu and Ye, 2015). In contrast to these findings, Cmr1 was reported to be essential for the activity of the complex (Hale et al., 2014). Further detailed studies will be necessary to elucidate its requirement for the function of the Cmr complex. Since the PfCmr proteins, except for PfCmr1, form a stable complex, we tried to crystallize PfCmrD1. However, we failed to obtain any crystals of the complex. Since the Archaeoglobus fulgidus genome also encodes the cmr genes with a similar arrangement to the P. furiosus 2 Molecular Cell 58, 1–13, May 7, 2015 ª2015 Elsevier Inc.

counterparts (Figure 1A), we constructed the coexpression and purification systems of the A. fulgidus Cmr2dHD-Cmr3 (AfCmr2dHD-Cmr3) and Cmr4-Cmr5-Cmr6 (AfCmr4-Cmr5Cmr6) subcomplexes. However, the yield of AfCmr2dHD-Cmr3 was too poor to reconstitute a sufficient amount of the A. fulgidus Cmr (AfCmr) complex for the crystallization experiment. Since the AfCmr proteins share sequence identities (27.1%–42.2%) with the PfCmr proteins (Figure 1A), we tested the interaction between PfCmr2dHD-Cmr3 and AfCmr4-Cmr5Cmr6. The interaction of these subcomplexes was confirmed by gel filtration chromatography (Figures 1B–1D). Next, we examined the RNA cleavage activity of the particle composed of PfCmr2dHD-Cmr3, AfCmr4-Cmr5-Cmr6, AfCmr1, and the 39-mer Pf7.01-crRNA, which is referred to as the chimeric Cmr (ChiCmr) complex. The AfCmr1-deficient particle (ChiCmrD1) was also analyzed. The ChiCmr and ChiCmrD1 complexes specifically degraded the 37-mer target RNA in a sequence-specific manner, and produced the same cleavage product as those by the PfCmr and PfCmrD1 complexes (Figures 1E and S1), suggesting that the chimeric particles are functional and adopt the physiologically relevant architectures. To reveal the RNA-silencing mechanism of the type III system, we crystallized ChiCmrD1 in complex with a 31-mer singlestranded DNA (ssDNA) complementary to the crRNA guide (Figure 2A; Table S1). The ChiCmrD1-ssDNA complex structure was determined by the MR-SAD method, and was refined to Rwork/ Rfree of 0.208/0.246 at 2.1 A˚ resolution (Table 1). Structure of the ChiCmrD1-ssDNA Complex The asymmetric unit contains one ChiCmrD1-ssDNA complex, with the protein stoichiometry of Cmr2131435261. As in the previous cryo-EM analyses of the Cmr complexes (Spilman et al., 2013; Staals et al., 2013), the Cmr proteins in ChiCmrD1 are helically arranged and form a groove that wraps around the crRNAtarget duplex (Figures 2B and 2C). The 50 -terminal region of the crRNA contacts Cmr3 and resides at the ‘‘base’’ of ChiCmrD1, while its 30 -terminal region (nucleotides 33–39) protrudes from the complex and is disordered (Figure 2) because of the absence of Cmr1, which reportedly constitutes the ‘‘head’’ of the complex and caps the 30 end of the crRNA (Spilman et al., 2013; Staals et al., 2013). In contrast to the previously reported results, in which several regions were disordered in the isolated Cmr3 (residues 10–31, 141–170, and 197–201) and Cmr4 (residues 260– 284) proteins (Benda et al., 2014; Osawa et al., 2013; Ramia et al., 2014; Shao et al., 2013), these segments in ChiCmrD1 were fully structured upon crRNA binding, indicating the functional role of the crRNA in complex assembly. The helical backbone of ChiCmrD1 buries a large portion of the crRNA guide, and its solvent-exposed bases form Watson-Crick base pairs with the target. In contrast, the target is fully exposed to the solvent, which may facilitate target association with and product dissociation from the complex. The interactions between the Cmr proteins and the crRNA-ssDNA duplex are shown schematically in Figure S2. Two divalent cations, Zn2+ and Mg2+, were observed in the complex structure. The zinc ion is coordinated by four cysteine residues in the D1 domain of Cmr2 (Figure S3A), as reported previously (Cocozaki et al., 2012). The Mg2+ is located at the


Figure 1. Reconstitution of the Chimeric Cmr Complex (A) The arrangements of the cmr genes in P. furiosus and A. fulgidus. Sequence identities between the respective proteins of these two species are shown. (B) Reconstitution of the hybrid complex composed of PfCmr2dHD-Cmr3 and AfCmr4-Cmr5-Cmr6. (C) Gel filtration chromatogram of the reconstituted chimeric Cmr23456. (D) SDS-PAGE analysis of the gel filtration fractions from (C). The pooled fractions are indicated. MK represents molecular markers. (E) RNA cleavage activity of the reconstituted chimeric Cmr complex. The 39-mer Pf7.01-crRNA (red and black) and 37-mer target RNA (blue), which bears a 6 nt overhang at the 50 end, were used. See also Figures S1 and S4 and Table S1.

