key: cord-291677-zcbyhsf1
authors: Wilamowski, M.; Sherrell, D.A.; Minasov, G.; Kim, Y.; Shuvalova, L.; Lavens, A.; Chard, R.; Maltseva, N.; Jedrzejczak, R.; Rosas-Lemus, M.; Saint, N.; Foster, I.T.; Michalska, K.; Satchell, K.J.F.; Joachimiak, A
title: Methylation of RNA Cap in SARS-CoV-2 captured by serial crystallography
date: 2020-08-16
journal: bioRxiv
DOI: 10.1101/2020.08.14.251421
sha: 
doc_id: 291677
cord_uid: zcbyhsf1

The genome of the SARS-CoV-2 coronavirus contains 29 proteins, of which 15 are nonstructural. Nsp10 and Nsp16 form a complex responsible for the capping of mRNA at the 5′ terminus. In the methylation reaction the S-adenosyl-L-methionine serves as the donor of the methyl group that is transferred to Cap-0 at the first transcribed nucleotide to create Cap-1. The presence of Cap-1 makes viral RNAs mimic the host transcripts and prevents their degradation. To investigate the 2′-O methyltransferase activity of SARS-CoV-2 Nsp10/16, we applied fixed-target serial synchrotron crystallography (SSX) which allows for physiological temperature data collection from thousands of crystals, significantly reducing the x-ray dose while maintaining a biologically relevant temperature. We determined crystal structures of Nsp10/16 that revealed the states before and after the methylation reaction, for the first time illustrating coronavirus Nsp10/16 complexes with the m7GpppAm2′-O Cap-1, where 2′OH of ribose is methylated. We compare these structures with structures of Nsp10/16 at 297 K and 100 K collected from a single crystal. This data provide important mechanistic insight and can be used to design small molecules that inhibit viral RNA maturation making SARS-CoV-2 sensitive to host innate response.

Five coronaviruses can induce clusters of severe respiratory diseases in humans: 229E, OC43, SARS, MERS and SARS-CoV-2 1,2 . The outbreak of Severe Acute Respiratory Syndrome (SARS-CoV) in 2003 and Middle East Respiratory Syndrome (MERS-CoV) in 2012, showed a high fatality rate of 11% and 34%, respectively, but had limited geographical spread 3, 4 . In contrast, in the past 7 months SARS-CoV-2 (the cause of COVID-19) has spread rapidly around the world and infected millions of people, killing over half a million. Though, it has a significantly lower fatality rate (estimated at 0.5 -2%) than MERS and SARS the amount of people infected is hundreds of times higher, and it caused significant economic hardship and extraordinary social restrictions. In many countries, after six months from the first reports of COVID-19, the number of cases is still increasing 5 . In the absence of herd immunity, vaccines, or drugs against the virus, the situation is frightful. Since the beginning of the worldwide outbreak of COVID-19, the scientific community has joined efforts to understand SARS-CoV-2 biology and to find chemical compounds that either block virus replication or enhance human immunological response. Initial focus on repurposing existing drugs that potentially could provide immediate treatment has thus far resulted only in limited success. Finding inhibitors of a biological cycle of SARS-CoV-2 or vaccines is, therefore, critical for global health, safety, and wellbeing. SARS-CoV-2 β-coronavirus has a large (~ 30 kb) and complex (29 proteins) (+) sense singlestranded RNA genome 6 . The RNA present in the mature virion resembles human mRNA: (i) it is capped on its 5′-end, (ii) contains a 3′-poly-A tail, and (iii) after infection can be directly translated to the two polyproteins Pp1a and Pp1ab using host machinery. These polyproteins are then matured into 15 non-structural proteins (Nsp), that assemble into a large replication-transcription complex.

