The CRISPR (clustered regularly interspaced short palindromic repeats) immune system in prokaryotes uses small guide RNAs to neutralize invading viruses and plasmids. In Escherichia coli, immunity depends on a ribonucleoprotein complex called Cascade. Here we present the composition and low-resolution structure of Cascade and show how it recognizes double-stranded DNA (dsDNA) targets in a sequence-specific manner. Cascade is a 405-kDa complex comprising five functionally essential CRISPR-associated (Cas) proteins (CasA(1)B(2)C(6)D(1)E(1)) and a 61-nucleotide CRISPR RNA (crRNA) with 5'-hydroxyl and 2',3'-cyclic phosphate termini. The crRNA guides Cascade to dsDNA target sequences by forming base pairs with the complementary DNA strand while displacing the noncomplementary strand to form an R-loop. Cascade recognizes target DNA without consuming ATP, which suggests that continuous invader DNA surveillance takes place without energy investment. The structure of Cascade shows an unusual seahorse shape that undergoes conformational changes when it binds target DNA.

Prokaryotes have evolved multiple versions of an RNA-guided adaptive immune system that targets foreign nucleic acids. In each case, transcripts derived from clustered regularly interspaced short palindromic repeats (CRISPRs) are thought to selectively target invading phage and plasmids in a sequence-specific process involving a variable cassette of CRISPR-associated ( cas ) genes. The CRISPR locus in Pseudomonas aeruginosa (PA14) includes four cas genes that are unique to and conserved in microorganisms harboring the Csy-type (CRISPR system yersinia) immune system. Here we show that the Csy proteins (Csy1–4) assemble into a 350 kDa ribonucleoprotein complex that facilitates target recognition by enhancing sequence-specific hybridization between the CRISPR RNA and complementary target sequences. Target recognition is enthalpically driven and localized to a “seed sequence” at the 5′ end of the CRISPR RNA spacer. Structural analysis of the complex by small-angle X-ray scattering and single particle electron microscopy reveals a crescent-shaped particle that bears striking resemblance to the architecture of a large CRISPR-associated complex from Escherichia coli , termed Cascade. Although similarity between these two complexes is not evident at the sequence level, their unequal subunit stoichiometry and quaternary architecture reveal conserved structural features that may be common among diverse CRISPR-mediated defense systems.

CRISPR-Cas is a prokaryotic adaptive immune system that provides sequence-specific defense against foreign nucleic acids. Here we report the structure and function of the effector complex of the Type III-A CRISPR-Cas system of Thermus thermophilus: the Csm complex (TtCsm). TtCsm is composed of five different protein subunits (Csm1-Csm5) with an uneven stoichiometry and a single crRNA of variable size (35-53 nt). The TtCsm crRNA content is similar to the Type III-B Cmr complex, indicating that crRNAs are shared among different subtypes. A negative stain EM structure of the TtCsm complex exhibits the characteristic architecture of Type I and Type III CRISPR-associated ribonucleoprotein complexes. crRNA-protein crosslinking studies show extensive contacts between the Csm3 backbone and the bound crRNA. We show that, like TtCmr, TtCsm cleaves complementary target RNAs at multiple sites. Unlike Type I complexes, interference by TtCsm does not proceed via initial base pairing by a seed sequence.

Summary The CRISPR-Cas system is a prokaryotic host defense system against genetic elements. The Type III-B CRISPR-Cas system of the bacterium Thermus thermophilus, the TtCmr complex, is composed of six different protein subunits (Cmr1-6) and one crRNA with a stoichiometry of Cmr112131445361:crRNA1. The TtCmr complex co-purifies with crRNA species of 40 and 46 nt, originating from a distinct subset of CRISPR loci and spacers. The TtCmr complex cleaves the target RNA at multiple sites with 6 nt intervals via a 5’ ruler mechanism. Electron microscopy revealed that the structure of TtCmr resembles a ‘sea worm’ and is composed of a Cmr2-3 heterodimer ‘tail’, a helical backbone of Cmr4 subunits capped by Cmr5 subunits, and a curled ‘head’ containing Cmr1 and Cmr6. Despite having a backbone of only four Cmr4 subunits and being both longer and narrower, the overall architecture of TtCmr resembles that of Type I Cascade complexes.