interface between Cmr3 and the 50 tag of the crRNA, where it is octahedrally coordinated by the side chain of Glu200 from Cmr3, the N7 atom of A6, and four water molecules (Figure S3B). However, these interactions are dispensable, since we found that the E200A mutation of Cmr3 and the A6C mutation of the crRNA did not affect the RNA cleavage activity of ChiCmrD1 (Figure S3C and see below, respectively). ChiCmrD1 is a hybrid complex composed of the PfCmr2dHDCmr3 and AfCmr4-Cmr5-Cmr6 subcomplexes. Many residues at the interfaces between PfCmr3 and AfCmr4, and PfCmr2 and AfCmr5, are identical or highly conserved in these two archaeal species (Figures S4 and S5). Therefore, similar interactions are expected to be observed in the respective PfCmr and AfCmr complexes. These findings, together with the conservation of the 50 tag sequence between the P. furiosus and A. fulgidus crRNAs, show that ChiCmrD1 represents a physiologically relevant complex structure with the native interactions.

Subunit Interactions The Cmr4 structure, which contains the modified RNA-recognition motif (RRM), resembles Cas7, a component of Cascade (Jackson et al., 2014; Mulepati et al., 2014; Zhao et al., 2014) (Figure S6). The molecular shape of Cmr4 is similar to a right hand, and is divided into the palm (residues 1–65, 122–258, and 286–355), finger (residues 66–121), and thumb (residues 259–285) regions (Figures 3A and S6). The three Cmr4 subunits (Cmr4.1–Cmr4.3) in the complex adopt essentially identical conformations, and are arranged in a head-to-tail fashion to form a helical stack (Figure 3B). The thumb is a b-hairpin protrusion, and those of Cmr4.1 and Cmr4.2 are situated close to the fingers of Cmr4.2 and Cmr4.3, respectively (Figure 3B). The Cmr4 stack is capped at one end by Cmr6, which also contains the modified RRM (Figure 3B). Similar to Cmr4, Cmr6 folds into a structure shaped like the right hand and wrist, but the thumb is partially disordered (Figure 3A). The Cmr4.3 thumb extends toward the Cmr6 finger, resulting in the similar arrangements of Molecular Cell 58, 1–13, May 7, 2015 ª2015 Elsevier Inc. 3


Table 1. Data Collection and Refinement Statistics ChiCmrD1-ssDNA Complex/SeMet

ChiCmrD1 Mutant-ssDNA Complex/SeMet

ChiCmrD1-ssDNA Complex/Native

Space Group

P1

P1

P1

Cell Dimensions a, b, c (A˚)

79.1, 79.4, 142.9

75.9, 76.5, 140.5

75.5, 76.2, 139.2

90.7, 103.4, 115.5

88.5, 103.4, 117.3

90.3, 104.8, 118.6

Wavelength (A˚) Resolution (A˚)

0.97919

0.97850

0.98000

50–3.4 (3.60–3.39)

50–2.4 (2.54–2.40)

50–2.1 (2.22–2.10)

Rmerge

0.087 (0.438)

0.114 (0.498)

0.075 (0.505)

I/sI

10.6 (2.9)

12.3 (3.5)

18.3 (4.4)

Completeness (%)

97.5 (94.2)

97.9 (95.9)

97.4 (94.4)

Redundancy

2.9 (2.9)

3.9 (3.9)

7.6 (7.5)

Data Collection

a, b, g ( )

Refinement Resolution (A˚)

44.3–2.1

Number of reflections

148,827

Rwork/Rfree (%)

20.8/24.6

Number of atoms Protein

18,693

crRNA

679

ssDNA

448

Ion

2

Water

130

B factors (A˚2) Protein

38.1

crRNA

34.3

ssDNA

46.2

Ion

27.7

Water

27.9

Rmsd Bond lengths (A˚)

0.003

Bond angles ( )

0.627

Highest-resolution shell is shown in parentheses.

Cmr4.3 and Cmr6 to those in the neighboring Cmr4 subunits (Figure 3B). At the base, the Cmr4 stack is covered by Cmr3, bearing the Nand C-terminal modified RRM domains (Cmr3N and Cmr3C, respectively) (Osawa et al., 2013; Shao et al., 2013), which both also resemble the right hand but lack the finger (Figures 3A and 3B). Cmr3N is assembled with Cmr4.1 in a similar fashion to the arrangements between the neighboring Cmr4 subunits. This interaction directs the Cmr3N thumb, which is disordered in the isolated Cmr2dHD-Cmr3 structures (Osawa et al., 2013; Shao et al., 2013), toward the Cmr4.1 finger (Figure 3B). Consequently, Cmr3, Cmr4.1–Cmr4.3, and Cmr6 become interdigitated and form the helical filament responsible for crRNA binding (Figure 3B). The structure further revealed that the two copies of Cmr5 (Cmr5.1 and Cmr5.2) and the D4 domain of Cmr2, which is structurally similar to Cmr5, assemble in a head-to-tail fashion, forming the second helical filament of the complex (Figure 3C). This helical stack is capped by the wrist of Cmr6 (Figure 3C). The two Cmr5 subunits and the D2 and D4 domains of Cmr2 create 4 Molecular Cell 58, 1–13, May 7, 2015 ª2015 Elsevier Inc.