The RNA is also used as a template for biosynthesis of (-) sense RNA that serves to make additional copies of (+) sense RNA and several sub-genomic RNAs for the translation of four structural and 9-10 accessory proteins 6 . For these RNAs to serve as mRNAs, they must undergo post-transcription modifications to resemble human mRNA. The RNA maturation involves several enzymatic steps performed by viral Nsps; Nsp13 is a bifunctional RNA/NTP triphosphatase (TPase) and helicase; Nsp14 is a bifunctional 3′-5′ exonuclease and guanine N7 methyltransferase;

and Nsp16 is Mg 2+ dependent ribose 2′-O methyltransferase and an elusive guanylyltransferase.

In eukaryotes, the attachment of the m7 G Cap at the 5′-end of RNA protects the transcript from mRNA turnover in the pathway dependent on 5′ → 3′ exoribonucleases activity, such as XRNs 7 , and is required for several cellular processes: maturation of mRNA, pre-mRNA nuclear export, and protein synthesis 8 . The Cap at the 5′ terminus of eukaryotic transcripts starts with 7methylguanosine at the reversed position and is connected through an uncommon 5′ to 5′ triphosphate bridge, which is attached to a nucleotide that undergoes methylation at 2′-O-ribose.

The canonical pathway of the mRNA capping mechanism requires the subsequent action of multiple enzymes 8 . At first, RNA 5′-triphosphatase (RTPase) cleaves the 5′-terminal γ-β phosphoanhydride bond of the nascent mRNA and enables further modification of the newly synthesized transcript at its 5′-terminus. Secondly, the diphosphate mRNA is capped using GMP by guanylyltransferase (GTase). Next, the (guanine-N7)-methyltransferase adds the methyl group to the 5′-terminal guanosine creating a precursor: Cap-0. Additionally, the 2′-O methyltransferase transfers the methyl group to the 2′-O-ribose of the first transcribed nucleotide at mRNA, making the m7 GpppNm2′-O (Cap-1) structure 9 . S-adenosyl-L-methionine (SAM) is a donor of the methyl group and is converted to S-adenosyl-L-homocysteine (SAH) during the reaction 10 . In humans and other higher eukaryotes, the second nucleotide of mRNA then undergoes methylation to produce Cap-2 11 .

The post-transcriptional modification of RNA is also essential for efficient translation of viral transcript in a eukaryotic host 12 . Influenza, Ebola, measles, poxvirus, and coronaviruses attach Cap to their genomic and sub-genomic RNA transcripts to mimic host molecules and escape from the innate immunological response 13 . Studies done in the last decade show that the 2′-O methylation of the Cap-0 facilitates this process by preventing activation of type I interferon that is induced by the cytoplasmic RNA sensors, melanoma differentiation-associated protein 5 (MDA5), and retinoic acid inducible gene-I (RIG-1) [14] [15] [16] they all utilize a Lys-Asp-Lys-Glu catalytic tetrad essential for the enzymatic activity 22, 23 .

It has been shown that the Nsp10/16 complex methylates the Cap-0 ( m7 GpppA2′-OH) to form Cap-1 ( m7 GpppAm2′-O) by adding a methyl group to the ribose 2′-O of the first nucleotide (usually adenosine, in CoVs) of the nascent mRNA using SAM as the methyl group donor 24 . The Nsp16 activity is necessary for Nsps' translation from new copies of viral (+) sense RNA as well as structural and accessory proteins translation from sub-genomic RNA which are all transcribed from the viral (-) sense RNA template. Vaccination with Nsp16 defective SARS-CoV or an immunogenic disruption of the Nsp10/16 interface protects mice from a lethal SARS challenge 25 .

Therefore, blocking Nsp16 activity should reduce viral proliferation, making the protein an attractive drug target.

