A high-transparency, micro-patternable chip for X-ray diffraction analysis of microcrystals under native growth conditions

Murray, Thomas D.; Lyubimov, A.Y.; Ogata, Craig M.; Vo, Huy T.; Uervirojnangkoorn, Monarin; Brunger, Axel T.; Berger, James M.

doi:10.1107/s1399004715015011

Cited by 74 publications

(63 citation statements)

References 83 publications

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“…The resulting devices can then be sealed with $50 to 500 nm-thick silicon nitride windows. 34,46,[141][142][143][144] This approach allows for the creation of extremely small features, such as wells to capture individual microcrystals. However, the devices are relatively time and labor intensive and expensive to produce.…”

Section: B Device Materials For Microcrystallographymentioning

confidence: 99%

Microfluidics: From crystallization to serial time-resolved crystallography

Sui

Perry

2017

Structural Dynamics

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Capturing protein structural dynamics in real-time has tremendous potential in elucidating biological functions and providing information for structure-based drug design. While time-resolved structure determination has long been considered inaccessible for a vast majority of protein targets, serial methods for crystallography have remarkable potential in facilitating such analyses. Here, we review the impact of microfluidic technologies on protein crystal growth and X-ray diffraction analysis. In particular, we focus on applications of microfluidics for use in serial crystallography experiments for the time-resolved determination of protein structural dynamics.

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Section: B Device Materials For Microcrystallographymentioning

confidence: 99%

Microfluidics: From crystallization to serial time-resolved crystallography

Sui

Perry

2017

Structural Dynamics

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“…These are generally based on a goniometer ‘borrowed’ from a synchrotron beamline (Cohen et al , 2014, Hirata et al , 2014) and successes with this approach have led to the emergence of serial crystallography at synchrotrons (Gati et al , 2014, Coquelle et al , 2015, Botha et al , 2015). On-going work in new sample mounts, such as grid-based supports (Huang et al , 2015, Roedig et al , 2015, Murray et al , 2015, Mueller et al , 2015, Baxter et al , 2016) and microfluidic flow cells (Lyubimov et al , 2015, Dhouib et al , 2009, Heymann et al , 2014), may see sample delivery techniques optimal for use at both FELs and synchrotrons.…”

Section: Endstation and Experimentsmentioning

confidence: 99%

Current advances in synchrotron radiation instrumentation for MX experiments

Owen

Juanhuix

Fuchs

2016

Archives of Biochemistry and Biophysics

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Following pioneering work 40 years ago, synchrotron beamlines dedicated to macromolecular crystallography (MX) have improved in almost every aspect as instrumentation has evolved. Beam sizes and crystal dimensions are now on the single micron scale while data can be collected from proteins with molecular weights over 10 MDa and from crystals with unit cell dimensions over 1000 Å. Furthermore it is possible to collect a complete data set in seconds, and obtain the resulting structure in minutes. The impact of MX synchrotron beamlines and their evolution is reflected in their scientific output, and MX is now the method of choice for a variety of aims from ligand binding to structure determination of membrane proteins, viruses and ribosomes, resulting in a much deeper understanding of the machinery of life. A main driving force of beamline evolution have been advances in almost every aspect of the instrumentation comprising a synchrotron beamline. In this review we aim to provide an overview of the current status of instrumentation at modern MX experiments. The most critical optical components are discussed, as are aspects of endstation design, sample delivery, visualization and positioning, the sample environment, beam shaping, detectors and data acquisition and processing.

