Mass: 42971.613 Da / Num. of mol.: 2 / Mutation: YES Source method: isolated from a genetically manipulated source Details: GDP-BETA-L-GULOSE WAS REFINED USING GMP (DEFINED AS GDP, MODIFIED IN THE CIF FILE TO REMOVE THE SECOND PHOSPHATE GROUP) AND GULOSE-MONOPHOSPHATE (GUL), LINKING THE GDP O3A TO THE GUL PB. GDP- ...Details: GDP-BETA-L-GULOSE WAS REFINED USING GMP (DEFINED AS GDP, MODIFIED IN THE CIF FILE TO REMOVE THE SECOND PHOSPHATE GROUP) AND GULOSE-MONOPHOSPHATE (GUL), LINKING THE GDP O3A TO THE GUL PB. GDP-BETA-L-4-KETO-GULOSE WAS REFINED USING GMP (DEFINED AS GDP, MODIFIED IN THE CIF FILE TO REMOVE THE SECOND PHOSPHATE GROUP) AND 4-KETO-GULOSE- MONOPHOSPHATE (4KG), LINKING THE GDP O3A TO THE 4KG PB NAD WAS REFINED USING ADP AND A NICOTINAMIDE RING (NI3), LINKING THE ADP PB TO THE NI3 O5* NADH WAS REFINED USING ADP AND A SKEWED REDUCED NICOTINAMIDE RING (NDH), LINKING THE ADP PB TO THE NDH O5* Source: (gene. exp.) Arabidopsis thaliana (thale cress) / Plasmid: PHISTEV-GME-K178R / Production host: Escherichia coli BL21(DE3) (bacteria) / Variant (production host): Rosetta / References: UniProt: Q93VR3, GDP-mannose 3,5-epimerase
Mass: 18.015 Da / Num. of mol.: 944 / Source method: isolated from a natural source / Formula: H2O
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Details
Compound details
ENGINEERED RESIDUE IN CHAIN A, LYS 178 TO ARG ENGINEERED RESIDUE IN CHAIN B, LYS 178 TO ARG
Sequence details
THE FIRST TWO RESIDUES (GA) IN THE SPECIFIED SEQUENCE ARE A REMNANT FROM A CLEAVED HIS-TAG (AND ARE ...THE FIRST TWO RESIDUES (GA) IN THE SPECIFIED SEQUENCE ARE A REMNANT FROM A CLEAVED HIS-TAG (AND ARE NOT IN THE UNIPROT SEQUENCE), THE RESIDUE NUMBERING IN THE STRUCTURE USES THE THIRD RESIDUE (M) AS RESIDUE 1.
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Experimental details
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Experiment
Experiment
Method: X-RAY DIFFRACTION / Number of used crystals: 1
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Sample preparation
Crystal
Density Matthews: 1.95 Å3/Da / Density % sol: 37 % Description: INITIAL MODEL FOR MOLECULAR REPLACEMENT BY PHASER DETERMINED BY MAD OF SELENO-METHIONINE PROTEIN. DATA COLLECTED ON BM14, ANALYSED WITH SOLVE AND RESOLVE.
Crystal grow
Method: vapor diffusion, sitting drop / pH: 7.4 Details: PROTEIN CRYSTALISED IN 100MM HEPES PH 7.4, 2.16 M AMMONIUM SULPHATE, VAPOUR DIFFUSION, SITTING DROP. CRYOPROTECTED WITH 4M SODIUM FORMATE.
Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelength
Wavelength: 0.934 Å / Relative weight: 1
Reflection
Resolution: 1.5→23.3 Å / Num. obs: 105071 / % possible obs: 99.9 % / Redundancy: 3.7 % / Biso Wilson estimate: 13.37 Å2 / Rmerge(I) obs: 0.05 / Net I/σ(I): 9.6
Reflection shell
Resolution: 1.5→1.54 Å / Redundancy: 3.6 % / Rmerge(I) obs: 0.21 / Mean I/σ(I) obs: 3.6 / % possible all: 99.9
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Processing
Software
Name
Version
Classification
REFMAC
5.2.0019
refinement
MOSFLM
datareduction
SCALA
datascaling
PHASER
phasing
Refinement
Method to determine structure: MOLECULAR REPLACEMENT / Resolution: 1.5→64.55 Å / Cor.coef. Fo:Fc: 0.978 / Cor.coef. Fo:Fc free: 0.959 / SU B: 2.281 / SU ML: 0.04 / Cross valid method: THROUGHOUT / σ(F): 0.96 / ESU R: 0.082 / ESU R Free: 0.069 / Stereochemistry target values: RESTRAINED Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS. EACH MONOMER OF GME K178R CONTAINS A MIXTURE OF COMPOUNDS IN THE ACTIVE SITE -NAD AND NADH IN THE NUCLEOTIDE BINDING SITE, GDP-BETA-L- ...Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS. EACH MONOMER OF GME K178R CONTAINS A MIXTURE OF COMPOUNDS IN THE ACTIVE SITE -NAD AND NADH IN THE NUCLEOTIDE BINDING SITE, GDP-BETA-L-GULOSE AND GDP-BETA-L-4- KETO-GULOSE IN THE NUCLEOTIDE SUGAR BINDING SITE. DURING REFINEMENT THE CONSTANT PORTION OF EACH MOLECULE (ADP FOR NAD, GMP FOR THE GDP-SUGARS, AS THE SECOND PHOSPHATE GROUP MOVES WITH DIFFERENT LIGANDS) WAS FIXED WITH OCCUPANCY OF 1 AND LINKED TO TWO VARIABLE PORTIONS WITH PARTIAL OCCUPANCY.
Rfactor
Num. reflection
% reflection
Selection details
Rfree
0.167
5247
5.05 %
RANDOM
Rwork
0.118
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obs
0.121
105045
99.9 %
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Solvent computation
Ion probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.4 Å / Solvent model: MASK BULK SOLVENT