the binding surface for the target and, together with the first helical filament, form a helical groove for binding the crRNA-target duplex (Figure 2B). Cleavage Sites with 6 nt Intervals The crRNA-ssDNA duplex adopts an unwound ribbon-like structure, instead of the canonical helix (Figure 4A). This is caused by the interactions with the Cmr4 thumbs, which periodically intercalate within the duplex (Figure 4B). These interactions kink the crRNA and ssDNA, causing the base flipping of each complementary nucleotide from the duplex at 6 nt intervals (Figures 4A and 4B). Consequently, the complementary nucleotides at positions 14, 20, and 26 from the 50 end of the crRNA (at positions 26, 20, and 14 from the 50 end of the target, respectively) are rotated by about 90 from the duplex axis, forming three 5 bp segments between the kinks (Figure 4A). This structural feature of the duplex is similar to that observed in Cascade, where Cas7 (an ortholog of Cmr4), which constitutes a helical backbone of Cascade, deforms the guide-target duplex at every sixth base


Figure 2. Crystal Structure of the ChiCmrD1-ssDNA Complex (A) Schematic representation of the crRNA-ssDNA duplex in the crystal. The base pairs within the duplex observed in the crystal are depicted by lines. Disordered nucleotides are colored green. (B) Overall structure of ChiCmrD1 bound to ssDNA. The 50 and 30 ends of the crRNA are shown. The color codes of each molecule are indicated. (C) Schematic representation of the complex with the same color codes. See also Figures S2, S3, and S5.

pair, causing an unwound ribbon-like structure that deviates drastically from the standard forms (Mulepati et al., 2014). The Cmr complex reportedly cleaves the target RNA at multiple positions with a 6 nt periodicity (Hale et al., 2014; Staals et al., 2013). Based on these findings, we hypothesized that the ChiCmr complex cleaves the 30 side phosphate bond of the nucleotides at positions 14, 20, and 26 of the target RNA. To explore the cleavage sites, we designed three synthetic RNAs (named 14d, 14d-20d, and 14d-20d-26d) that contain 20 -deoxyribonucleotide(s) at position(s) 14, 20, and 26 of the target RNA (Figure 4C; Table S1). In contrast to the 37-mer target RNA, which was degraded to the 20 nt product, the 26 and 32 nt degradation products were observed with the 14d and 14d20d substrates, respectively (Figure 4D). The 14d-20d-26d substrate was not degraded (Figure 4D). These results demonstrated that the ChiCmr complex cleaves the target RNA at the three sites deduced from the ChiCmrD1-ssDNA complex structure. Therefore, we concluded that the ChiCmr complex degrades the 37-mer target RNA to the observable 20 nt product, as well as 5 and 6 nt fragments that are undetectable in this assay (Figure 4C). Since the 50 tag of the crRNA is recognized in ChiCmrD1 (see below), our results showed that the cleavage

sites are determined by a 50 ruler mechanism, as suggested previously (Hale et al., 2014; Staals et al., 2013). Active Sites of the Cmr Complex The biochemical experiment using synthetic RNAs with 20 -deoxyribonucleotides revealed that ChiCmrD1 cleaved the three phosphate bonds between nucleotides 14 and 15, 20 and 21, and 26 and 27 in the target RNA (Figures 4C and 4D). These scissile bonds are surrounded on three sides by the Cmr4 thumb, the loop (residues 16–43) in the Cmr4 palm, and either Cmr5 or the D4 domain of Cmr2 (Figure 4E). The strictly conserved Asp31 from Cmr4 is located near (3.7–5.7 A˚) the scissile bonds (Figure 4F). The D31A mutation of Cmr4 drastically affected the target cleavage reaction by the complex (Figure 4G). The D31E and D31N mutants also lost the activity (Figure 4G), demonstrating that Asp31 of Cmr4 constitutes the active sites of the Cmr complex. The Asp31 residues are separated by about 23– 24 A˚ (Figure 4F), in which their arrangements accommodate the 6 nt length in the extended conformation of the nucleic acid. These findings provide insight into the mechanism by which the target RNA is degraded at multiple sites with 6 nt intervals. Recently, the importance of the corresponding aspartate Molecular Cell 58, 1–13, May 7, 2015 ª2015 Elsevier Inc. 5


Figure 3. Subunit Assemblies of the Cmr Proteins (A) Ribbon representations of Cmr3, Cmr4, and Cmr6, colored as follows: palm (gray), finger (cyan), thumb (red), and wrist (blue). The Cmr6 thumb, which is disordered in ChiCmrD1, is depicted by a dashed line. (B) Helical filament composed of Cmr3, Cmr4.1–Cmr4.3, and Cmr6. Each subunit is color coded as in Figure 2. The thumbs of Cmr3N and all Cmr4 subunits are highlighted in red. (C) Helical filament composed of Cmr2, Cmr5.1, Cmr5.2, and Cmr6. For clarity, the D1–D3 domains of Cmr2 and the regions other than the Cmr6 wrist are colored gray. See also Figure S6.