While several structures of Nsp10/16 in a complex with Cap-0 have been determined by crystallography (PDB entries: 6WQ3, 6WRZ, 6WVN, 6WKS), none have captured the postmethylation state. We conducted serial synchrotron crystallography (SSX) experiments at 297 K to test whether low radiation dose could help uncover the structure of Nsp10/16 in a complex with Cap-1. Previously, x-ray free-electron laser (XFEL) serial crystallography (SFX) had established methods to study molecular dynamics and chemical reactions in protein crystals occurring at femtosecond to millisecond timescales [26] [27] [28] . The XFEL-based protocol enabled to analyze protein structures with no radiation damage, in the so-called "diffraction before destruction" mode 29, 30 . SFX has been successfully applied to determine the structure of the membrane protein complex of photosystem I and of several other macromolecules using time-resolved approach 29 .

The synchrotron equivalent, SSX, uses a similar approach to SFX but can access longer, biologically relevant time scales. Moreover SSX is a much more accessible technique at a number of light sources and less sample consuming than SFX 31 . Nonetheless, delivering hundreds of thousands of batch-grown crystals to the x-ray beam still presents challenges. Two approaches are common: the first uses a liquid injector of microcrystals 29, 32, 33 , and the second uses a fixed-target system where crystals are deposited on a chip and scanned through the x-ray beam 34, 35 . Here we present three crystal structures of the Nsp10/16 complex determined by fixed-target SSX at 297 K:

in the presence of SAM, Cap-0/SAM, and with Cap-1/SAH generated by metal-dependent conversion of Cap-0/SAM substrates. We compare these structures with structures of Nsp10/16 at 297 K and 100 K collected from a single crystal. We observe the state of the molecules in the crystal before and after methylation. The uniqueness of the Cap-1 structure shows the advantages of the SSX method in structural studies: allowing the use of larger crystals, reducing radiation damage, and being closer to physiological temperatures.

We collected SSX data for the Nsp10/16 crystals using the fixed-target SSX system (Advanced Lightweight Encapsulation Crystallography (ALEX) mesh-holder) as depicted in Figure 1A For initial data processing, we used the Kanzus automated data pipeline. Kanzus integrates the 19-ID data collection system with the Argonne Leadership Computing Facility (ALCF), using the Theta supercomputer for high-speed on-demand data analysis. Extended Figure 3A presents an image with labeled indexed reflections and shows that most observed reflections of Nsp10/16 crystals were indexed. PRIME identified inadequate low-resolution statistics from the integration files, thus we subjected them to outlier rejection. Integration files with resolution lower than 3.5 Å, containing less than 90 reflections, or with a unit cell volume 4.5% different from the average were removed from the scaling process (Extended Data Tab. 1, Fig. 2C, 2D ). The total number of integrated files that went into PRIME for Nsp10/16 bound with SAM, Cap-1/SAH, and Cap-0/SAM were 7,951; 5,038 and 4,704 respectively. The overall hit-rate (images used for structure divided by images taken) for the five meshes was 7%. Some positions on the mesh had multiple crystals, 11% of 'hits' had two lattices, and fewer than 1% had three (Extended Data Tab. 1, Fig. 3B ). For structure determination we used all indexed lattices in each diffraction image.

Two structures from SSX were solved at 2.25 Å (Nsp10/16/SAM and Nsp10/16/Cap-1/SAH), and a third at 2.18 Å (Nsp10/16/Cap-0/SAM) (Extended Data Tab. 1). The highest resolution shell in PRIME analysis was specified using a CC1/2 cut-off below 0.40. Completeness for the SSX data was close to 100% and Rwork was 20.65%, 22.31%, and 21.70% for Nsp10/16/SAM, Nsp10/16/Cap-1/SAH, and Nsp10/16/Cap-0/SAM, respectively (Extended Data Tab. 2). For a comparison to SSX data, we also collected data from a single crystal of the Nsp10/16 with Cap-0/SAM and Cap-1/SAH in a capillary at 297 K. These data could only beprocessed to 2.65 Å with reasonable statistics (Extended Data Tab. 1, 2). Using RADDOSE-3D 37 , we estimated the accumulated dose of x-ray for Nsp10/16 from SSX to be 0.12 MGy, whereas the dose for the capillary-mounted crystal was 4.97 MGy over 40 times higher.

