From Initial Hit to Crystal Optimization with Microseeding of Human Carbonic Anhydrase IX—A Case Study for Neutron Protein Crystallography

Koruza, Katarina; Lafumat, Bénédicte; Nyblom, Maria; Knecht, Wolfgang; Fisher, Zoë

doi:10.3390/cryst8110434

Cited by 6 publications

(6 citation statements)

References 27 publications

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“…The largest CA IX SV crystal that we obtained was 0.8 mm 3 and efforts to scale up and increase the volume continue (see Fig. 5 in Koruza, Lafumat, Nyblom et al, 2018). We obtained crystals in space group P2 1 with two apparently different unit cells labelled 'small' (unit-cell parameters a = 44.5, b = 65.4, c = 46.7 Å , = 115.1 ) and 'big' (unit-cell parameters a = 48.9, b = 65.1, c = 76.3 Å , = 92.86 ).…”

Section: Crystallographymentioning

confidence: 91%

“…Crystallization drops were prepared using both the hangingdrop and sitting-drop vapour-diffusion methods after a lengthy optimization procedure as described elsewhere (Koruza, Lafumat, Nyblom et al, 2018). Briefly, crystals were initially grown for the preparation of seed stocks using a 1:1 ratio of protein solution (17 mg ml À1 H/H CA IX SV ) and 30%(w/v) PEG 4000, 0.1 M Tris-HCl pH 8.5, 0.2 M sodium acetate or 0.2 M ammonium formate.…”

Section: Ca IX Sv Crystallization Optimization and H/d Exchangementioning

confidence: 99%

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Structural comparison of protiated, H/D-exchanged and deuterated human carbonic anhydrase IX

Koruza

Lafumat

Nyblom

et al. 2019

Acta Crystallogr D Struct Biol

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Human carbonic anhydrase IX (CA IX) expression is upregulated in hypoxic solid tumours, promoting cell survival and metastasis. This observation has made CA IX a target for the development of CA isoform-selective inhibitors. To enable structural studies of CA IX–inhibitor complexes using X-ray and neutron crystallography, a CA IX surface variant (CA IXSV; the catalytic domain with six surface amino-acid substitutions) has been developed that can be routinely crystallized. Here, the preparation of protiated (H/H), H/D-exchanged (H/D) and deuterated (D/D) CA IXSV for crystallographic studies and their structural comparison are described. Four CA IXSV X-ray crystal structures are compared: two H/H crystal forms, an H/D crystal form and a D/D crystal form. The overall active-site organization in each version is essentially the same, with only minor positional changes in active-site solvent, which may be owing to deuteration and/or resolution differences. Analysis of the crystal contacts and packing reveals different arrangements of CA IXSV compared with previous reports. To our knowledge, this is the first report comparing three different deuterium-labelled crystal structures of the same protein, marking an important step in validating the active-site structure of CA IXSV for neutron protein crystallography.

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Section: Crystallographymentioning

confidence: 91%

Section: Ca IX Sv Crystallization Optimization and H/d Exchangementioning

confidence: 99%

Structural comparison of protiated, H/D-exchanged and deuterated human carbonic anhydrase IX

Koruza

Lafumat

Nyblom

et al. 2019

Acta Crystallogr D Struct Biol

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“…This crystallization phase diagram was deduced from the raw data of Figure S2. This crystallization phase diagram is unique for RuBisCO from A. eutrophus and is not consistent with either the diagram of human carbonic anhydrase IX by Koruza K. et al [24] or nucleation and growth of protein crystals: general principles and assays in Methods in Enzymology [25]. We can understand easily from this canoe-shaped crystallization phase transition diagram both the salt-in effect and the salting out effect simultaneously which are required to crystallize a protein with precipitating agent as salts.…”

Section: Effect Of Protein Concentration and Dependence On The Volume Of The Crystallization Dropletmentioning

confidence: 56%

New Insight into the Effects of Various Parameters on the Crystallization of Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase (RuBisCO) from Alcaligenes eutrophus