(Asp26 in PfCmr4) for the RNA cleavage reaction by the PfCmr complex was also suggested (Benda et al., 2014; Ramia et al., 2014; Zhu and Ye, 2015). Furthermore, the aspartate is also conserved in Csm3, an ortholog of Cmr4, which oligomerizes to form the helical filament along the crRNA in the Csm complex (Hatoum-Aslan et al., 2013; Makarova et al., 2011a; Rouillon et al., 2013; Staals et al., 2014). This aspartate in Streptococcus thermophilus Csm3 was suggested to constitute the active site of the complex (Tamulaitis et al., 2014). Therefore, the Csm and Cmr complexes in the type III CRISPR-Cas system define the periodic cleavage sites in the same manner. The present study also demonstrated that the 20 -hydroxyl group of the target RNA is crucial for the cleavage activity by the Cmr complex, suggesting that deprotonation of the 20 -hydroxyl group by the general base catalyst facilitates the nucleophilic attack of the scissile bond. This is supported by the finding that the Cmr complex degrades the target RNA into fragments with 30 -phosphate (or 20 , 30 -cyclic phosphate) and 50 -hydroxyl termini (Hale et al., 2009). These products resemble those of the metal ion independent endoribonucleases, such as RNase A and RNase T1. Our structure of the ChiCmrD1-ssDNA complex suggested that Asp31 of Cmr4, the scissile phosphate, and the 20 -hydroxyl group of the target are almost linearly aligned. Given that RNA cleavage proceeds via general acid-base catalysis, Asp31 of Cmr4 might serve as a general acid catalyst to protonate the 50 terminus of the cleavage product during the reaction, since this residue is located on the opposite side of the putative 6 Molecular Cell 58, 1–13, May 7, 2015 ª2015 Elsevier Inc.

20 -hydroxyl group relative to the scissile bond. In contrast, in the present complex structure, no conserved residue is situated near the putative 20 -hydroxyl group. Therefore, we could not identify the candidate for the general base catalyst. In contrast to this characteristic feature of the metal ion independent endoribonucleases, the Cmr complex exhibited Mg2+ dependency for the target RNA degradation (Hale et al., 2009; Staals et al., 2013), which was also confirmed using ChiCmrD1 (data not shown). We did not observe any divalent cation around the scissile bonds of the target, although the complex was crystallized in the presence of 5 mM Mg2+ ion. Furthermore, there is no metal ion-coordinating environment around each cleavage site in the ChiCmrD1-ssDNA complex structure. Therefore, the role of the divalent cation in the Cmr complex-catalyzed reaction has remained elusive. Alternatively, the target RNA may be cleaved through an autocatalytic mechanism, in which Asp31 of Cmr4 is involved in the formation of a suitable environment for the reaction, as suggested previously (Ramia et al., 2014). The structure determination of the Cmr complex bound to a target RNA might be required to reveal the catalytic mechanism and Mg2+ dependency of the complex. Mechanism of 6 nt Length Measurement by the Cmr Complex The backbone phosphates of the crRNA guide basically bind the main-chain amides (Gly22 and Asp333) and the side chain


Figure 4. Mechanism for Specifying the Multiple Cleavage Sites with 6 nt Intervals (A) Ribbon representation of the crRNA-ssDNA duplex, showing the periodic nucleotide displacements of both strands, which form 5 bp segments between kinks. The displaced nucleotides in the crRNA and ssDNA are shown in blue and green, respectively. (B) Intercalation of the Cmr4 thumbs (red) into the duplex with 6 nt intervals. (C) Schematic representations of the 20 -deoxy-substituted RNAs at position(s) 14, 20, and 26 of the targets. The nucleotide lengths of the observable products from each substrate are indicated. (D) Target cleavage reactions with the 20 -deoxy-substituted RNAs as substrates, revealing the cleavage sites in the target RNAs. The dotted line indicates noncontiguous lanes. (E) The scissile bonds between nucleotides 14 and 15 (right), 20 and 21 (middle), and 26 and 27 (left) are sandwiched between Cmr4.3 and Cmr5.2, Cmr4.2 and Cmr5.1, and Cmr4.1 and the D4 domain of Cmr2, respectively. (F) Periodic arrangements of the crucial aspartate in Cmr4, in close proximity to the scissile bonds. The distances between the Asp31 side chains and the target sites are indicated. (G) RNA cleavage reaction by the mutant protein-containing Cmr complex.

(Ser49) from one Cmr4 subunit (cluster A residues), the main chain of Gly121 and the side chains of Lys51 and Arg55 from the following Cmr4 subunit (cluster B residues), and the main chain of Ile267 from the preceding Cmr4 subunit (Figures 5A, 5B, and S7). The K51A/R55A double mutations in Cmr4 abolished the RNA cleavage activity (Figure 4G). The main-chain amide of Gly238 and the side chains of Lys140 and Lys144 from Cmr6 interact with the phosphate backbone of the crRNA guide in the third 5 bp segment, in an almost identical manner to the cluster B residues of Cmr4 (Figures 5C and S7), revealing their common role in crRNA binding. Therefore, the Cmr complex

measures the 6 nt length of the crRNA guide by repetitive interactions. The crRNA guide binds the helical filament in a nonsequence-specific manner (Figure S2). This is the reason why the Cmr complex is programmable with any guide sequence (Hale et al., 2012). The ChiCmrD1 structure revealed that Cmr4 serves as the molecular ruler for defining 6 nt stretches in the crRNA guide. This finding suggests that the difference in the numbers of Cmr4 subunits in the complex is related to the presence of the two different crRNA lengths in the type III-B system, which differ by 6 nt (Hale et al., 2009, 2012; Staals et al., 2013). In other words, the Molecular Cell 58, 1–13, May 7, 2015 ª2015 Elsevier Inc. 7