The Nsp10/16 complex is an a/b heterodimer (Fig. 2) . The 139 amino acid long Nsp10 has unique fold formed by 5 α-helices and a pair of antiparallel β-strands which are facing Nsp16 in the complex. Nsp10 possesses two zinc ions coordinated by the CCHC and CCCC motifs that are 100% conserved in β-coronaviruses 20 . Nsp10 is involved in forming the complexes with multiple SARS-CoV-1 and -2 proteins. The best characterized is the complex with Nsp14 3′-5′ exoribonuclease (and guanine N7 methyltransferase) and Nsp16 that is 2′-O-ribose methylase.

Previous research showed that Nsp16 is only active in the presence of Nsp10 19 . Interestingly, the activity of 2′-O MTase of the Nsp10/16 complex was reduced by using a peptide compound based on the Nsp10 sequence 38 . Nsp10 is a molecular co-factor for various SARS-CoV-2 enzymes and therefore a good candidate for design of molecules that will affect its structure or disrupt the interaction with Nsp14 or Nsp16 2′-O MTases.

The structure of Nsp16 is characterized by a Rossmann fold, with a large β-sheet surrounded by α-helices, β-strands and loops. Nsp16 contains 11 α-helices, 12 β-strands and 7 loops. Nsp16 has a centrally positioned β-sheet (β3↑,β2↑,β1↑,β4↑,β5↑,β7↓,β6↑) with only one antiparallel strand β7. The SAM and the Cap binding sites are located at the surface of Nsp16. None of the Nsp10 residues have a direct connection with the ligands' binding sites ( Fig. 2A, 2C ), but Nsp10 is required for Nsp16 MTase activity 21 . Additionally, we observed partial electron density for the 7-methyl-guanosite-5′-triphosphate ( m7 Gppp) of the Cap in the possible allosteric site of the We also compared the two structures of Nsp10/16/SAM determined at 297 K (this work PDB entry 7JIB) and 100 K (PDB entry 6W4H) (Cα RMSD: 0.35 Å) and observed significant differences: one loop from the Cap binding pocket is shifted approximately 1.4 Å in 297 K structures, which makes the Cap binding site more accessible (Extended Data Fig. 4D) . Therefore, the 297 K structures of Nsp10/16 in a complex with SAM, Cap-0/SAM, and Cap-1/SAH, as determined by SSX, depict states important for molecular modeling studies and structure-based drug design.

To better understand Nsp16 activity from SARS-CoV-2, we compared it with NS5 2′-O MTase from Dengue virus (PDB entry 5DTO) (Fig. 3) . We aligned the structure of Nsp16 with the Nterminal domain of NS5 that spans 298 residues from N-terminus. Both Nsp16 and NS5 2′-O MTases assume a Rossmann fold with a large β-sheet decorated with α-helices (Fig. 3B, D) .

MTases contain a central β-sheet that is composed of six parallel and one antiparallel strands.

Despite low sequence identity (13.6%) the RMSD between the two MTases is reasonable (3.01 Å).

Both enzymes possess a canonical 2′-O MTase catalytic tetrad Lys-Asp-Lys-Glu with the aspartic acid residue forming a hydrogen bond with a water molecule potentially relevant for catalysis ( Fig. 3A, C) . The SAM binding sites share high similarity but there are major differences in the mRNA Cap binding sites. The Mg 2+ or Mn 2+ ion is necessary for Nsp16 activity 39 , however is not localized near the active site in the currently available structures of Nsp10/16 from SARS-CoV-1 and SARS-CoV-2. The Mg 2+ ion in the N-terminal domain of NS5 2′-O MTase is coordinated by the phosphate oxygens of the Cap 5′ to 5′ triphosphate linker and three water molecules that bridge to three bases (A1-G2-U3) on the 5′-end.

The SARS-CoV-2 Nsp10/16 2′-O MTase complex provides a molecular arrangement for binding of the mRNA Cap-0 and subsequent methylation of the first transcribed nucleotide.