Choe

Kim

2022

Crystals

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Crystallization remains a bottleneck for determining the three-dimensional X-ray structure of proteins. Many parameters influence the complexity of protein crystallization. Therefore, it is not easy to systematically examine all of these parameters individually during crystallization because of a limited quantity of purified protein. We studied several factors that influence crystallization including protein concentration, pH, temperature, age, volume of crystallization, inhibitors, metal ions, seeding, and precipitating agents on RuBisCO samples from Alcaligenes eutrophus which are not only freshly purified, but are also dissolved both individually and in combination from microcrystals and precipitated droplets of recycled RuBisCO. Single-, twin-, and/or microcrystals are dependent upon the concentration of RuBisCO by both RuBisCO samples. The morphology, either orthorhombic- or monoclinic-space group, depends upon pH. Furthermore, ammonium sulfate((NH4)2SO4) concentration at 20 °C (22% saturated) and/or at 4 °C (28% saturated) affected the crystallization of RuBisCO differently from one another. Finally, the age of RuBisCO also affected more uniformity and forming sharp edge during crystallization. Unexpected surprising monoclinic RuBisCO crystals were grown from dissolved microcrystals and precipitated droplets recycled RuBisCO samples. This quaternary RuBisCO single crystal, which contained Mg2+ and HCO3 for an activated ternary complex and is inhibited with a transition substrate analogue, CABP (2-carboxyarabinitol-1,5-bisphosphate)−, diffracts better than 2.2 Å. It is different from Hansen S. et al. reported RuBisCO crystals which were grown ab initio in absence of Mg2+, HCO3− and CABP, a structure which was determined at 2.7 Å resolution.

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“…The procedure for algal autolysis was adjusted and modified from literature descriptions. , Microalgae B. braunii (UTEX Showa strain, Culture Collection of Algae at the University of Texas at Austin) were continuously cultivated in perdeuterated modified Bolds 3 N medium .…”

Section: Methodsmentioning

confidence: 99%

Perdeuterated GbpA Enables Neutron Scattering Experiments of a Lytic Polysaccharide Monooxygenase

Sørensen,

Montserrat-Canals,

Loose

et al. 2023

ACS Omega

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Lytic polysaccharide monooxygenases (LPMOs) are surface-active redox enzymes that catalyze the degradation of recalcitrant polysaccharides, making them important tools for energy production from renewable sources. In addition, LPMOs are important virulence factors for fungi, bacteria, and viruses. However, many knowledge gaps still exist regarding their catalytic mechanism and interaction with their insoluble, crystalline substrates. Moreover, conventional structural biology techniques, such as X-ray crystallography, usually do not reveal the protonation state of catalytically important residues. In contrast, neutron crystallography is highly suited to obtain this information, albeit with significant sample volume requirements and challenges associated with hydrogen’s large incoherent scattering signal. We set out to demonstrate the feasibility of neutron-based techniques for LPMOs using N-acetylglucosamine-binding protein A (GbpA) from Vibrio cholerae as a target. GbpA is a multifunctional protein that is secreted by the bacteria to colonize and degrade chitin. We developed an efficient deuteration protocol, which yields >10 mg of pure 97% deuterated protein per liter expression media, which was scaled up further at international facilities. The deuterated protein retains its catalytic activity and structure, as demonstrated by small-angle X-ray and neutron scattering studies of full-length GbpA and X-ray crystal structures of its LPMO domain (to 1.1 Å resolution), setting the stage for neutron scattering experiments with its substrate chitin.

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From Initial Hit to Crystal Optimization with Microseeding of Human Carbonic Anhydrase IX—A Case Study for Neutron Protein Crystallography

Cited by 6 publications

References 27 publications

Structural comparison of protiated, H/D-exchanged and deuterated human carbonic anhydrase IX

Structural comparison of protiated, H/D-exchanged and deuterated human carbonic anhydrase IX

New Insight into the Effects of Various Parameters on the Crystallization of Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase (RuBisCO) from Alcaligenes eutrophus

Perdeuterated GbpA Enables Neutron Scattering Experiments of a Lytic Polysaccharide Monooxygenase

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