Figure 5. Repetitive Interactions with the Guide-Target Duplex (A) The cluster A residues from Cmr4.2 bind the phosphate backbone of the crRNA in the second 5 bp segment (stereoview). The hydrophobic residues (Ala263, Val265, and Leu279) from the Cmr4.1 thumb stabilize the 5 bp segment at one end. Hydrogen bonds are indicated as dotted lines. The nucleotide numbers of the crRNA are indicated. The coloring scheme is the same as in Figure 2. (B) Stereoview of the interactions between the cluster B residues from Cmr4.3 and the crRNA backbone of the second 5 bp segment. Trp280 from the Cmr4.2 thumb locks the 5 bp segment at the other end. (C) Binding mode between Cmr6 and the third 5 bp segment (stereoview). (D) Stereoview of the interactions between the Cmr3N thumb and the first 5 bp segment. See also Figures S2, S7, and S8.

8 Molecular Cell 58, 1–13, May 7, 2015 ª2015 Elsevier Inc.


Figure 6. Mechanisms of the 50 Tag Recognition and Target Cleavage (A) The 50 tag of the crRNA is buried by Cmr3 and Cmr4.1, where the thumbs of Cmr3N and Cmr3C as well as the L-loop stabilize its curved, S-shaped structure. The coloring scheme is the same as in Figure 2, except for the L-loop and the thumbs of Cmr3N and Cmr3C, which are highlighted in gray, pink, and blue, respectively. (B) Stereoview of the interactions between Cmr3 and the first two nucleotides of the 50 tag, revealing the importance of U2 in Cmr assembly. Hydrogen bonds are indicated as dotted lines. (C) Stabilization of the quadruple base stack by Cmr3 (stereoview). (D) RNA cleavage activity of the mutant crRNA-containing Cmr complex. (E) Schematic representation of the target RNA cleavage mechanism. The Cmr complex cleaves it at multiple sites with 6 nt intervals, beginning 5 nt downstream from the 50 tag. The crRNA 50 tag is shown in white. The crucial aspartate residues are also indicated. See also Figure S2.

complexes with the protein stoichiometries of Cmr112131435261 and Cmr112131445361 may bind the 39-mer and 45-mer crRNAs, respectively. In fact, the Cmr112131445361 model fits well into the EM density of the 45-mer crRNA-containing PfCmr complex (Spilman et al., 2013) (Figure S8), while a vacant space exists when the Cmr112131435261 model is fitted. Therefore, Cmr4 and Cmr5 are involved in differentiating the length of the particle by adjusting their subunit numbers, which explains the crRNA length variation in the type III-B system (Hale et al., 2009, 2012; Staals et al., 2013). Recent studies also suggested the similar roles of these two proteins as determinants of the crRNA length (Benda et al., 2014; Hale et al., 2014; Zhu and Ye, 2015). The presence of two types of crRNAs was also reported in the Csm complex, where Csm3, an ortholog of Cmr4, determines the size of the crRNA (Hatoum-Aslan et al., 2011, 2013). Stabilization of the 5 bp Segments The 5 bp segments contact the helical filament formed by Cmr3, Cmr4.1–Cmr4.3, and Cmr6. The first 5 bp segment is stabilized by the Cmr3N thumb (Ile147, Ile149, Val158, and Leu163), and thus the start position of the guide-target duplex is defined by the complex (Figure 5D). This finding provides the first structural view that specifies the target cleavage sites measured from the crRNA 50 tag. Analogous interactions are observed between the other two 5 bp segments and the thumbs of Cmr4.1 and

Cmr4.2 (Ala263, Val265, and Leu279) (Figure 5A). The opposite ends of every 5 bp segment are capped by the Trp280 residues in the Cmr4 thumbs (Figures 5A–5C and S7). Consistently, the W280A mutation of Cmr4 impaired the target degradation activity (Figure 4G), confirming the importance of this residue in stabilizing the 5 bp segments. The present study has revealed the important roles of the Cmr4 thumbs in deforming the structure of the guide-target duplex with 6 nt intervals and stabilizing the resulting conformation (Figures 4B and 5A–5C). The previously reported mutations of His15 and Glu227 in PfCmr4 (corresponding to His20 and Glu282 in AfCmr4) markedly reduced the activity of the Cmr complex (Benda et al., 2014). In the present structure, the side chains of His20 and Glu282 form intramolecular hydrogen bonds with the main-chain carbonyl of Glu283 and the side chains of Arg264 and Trp280, respectively, which are all located in the Cmr4 thumb. Therefore, the structural integrity of the Cmr4 thumb is crucial for the efficient reaction by the Cmr complex. Recognition of the crRNA 50 Tag in the Cmr Complex The 50 tag of the crRNA, which adopts an S-shaped curve, is clamped into the pocket formed by Cmr3 and Cmr4.1 (Figure 6A). The thumbs of Cmr3N and Cmr3C, as well as a long loop designated as the L-loop (residues 9–26 in Cmr3), which resides between two Cmr3 thumbs, maintain the distinctive conformation Molecular Cell 58, 1–13, May 7, 2015 ª2015 Elsevier Inc. 9