The catalytic core is in the center of the Rossmann fold and it binds the SAM molecule in the deep, narrow grove that is buried inside the Nsp16 active site. We observed that Nsp16 had recruited SAM during expression in E. coli (PDB entry 6W61) 20 . However, addition of 2 mM SAM to the crystallization solution improves crystallization efficiency, reduces crystallization time, and increases the number of crystals -three highly desirable traits for SSX batch crystallization.

The SAM binding site is negatively charged and is formed by several Nsp16 residues: Asn43, Tyr47, Gly71, Ala72, Gly73, Ser 74, Gly81, Thr82, Asp99, Leu100, Asn101, Asp114, Cys115, Asp130, Met131, and Phe149 (Fig. 2, 4, Extended Data Fig. 5B ). The SAM carboxylate moiety binds to the positively charged N-terminus of the third a-helix spanning Pro80-Trp88. This interaction provides additional electrostatic stabilization. Nsp16 directly interacts with Cap-1 through several residues that form a positively charged, elongated binding groove accommodating the mRNA Cap-0 (Fig. 2, 4 , Extended Data 5A). The N7-metyl guanosine binding pocket is formed by Cys25, Asp26, Leu27, Tyr30, Thr172, Glu173, and Ser202. The 5′ to 5′ triphosphate bridge of the Cap is stabilized through interaction with Tyr30, Lys137, Thr172, His174, Ser201, Ser202, and Glu203. The first nucleotide of the mRNA Cap (adenosine in the presented structure)

is bound through Lys46, Asp130, Tyr132, Pro134, Lys170, Asn198, and Glu203.

SAM-dependent MTases share a conserved catalytic mechanism wherein the methyl group is transferred to the acceptor substrate via an SN2 reaction 40 , which requires a linear alignment of the acceptor substrate (nucleophile), methyl group (electrophile), and the sulfur atom (leaving group) of the SAH product 41 . It was proposed that Nsp16 2′-O MTases follow the same general mechanism 42 . In these enzymes, the reaction is facilitated by the catalytic tetrad Lys-Asp-Lys-Glu, where Lys170 sandwiched between the two acidic residues (Asp130 and Glu203) serves as a proton abstractor (Fig. 4A, B) . It was shown previously in biochemical studies that substitution to Ala of any residue of the 2′O-MTase catalytic tetrad results in an inactive enzyme 10, 43 . In SARS-CoV-2 Nsp16 the catalytic tetrad Lys46-Asp130-Lys170-Glu203, is superposing well with Lys61-D146-Lys180-Glu216 of the Dengue virus homolog (Fig. 3A, C) . In the 3D context, Lys46 binds to Glu203 which then binds to Lys170 interacting with Asp130, which further links to the amino group of SAM. In this network, a proton can be transferred between multiple residues, occupying different sites potentially depending on the reaction state (Fig. 4) . Lys170 is well-positioned to act as a general base deprotonating 2′-OH. Asp130 may serve multiple functions -as an acid deprotonating Lys170 (alternating with Glu203), and an anchoring point for the cofactor via interaction with its amino group, and as a stabilization for the sulfonium cation. However, the reaction does not occur in a presence of EDTA, as demonstrated by the ability to capture the Cap-0/SAM complex. It was reported previously using biochemical assays that the activity of Nsp10/16 2′-O MTase is magnesium dependent 39 . Though, we did not observe any trace of metal near the active site in the 297 K structures reported in this work, and any other CoV-2 Nsp10/16 structures reported to date, despite the presence of magnesium in the crystallization buffer. We hypothesize that the magnesium ion (or other metal ion) can transiently bind to the active site possibly replacing one of the water molecules (for example water 38) and promoting formation of a reactive conformation by changing electrostatics and geometry of the catalytic residues to stimulate methylation reaction. Magnesium has a compact and tight coordination sphere with strict octahedral geometry and a typically short Mg-O distance of 2.08 Å 44 . By coordinating 2′ oxygen and several water molecules magnesium could shortened the distance between 2′ oxygen and SAM methyl moiety thus promoting formation of transition state and methyl transfer. During the reaction a positively charged, sp2 planar transition state is formed and the methyl group inverts its stereochemistry.

