Native SAD phasing at room temperature

Greisman, Jack B.; Dalton, Kevin M.; Sheehan, Candice J.; Klureza, Margaret A.; Kurinov, Igor; Hekstra, Doeke R.

doi:10.1107/s2059798322006799

Cited by 11 publications

(13 citation statements)

References 53 publications

(84 reference statements)

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“…3). Another recently deposited RT structure of apo PTP1B, PDB entry 7rin (Greisman et al, 2022), agrees with PDB entry 6b8x in these respects, adopting the same open/closed WPD and L16 site coupling and partial occupancy of 7 (Fig. 3).…”

Section: Resultssupporting

confidence: 73%

Room-temperature serial synchrotron crystallography of the human phosphatase PTP1B

Sharma¹,

Ebrahim²,

Keedy³

2023

Acta Crystallogr F Struct Biol Commun

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Room-temperature X-ray crystallography provides unique insights into protein conformational heterogeneity, but obtaining sufficiently large protein crystals is a common hurdle. Serial synchrotron crystallography (SSX) helps to address this hurdle by allowing the use of many medium- to small-sized crystals. Here, a recently introduced serial sample-support chip system has been used to obtain the first SSX structure of a human phosphatase, specifically protein tyrosine phosphatase 1B (PTP1B) in the unliganded (apo) state. In previous apo room-temperature structures, the active site and allosteric sites adopted alternate conformations, including open and closed conformations of the active-site WPD loop and of a distal allosteric site. By contrast, in our SSX structure the active site is best fitted with a single conformation, but the distal allosteric site is best fitted with alternate conformations. This observation argues for additional nuance in interpreting the nature of allosteric coupling in this protein. Overall, our results illustrate the promise of serial methods for room-temperature crystallography, as well as future avant-garde crystallography experiments, for PTP1B and other proteins.

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Section: Resultssupporting

confidence: 73%

Room-temperature serial synchrotron crystallography of the human phosphatase PTP1B

Sharma¹,

Ebrahim²,

Keedy³

2023

Acta Crystallogr F Struct Biol Commun

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“…We expressed, purified, and crystallized ecDHFR as described previously [43], with one modification. In order to purify ecDHFR for the complex with 10-methylfolate, we modified the methotrexate-affinity chromatography to include a wash with 200 mM potassium phosphate buffer (pH 6.0) with 1 M potassium chloride, 1 mM ethylenediaminetetraacetic acid (EDTA), and 1 mM dithiothreitol (DTT) and elution the protein using a linear gradient with 50 mM potassium borate buffer (pH 10.15) and 2 M potassium chloride.…”

Section: Methods Protein Purification and Crystallizationmentioning

confidence: 99%

“…[9,15,16]). However, the advances described here, building on improvements in hardware [23,42], data collection strategies [7,43], and analysis methods [44][45][46], enabled elucidation of the correlated motions of an enzyme in atomic detail. We expect the presented methods and strategy will likewise permit identification of the motions that underlie the function of a wide range of proteins, promoting the development of new mechanistic models to explain protein function and its allosteric regulation.…”

Section: Identifying Functional Network Of Residuesmentioning

confidence: 99%

Resolving conformational changes that mediate a two-step catalytic mechanism in a model enzyme

Greisman

Dalton

Brookner

et al. 2023

Preprint

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Enzymes catalyze biochemical reactions through precise positioning of substrates, cofactors, and amino acids to modulate the transition-state free energy. However, the role of conformational dynamics remains poorly understood due to lack of experimental access. This shortcoming is evident with E. coli dihydrofolate reductase (DHFR), a model system for the role of protein dynamics in catalysis, for which it is unknown how the enzyme regulates the different active site environments required to facilitate proton and hydride transfer. Here, we present ligand-, temperature-, and electric-field-based perturbations during X-ray diffraction experiments that enable identification of coupled conformational changes in DHFR. We identify a global hinge motion and local networks of structural rearrangements that are engaged by substrate protonation to regulate solvent access and promote efficient catalysis. The resulting mechanism shows that DHFR's two-step catalytic mechanism is guided by a dynamic free energy landscape responsive to the state of the substrate.

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“…Few room-temperature (RT) native phasing experiments have been reported following the pioneering work by Hendrickson (Hendrickson & Teeter, 1981) and Cianci (Cianci et al, 2004). With respect to SAD phasing at RT, the recent work by Greisman et al (2022) is of particular interest. The data-collection approach used by Greisman and coworkers is largely based on traditional methods for anomalous data collection using large crystals inside MicroRT sleeves (MiTeGen), 5 Â 30 mm (FWHM) helical data collection at 6.55 keV energy at a synchrotron and 26-35-fold redundant data sets.…”

Section: Introductionmentioning

confidence: 99%

Obtaining anomalous and ensemble information from protein crystals from 220 K up to physiological temperatures

Doukov

Herschlag

Yabukarski

2023

Acta Cryst Sect D Struct Biol

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X-ray crystallography has been invaluable in delivering structural information about proteins. Previously, an approach has been developed that allows high-quality X-ray diffraction data to be obtained from protein crystals at and above room temperature. Here, this previous work is built on and extended by showing that high-quality anomalous signal can be obtained from single protein crystals using diffraction data collected at 220 K up to physiological temperatures. The anomalous signal can be used to directly determine the structure of a protein, i.e. to phase the data, as is routinely performed under cryoconditions. This ability is demonstrated by obtaining diffraction data from model lysozyme, thaumatin and proteinase K crystals, the anomalous signal from which allowed their structures to be solved experimentally at 7.1 keV X-ray energy and at room temperature with relatively low data redundancy. It is also demonstrated that the anomalous signal from diffraction data obtained at 310 K (37°C) can be used to solve the structure of proteinase K and to identify ordered ions. The method provides useful anomalous signal at temperatures down to 220 K, resulting in an extended crystal lifetime and increased data redundancy. Finally, we show that useful anomalous signal can be obtained at room temperature using X-rays of 12 keV energy as typically used for routine data collection, allowing this type of experiment to be carried out at widely accessible synchrotron beamline energies and enabling the simultaneous extraction of high-resolution data and anomalous signal. With the recent emphasis on obtaining conformational ensemble information for proteins, the high resolution of the data allows such ensembles to be built, while the anomalous signal allows the structure to be experimentally solved, ions to be identified, and water molecules and ions to be differentiated. Because bound metal-, phosphorus- and sulfur-containing ions all have anomalous signal, obtaining anomalous signal across temperatures and up to physiological temperatures will provide a more complete description of protein conformational ensembles, function and energetics.

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Native SAD phasing at room temperature

Cited by 11 publications

References 53 publications

Room-temperature serial synchrotron crystallography of the human phosphatase PTP1B

Room-temperature serial synchrotron crystallography of the human phosphatase PTP1B

Resolving conformational changes that mediate a two-step catalytic mechanism in a model enzyme

Obtaining anomalous and ensemble information from protein crystals from 220 K up to physiological temperatures

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