of the 50 tag (Figure 6A). The importance of 50 tag stabilization by the Cmr3N thumb is supported by the recent study, in which the mutation of Asp86 in PfCmr4 (corresponding to Asp127 in AfCmr4), which forms bidentate hydrogen bonds with Arg146 in the Cmr3N thumb (Figure S5B), almost abolished the RNA cleavage activity (Zhu and Ye, 2015). In Cascade, Cas5 (an ortholog of Cmr3) and Cas7.6 (corresponding subunit of Cmr4.1) participate in the 50 tag recognition in a manner analogous to the respective Cmr proteins of ChiCmrD1 (Jackson et al., 2014; Mulepati et al., 2014; Zhao et al., 2014), demonstrating the structural and functional similarities of the proteins in the type I and III systems. The resemblance between the recognition modes also results in the conformational similarity of the 50 tags in the Cmr and Cascade complexes. In the ChiCmrD1 structure, the sugar-phosphate backbone of the 50 tag forms extensive hydrogen bonds with the two proteins (Figures 6B, 6C, and S2). Intriguingly, the Lys51 and Arg55 residues from Cmr4.1 contact the phosphate of A7 in the same manner as those from Cmr4.2 and Cmr4.3 bound to the crRNA guide (Figures 5B and 6C). The 50 -OH of A1 is anchored by the main-chain amide of Gly58 in the Cmr3N palm, and is snugly accommodated within the pocket (Figure 6B), which explains the finding that the addition of a phosphate moiety to the 50 end of the 50 tag prevented the function of the crRNA (Hale et al., 2014). The first two nucleotides (A1 and U2) of the 50 tag are sandwiched between the Cmr3N palm and Cmr3C thumb, and form hydrophobic interactions with their residues (Ala41, Phe47, Tyr48, and Met271) (Figure 6B). U2 is specifically recognized by hydrogen bonds with the main-chain carbonyl and amide of Thr196 and Gly198 of Cmr3N, respectively (Figure 6B). The U2A mutation in crRNA almost abolished the target RNA degradation activity (Figure 6D). These findings agree well with the conservation of this nucleotide in the 50 tag (Kunin et al., 2007). Intriguingly, U2 is also recognized in a base-specific manner by Cas5 in Cascade (Jackson et al., 2014; Mulepati et al., 2014; Zhao et al., 2014), thereby highlighting the critical role of this nucleotide in complex formation in the type I and III systems. Trp269 of the Cmr3C thumb forces U3 deeply into the pocket, and caps a quadruple base stack composed of G4, A5, A6, and A7 (Figure 6C). Consistently, the W269A mutation of Cmr3 abolished the RNA cleavage activity (Figure 4G), and the G4C mutation of the crRNA, which weakens the stacking interaction with Trp269, decreased the activity of the complex (Figure 6D). A6 is bound to the L-loop in Cmr3, where its Watson-Crick edge contacts the main-chains of Phe20 and Ala22 (Figure 6C). However, the A6C mutation of the 50 tag had no effect on the activity (Figure 6D), suggesting that the conformation of the L-loop is rearranged in response to the bound nucleotides. Tyr164 of the Cmr3N thumb stacks with A7 (Figure 6C), but this interaction is not involved in the activity, as revealed by the Y164A mutation (Figure 4G). Upon interacting with the Cmr3N thumb, G8 is rotated by 90 from the axis of the crRNA, in a manner similar to the three guide nucleotides displaced by the Cmr4 thumbs (Figures 4A and 6A), showing the analogous roles of these thumbs in the structural rearrangement of the crRNA. The present study demonstrated that the 50 tag mutants, except for the U2A mutation, retained the RNA degradation ac10 Molecular Cell 58, 1–13, May 7, 2015 ª2015 Elsevier Inc.