After the methyl-transfer reaction is completed the product is released from the active site. This is consistent with the structure of Cap-1/SAH where several active site residues move, these include Tyr132, the entire α-helix spanning Pro134-Lys137, and Tyr30 on the opposite site ( Fig. 4A, Extended Data Fig. 4C ). All these residues are involved in interactions with Cap-0 and observed changes perhaps allow Cap-1 to leave. Interestingly, the SAM/SAH binding site remains virtually unchanged in all three structures (Extended Data Fig. 4) , suggesting that the SAH exchange with SAM may require dissociation of Nsp10 that controls conformation of important loop Gly73-Gly77. Opening this loop may help SAH to leave and then a new SAM molecule can bind.

The biochemical assays show that optimal pH for MERS-CoV Nsp10/16 activity is approximately 8 -8.5 39 . Additionally, the 2′-O MTase activity of Nsp10/16 from the MERS is significantly reduced at pH below 7. Assuming that Nsp10/16 2′-O MTase efficiency is pH dependent, we performed the 297 K SSX data collection using a buffer with pH 6.5 that could potentially slow down the reaction. Comparison of the 100 K and the 297 K structures revealed clear methylation only at 297 K, we hypothesize that Cap-1 could be sensitive to radiation damage and thus can be only observed in low x-ray dose experiments. Interestingly, the 297 K structure of Nsp10/16 obtained from a single crystal has a mixture of states with 30% of the Cap-1/SAH products and 70% of the Cap-0/SAM substrates. It is well known that radiation damage can impact redox systems. High x-ray dose could affect electron density around amino acid residues and lead to photoreduction of metalloproteins 45, 46, 47 . These free radical reactions can occur in crystals under cryogenic conditions 48 .

Enzymes that catalyze transmethylation reactions using SAM as the methyl group donor have been described in many cellular processes involving nucleic acids, proteins, phospholipids, and This clearly needs further investigation because SSX low radiation dose may be advantageous in helping reveal subtle and diverse chemical transformations in enzymes that otherwise may be degraded during x-ray or electron diffraction experiments. Therefore, the structure of Nsp10/16 with Cap-1 presented here provides unique information for understanding the mechanism that allows SARS-CoV-2 to mimic mature eukaryotic mRNA and escape recognition by human innate immune response.

The recent developments in micro focusing x-ray beams at synchrotron light sources and improvement in sample delivery technology, data collection, detectors, and computing will allow rapid determination of new structures using SSX and revealing significant biological information.

The further development of SSX and implementation of time-resolved SSX crystallography is an approach that could visualize chemical processes and protein molecular dynamics -such as of the transfer of the methyl group catalyzed by Nsp10/16 2′O-MTase from SARS-CoV-2. Thus far the Cap-1 was only observed in the structure obtained using SSX. For diffraction experiments conducted at 297 K, SSX provides significant advantages over data collected using a single crystal as it considerably increases resolution, use less sample in comparison with a liquid jet delivery system and reduces levels of x-ray dose. Studies of other enzymes can also significantly benefit from using this approach.

The recombinant proteins Nsp10 and Nsp16 from SARS-CoV-2 were expressed in E. coli and tube. These volumes allowed us to get the number of crystals suitable for data collection from one chip. We have prepared several batches using same conditions to get reproducible data collection and have the ability to merge data from multiple chips. The crystals grew to optimum sizes in 10 days at 21°C before the SSX data collection. To obtain the structure of Nsp10/16/Cap-0/SAM, the crystal batch was supplemented with 1 mM EDTA two days before data collection.

The crystals from batch crystallization were centrifuged at 100 RCF for 2 minutes at 297 K.