The structural analysis of macromolecular functional protein assemblies by contemporary high resolution structural biology techniques (such as nuclear magnetic resonance, X-ray crystallography, and electron microscopy) is often still challenging. The potential of a rather new method to generate structural information, native mass spectrometry, in combination with ion mobility mass spectrometry (IM-MS), is highlighted here. IM-MS allows the assessment of gas phase ion collision cross sections of protein complex ions, which can be related to overall shapes/volumes of protein assemblies, and thus be used to monitor changes in structure. Here we applied IM-MS to study several (intermediate) chaperonin complexes that can be present during substrate folding. Our results reveal that the protein assemblies retain their solution phase structural properties in the gas phase, addressing a long-standing issue in mass spectrometry. All IM-MS data on the chaperonins point toward the burial of genuine substrates inside the GroEL cavity being retained in the gas phase. Additionally, the overall dimensions of the ternary complexes between GroEL, a substrate, and cochaperonin were found to be similar to the dimensions of the empty GroEL-GroES complex. We also investigated the effect of reducing the charge, obtained in the electrospray process, of the protein complex on the global shape of the chaperonin. At decreased charge, the protein complex was found to be more compact, possibly occupying a lower number of conformational states, enabling an improved ion mobility separation. Charge state reduction was found not to affect the relative differences observed in collision cross sections for the chaperonin assemblies.

The molecular complexity of biopharmaceuticals puts severe demands on the bioanalytical techniques required for their comprehensive structural characterization. Mass spectrometry (MS) has gained importance in the analysis of biopharmaceuticals, taking different complementary approaches ranging from peptide-based sequencing to direct analysis of intact proteins and protein assemblies. In this protocol, we describe procedures optimized to perform the analysis of monoclonal antibodies (mAbs) at the intact protein level under pseudo-native conditions, using native MS. Some of the strengths of native MS in the analysis of biopharmaceuticals are its analysis speed, sensitivity and specificity: for most experiments, the whole protocol requires one working day, whereby tens of samples can be analyzed in a multiplexed manner, making it suitable for high-throughput analysis. This method can be used for different applications such as the analysis of mixtures of mAbs, drug-antibody conjugates and the analysis of mAb post-translational modifications, including the qualitative and quantitative analysis of mAb glycosylation.

Significance CRISPR-Cas systems provide prokaryotic adaptive immunity against invading genetic elements. For immunity, fragments of invader DNA are integrated into CRISPR arrays by Cas1 and Cas2 proteins. Type I-F systems contain a unique fusion of Cas2 to Cas3, the enzyme responsible for destruction of invading DNA. Structural, biophysical, and biochemical analyses of Cas1 and Cas2-3 from Pectobacterium atrosepticum demonstrated that they form a 400-kDa complex with a Cas1 4 :Cas2-3 2 stoichiometry. Cas1–Cas2-3 binds, processes, and catalyzes the integration of DNA into CRISPR arrays independent of Cas3 activity. The arrangement of Cas3 in the complex, together with its redundant role in processing and integration, supports a scenario where Cas3 couples invader destruction with immunization—a process recently demonstrated in vivo.

Proteomics applications performed on the popular benchtop Q Exactive Orbitrap mass spectrometer have so far relied exclusively on higher collision-energy dissociation (HCD) fragmentation for peptide sequencing. While this fragmentation technique is applicable to a wide range of biological questions, it also has limitations, and all questions cannot be addressed equally well. Here, we demonstrate that the fragmentation capabilities of the Q Exactive mass spectrometer can be extended with ultraviolet photodissociation (UVPD) fragmentation, complete with synchronization triggering to make it compatible with liquid chromatography (LC)/tandem mass spectrometry (MS/MS) workflows. We show that UVPD not only is directly compatible with LC/MS workflows but also, when combined with these workflows, can result in higher database scores and increased identification rates for complex samples as compared to HCD methods. UVPD as a fragmentation technique offers prompt, high-energy fragmentation, which can potentially lead to improved analyses of labile post-translational modifications. Techniques like HCD result in substantial amounts of modification losses, competing with fragmentation pathways that provide information-rich ion fragments. We investigate here the utility of UVPD for identification of phosphorylated peptides and find that UVPD fragmentation reduces the extent of labile modification loss by up to ∼60%. Collectively, when integrated into a complete workflow on the Q Exactive Orbitrap, UVPD provides distinct advantages to the analysis of post-translational modifications and is a powerful and complementary addition to the proteomic toolbox.