tivity of the complex (Figure 6D), thus revealing that the Cmr complex only recognizes U2 in a sequence-specific manner. However, these observations raise the question about how crRNAs, but not noncognate RNAs, are specifically loaded into the Cmr complex in vivo. Recent biochemical experiments suggested that Cas6, which cleaves the CRISPR transcript within the repeat (Carte et al., 2008, 2010), plays an important role for the specific incorporation of crRNAs into the downstream interference complexes in the type III system, possibly by transiently interacting with the protein subunit(s) of the interference complex on the repeat (Rouillon et al., 2013; Sokolowski et al., 2014; Zhang et al., 2012). This finding explains why the Cmr complex specifically contains the crRNA, despite the lack of apparent sequence specificity, except for U2 in the 50 tag. RNA Silencing Mechanism in the Type III System The present ChiCmrD1-ssDNA complex structure clearly showed that the 50 tag of the crRNA is recognized and the start position of the duplex is strictly defined in the complex (Figures 5D and 6A). Together with the identification of the target cleavage sites (Figure 4C), this study reveals the detailed structural basis for the RNA degradation mechanism by the Cmr complex, as follows (Figure 6E). The crRNA in the Cmr complex, with guide nucleotides flipped out at every sixth nucleotide, adopts the competent structure for facilitating the hybridization of the target strand, to form the unwound ribbon-like structure of the duplex. Once the target RNA is captured in a base-complementary manner, the target sites are expelled from the duplex, due to the steric clashes with the Cmr4 thumbs. Consequently, the target nucleotides become conformationally labile and are placed into the respective active sites of the complex, where the strictly conserved aspartate from Cmr4 is located. Then, the Cmr complex cleaves it at multiple sites with 6 nt intervals, which are specified by the 50 ruler mechanism that defines the nearest cleavage site to be 5 nt downstream of the 50 tag. Since Csm4, a Cas5 family protein in the Csm complex (Makarova et al., 2011a), structurally resembles Cmr3 (Numata et al., 2015), it may bind the 50 tag and specify the start position of the guide-target duplex in a manner similar to that observed in the present structure. Together with the fact that Csm3 is the nuclease subunit (Tamulaitis et al., 2014), the Csm complex may trap the target RNA, explore the cleavage sites, and degrade it using almost the same mechanisms as those of the Cmr complex, as revealed in detail by the present study. Therefore, the mechanism of the crRNA-guided RNA silencing may be conserved across the subtypes in the type III CRISPR-Cas system. Evolution of the Type I and III Interference Complexes The structure of the Cmr complex, which adopts an arc-like architecture, resembles that of Cascade (Figure 7A). This is attributable to the presence of similar proteins in these interference complexes (Makarova et al., 2011a). Cmr3 and Cmr4 are the orthologs of Cas5 and Cas7 in Cascade, respectively (Makarova et al., 2011a). This is consistent with their common features in the complexes. Namely, Cmr4 and Cas7 form the helical filament, which enables one copy of each protein to correspond exactly to the 6 nt length of the crRNA guide (Jackson et al., 2014;


Figure 7. Structural and Functional Similarities between the Cmr and Cascade Complexes (A) Overall structures of the ChiCmrD1-ssDNA (left) and E. coli Cascade-ssDNA (right) complexes. (B) Mechanism for defining the 6 nt length of the crRNA guide by using the thumbs of Cmr4 (left) and Cas7 (right), by which the complementary nucleotides of the guide-target duplex are displaced with 6 nt intervals. (C) Interactions showing the functional similarities of Cmr3 (left) and Cas5 (right), which both cap the helical filament, bind the 50 tag of the crRNA, and stabilize the start position of the duplex. The nucleotide numbers of the 50 tag are indicated. (D) The second helical filaments observed in ChiCmrD1 (left) and Cascade (right). See also Figure S6.

Mulepati et al., 2014; Zhao et al., 2014) (Figure 7B). Cmr3 and Cas5 cap the helical filament, bind the 50 tag of the crRNA, and stabilize the start position of the duplex (Jackson et al., 2014; Mulepati et al., 2014; Zhao et al., 2014) (Figure 7C). In contrast, Cmr2 and Cmr5 lack sequence identities with Cse1 and Cse2, respectively. However, these two proteins in the Cmr complex reside in similar locations to Cse1 and Cse2 in Cascade, respectively, which form the second filament in the complex (Jackson et al., 2014; Mulepati et al., 2014; Zhao et al., 2014) (Figure 7D). These common features not only organize the Cmr proteins into an architecture analogous to Cascade but also result in a similar mode of crRNA binding to the complexes. Considering the structural resemblance between the Cmr and Csm complexes (Rouillon et al., 2013; Staals et al., 2014), divergent evolution from a common ancestral complex may have occurred in the type I and III interference complexes. The nucleotide displacement of the guide-target duplex at 6 nt intervals, observed in the Cmr and Cascade complexes (Mulepati et al., 2014), is particularly interesting (Figure 7B). This characteristic feature facilitates the formation of the unwound ribbon-like arrangement of the guide-target duplex, which circumvents the topological problem

of the canonical double helix. In other words, by generating the unusual structure of the crRNA guide, the target strand is able to easily bind the interference complex, by avoiding helix winding during the hybridization process. This nucleotide displacement mechanism also plays a crucial role in the target cleavage reaction, by destabilizing the structure of the scissile bond in the type III-B Cmr complex. This is in striking contrast with Cascade, which recruits the nucleasehelicase Cas3 that degrades the target DNA by a distinct mechanism (Hochstrasser et al., 2014; Mulepati and Bailey, 2013; Sinkunas et al., 2013; Westra et al., 2012). In the Csm complex, Csm3 may also induce the nucleotide flipping from the duplex by using its putative thumb, which is disordered in the isolated Csm3 structures (Hrle et al., 2013; Numata et al., 2015) (Figure S6). The displaced nucleotides in the target may serve as landmarks to specify the cleavage sites in the type III system. In conclusion, this study revealed the mechanisms for Cmr complex assembly and target cleavage site specification with 6 nt periodicity. Our structure showed that the start position of the guide-target duplex is exactly defined within the complex, which leads to the 50 ruler mechanism for target degradation. The present work also paves the way toward elucidating the RNA-silencing mechanism by the Csm complex, which shares structural and functional similarities with the Cmr complex. EXPERIMENTAL PROCEDURES Sample Preparation The recombinant Cmr proteins from P. furiosus and A. fulgidus were produced and purified, and then the Cmr complex was reconstituted. Detailed methods for protein expression, purification, and complex formation can be found in the Supplemental Experimental Procedures.