The excess solution was removed with a pipette, until 15 µl was left. Crystals sedimented on the walls of Eppendorf polypropylene tube were gently resuspended using 20 µl pipette tips with a cut end to increase the diameter of the tip to minimize mechanical damage to crystals. Then 1.5 µl of 10 mM stock of the m7 GpppA (S1405L, New England Biolabs) was added to crystal slurry and the mix was loaded on a 60 µm grid made from nylon (NY6004700, Millipore) which was placed on a 6 µm layer of mylar polyester film. The nylon mesh was covered on the top with a second layer of 6 µm mylar and sealed with the ALEX magnetic holder, such that sample is hermetically sealed from the outside environment. Data collection was started approximately 20 minutes after assembly of the chip.

We collected SSX data for the Nsp10/16 crystals at the 19-ID beamline at the Advanced Photon Source using the fixed-target SSX (ALEX) mesh-holder developed at the Structural Biology Center (SBC) as depicted in Figure 1A and Extended Data Fig. 1 . A crystal slurry is deposited on a nylon mesh, which immobilizes the crystals; they are then encapsulated between two polyester films 36 .

The rod-shape crystals grew without seeding and the average crystal size was 120x25x25 µm (Extended Data Fig. 2A) . Serial data collection was collected using three SmarAct SLC-17 stages configured in an XYZ geometry, with each having sufficient movement range to cover the sample area of the specially designed ALEX holder (patent application serial #16/903,601). The beamline was configured at an energy of 12,662 eV, with collimator slit sizes set to 75 x 75 µm, and step size (distance between exposures) of 50 µm: overlapping exposed area to maximize crystal hits.

The five mesh-covered samples used a grid of 170 steps in the x-direction (columns) by 210 steps in the y-direction (rows), covering a total area of approximately 8.5 x 10.5 mm. The number of steps and the resulting area varied slightly per sample depending on the chip mount position or possible false-starts. Extended Data Table 1 contains details of number of chips and detector distances used for data collection for Nsp10/16 SSX structures.

Crispy, the data acquisition GUI for serial collection at sector 19, allows for quick alignment and acts as a source of information for downstream processing. Metadata in the form of JSON files and beamline/collection strategy parameters are input and passed into the system before collection.

These parameters include grid dimensions, detector distance/resolution, unit cell dimension, protein PDB coordinates, and a handful of others.

The Kanzus pipeline orchestrates SSX data acquisition, analysis, cataloging, and publishes processing metrics. Kanzus uses a cloud-hosted research automation system called Globus Automate to manage these multi-step data "flows" 54 . The first phase of the pipeline is integrated with the APS Data Management System at the beamline, which deposits each newly acquired image into an Globus-accessible storage system at the APS. As new images are acquired, Globus Automate "flows" are launched to process them as follow: 1) moves new files from APS to Theta by using the Globus Transfer service 55 ; 2) performs DIALS stills_process 56 on batches of 256 images by using funcX 57 , a function-as-a-service computation system (funcX uses Parsl 58 to abstract and acquire nodes on Theta as needed, and dispatches tasks to available nodes); 3) extracts metadata from files regarding identified diffractions, and generates visualizations (funcX)

showing the locations of positive hits on the mesh; and 4) publishes raw data, metadata, and visualizations to a portal on the ALCF Petrel data system 59 . The result of this automated process is an indexed, searchable data collection that provides full traceability from data acquisition to processed data, and that can be used to inspect and update the running experiment.

Images were collected at 7 Hz, meaning that a 256-image batch totaling 1.56 GB, was generated every 35 seconds. The data transfers to ALCF ran at up to 700 MB/s via Globus, and 30 Theta nodes processed images by using DIALS stills_process at 22 images per second in steadystate. As experimental configuration values were refined, reprocessing tasks were submitted as required. FuncX managed these tasks by expanding the number of Theta nodes being used to a maximum of 250, which enabled a processing rate of greater than 200 images a second.

Successfully processed images with diffraction-produced integration files were returned to the beamline computers and later refined and merged using PRIME 60 .

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