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“…Our methodology combines 1) the use of specialized multi-crystal holders (MCH) compatible with cryo-protectant or native crystallization conditions, 2) the identification of individual micro-crystals on MCHs through the analysis UV-microscopy images, and 3) the development of automated routines for serial positioning of the identified crystals during data collection. Implementation of these methods improved on similar reference and alignment schemes (25)(26)(27)(28)(29)(30)(31) enabling rapid mapping and positioning of multiple crystals in random locations on the holder, which consistently resulted in >80% crystal hit-rates and the highest indexing rates reported to date for any SFX experiment.…”

Section: Introductionmentioning

confidence: 99%

Molecular mimicry in deoxy-nucleotide catalysis: the structure ofEscherichia colidGTPase reveals the molecular basis of dGTP selectivity: New structural methods offer insight on dGTPases

Song

et al. 2018

Preprint

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Serial Femtosecond Crystallography | X-ray free electron laser | Metalloenzymes | dNTP Triphophohydrolase | Mutli-crystal Holder | Chemical cross-linking AbstractDeoxynucleotide triphosphate triphosphyohydrolyases (dNTPases) play a critical role in cellular survival and DNA replication through the proper maintenance of cellular dNTP pools by hydrolyzing dNTPs into deoxynucleosides and inorganic triphosphate (PPPi). While the vast majority of these enzymes display broad activity towards canonical dNTPs, exemplified by Sterile Alpha Motif (SAM) and Histidine-aspartate (HD) domain-containing protein 1 (SAMHD1), which blocks reverse transcription of retroviruses in macrophages by maintaining dNTP pools at low levels, Escherichia coli (Ec)-dGTPase is the only known enzyme that specifically hydrolyzes dGTP. However, the mechanism behind dGTP selectivity is unclear. Here we present the free-, ligand (dGTP)-and inhibitor (GTP)-bound structures of hexameric E. coli dGTPase. To obtain these structures, we applied UV-fluorescence microscopy, video analysis and highly automated goniometer-based instrumentation to map and rapidly position individual crystals randomlylocated on fixed target holders, resulting in the highest indexing-rates observed for a serial femtosecond crystallography (SFX) experiment. The structure features a highly dynamic active site where conformational changes are coupled to substrate (dGTP), but not inhibitor binding, since GTP locks dGTPase in its apo form. Moreover, despite no sequence homology, dGTPase and SAMHD1 share similar active site and HD motif architectures; however, dGTPase residues at the end of the substrate-binding pocket mimic Watson Crick interactions providing Guanine base specificity, while a 7 Å cleft separates SAMHD1 residues from dNTP bases, abolishing nucleotidetype discrimination. Furthermore, the structures sheds light into the mechanism by which long distance binding (25 Å) of single stranded DNA in an allosteric site primes the active site by conformationally "opening" a tyrosine gate allowing enhanced substrate binding. Significance StatementdNTPases play a critical role in cellular survival through maintenance of cellular dNTP. While dNTPases display activity towards dNTPs, such as SAMHD1 -which blocks reverse transcription of HIV-1 in macrophages-Escherichia coli (Ec)-dGTPase is the only known enzyme that specifically hydrolyzes dGTP. Here we use novel free electron laser data collection to shed light into the mechanisms of (Ec)-dGTPase selectivity. The structure features a dynamic active site where conformational changes are coupled to dGTP binding. Moreover, despite no sequence homology between (Ec)-dGTPase and SAMHD1, both enzymes share similar active site architectures; however, dGTPase residues at the end of the substrate-binding pocket provide dGTP specificity, while a 7 Å cleft separates SAMHD1 residues from dNTP.

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A high-transparency, micro-patternable chip for X-ray diffraction analysis of microcrystals under native growth conditions

Abstract: A highly X-ray-transparent, silicon nitride-based device has been designed and fabricated to harvest protein microcrystals for high-resolution X-ray diffraction data collection using microfocus beamlines and XFELs.

Cited by 74 publications

References 83 publications

Microfluidics: From crystallization to serial time-resolved crystallography

Microfluidics: From crystallization to serial time-resolved crystallography

Current advances in synchrotron radiation instrumentation for MX experiments

Molecular mimicry in deoxy-nucleotide catalysis: the structure ofEscherichia colidGTPase reveals the molecular basis of dGTP selectivity: New structural methods offer insight on dGTPases

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