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Crystallization and Data Collection The ChiCmrD1-ssDNA complex was crystallized by the hanging-drop vapor diffusion method at 20 C, under conditions containing 13%–15% (w/v) PEG3350 and 100 mM succinic acid (pH 7.0). X-ray diffraction data were collected at the beamline BL-17A of the Photon Factory (Ibaraki, Japan). Diffraction data were integrated and scaled with the program XDS (Kabsch, 2010). See the Supplemental Experimental Procedures for further details. Structure Determination and Refinement The ChiCmrD1-ssDNA complex structure was solved by a combination of the molecular replacement and single-wavelength anomalous dispersion (MR-SAD) methods with the program Phenix (Adams et al., 2010). The model was manually built with the program Coot (Emsley and Cowtan, 2004). The model was then improved by iterative cycles of refinement with the program Phenix and manual rebuilding with Coot, using the native data set that diffracted to 2.1 A˚ resolution. The stereochemistry of the structure was analyzed by PROCHECK (Laskowski et al., 1993). Details can be found in the Supplemental Experimental Procedures. RNA Cleavage Assay The RNA cleavage assay was performed to evaluate the activity of the Cmr complex. A detailed description is provided in the Supplemental Experimental Procedures. ACCESSION NUMBERS Atomic coordinates and structure factors have been deposited in the Protein Data Bank under ID code 3X1L. SUPPLEMENTAL INFORMATION Supplemental Information includes eight figures, one table, and Supplemental Experimental Procedures and can be found with this article at http://dx.doi. org/10.1016/j.molcel.2015.03.018. AUTHOR CONTRIBUTIONS T.O., H.I., and T.N. expressed and purified the proteins and their mutants to reconstitute the complexes. T.O. performed the crystallization, diffraction data collection, and structure determination. T.N. assisted with the structure determination. T.O. and T.N. carried out the biochemical analyses. C.S. performed the model fitting into the cryo-EM reconstruction. T.O. and T.N. wrote the manuscript. All authors discussed the results and commented on the manuscript. T.N. supervised all of the work. ACKNOWLEDGMENTS We thank Dr. Kozo Tomita (AIST) for valuable and critical comments and suggestions for this manuscript. We thank Professor Yoshizumi Ishino (Kyushu University) for providing the P. furiosus genomic DNA for the experiment. We also thank the beamline staff at BL-17A of the Photon Factory (Ibaraki, Japan) for technical assistance during data collection. This work was supported by a Grant-in-Aid for Scientific Research on Innovative Areas from the Ministry of Education, Culture, Sports, Science and Technology (MEXT) to T.N. and C.S. and by a Grant-in-Aid for Young Scientists from the Japan Society for the Promotion of Science (JSPS) and grants from the Kato Memorial Bioscience Foundation, the Kurata Memorial Hitachi Science and Technology Foundation, and the Institute for Fermentation, Osaka (IFO) to T.N. Received: January 15, 2015 Revised: February 18, 2015 Accepted: March 11, 2015 Published: April 23, 2015

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Molecular Cell 58, 1–13, May 7, 2015 ª2015 Elsevier Inc. 13

Structural biology. Crystal structure of a CRISPR RNA-guided surveillance complex bound to a ssDNA target.

Crystal structure and CRISPR RNA-binding site of the Cmr1 subunit of the Cmr interference complex.

Crystallization and preliminary X-ray diffraction analysis of the CRISPR-Cas RNA-silencing Cmr complex.

The Cmr complex: an RNA-guided endoribonuclease.

Structure of an Rrp6-RNA exosome complex bound to poly(A) RNA.

Structural biology. Structures of the CRISPR-Cmr complex reveal mode of RNA target positioning.

Crystal Structure of Cpf1 in Complex with Guide RNA and Target DNA.

Crystal structure of Cas9 in complex with guide RNA and target DNA.

RNA editing of microRNA prevents RNA-induced silencing complex recognition of target mRNA.

Crystal structure at 1.7 A of the bovine papillomavirus-1 E2 DNA-binding domain bound to its DNA target.

Crystal structures of the Lsm complex bound to the 3' end sequence of U6 small nuclear RNA.

Crystal structure of the N-myristoylated lipopeptide-bound MHC class I complex.

Crystal structure of rofecoxib bound to human cyclooxygenase-2.

Solution structure of cyclosporin A and a nonimmunosuppressive analog bound to fully deuterated cyclophilin.

Crystal structure of the HIV neutralizing antibody 2G12 in complex with a bacterial oligosaccharide analog of mammalian oligomannose.

Crystal Structure of a Transcribing RNA Polymerase II Complex Reveals a Complete Transcription Bubble.

Crystal structure of ATP-bound Get3-Get4-Get5 complex reveals regulation of Get3 by Get4.

Crystal structure of a 4-thiouridine synthetase-RNA complex reveals specificity of tRNA U8 modification.

Crystal structure of the adenosine A2A receptor bound to an antagonist reveals a potential allosteric pocket.

The crystal structure of a K+ complex of valinomycin.

The Paf1 complex represses small-RNA-mediated epigenetic gene silencing.

Intermolecular base stacking mediates RNA-RNA interaction in a crystal structure of the RNA chaperone Hfq.

Crystal structure of the FLT3 kinase domain bound to the inhibitor Quizartinib (AC220).

High-resolution crystal structure of the therapeutic antibody pembrolizumab bound to the human PD-1.