3KJR

Crystal structure of dihydrofolate reductase/thymidylate synthase from Babesia bovis determined using SlipChip based microfluidics


Experimental Data Snapshot

  • Method: X-RAY DIFFRACTION
  • Resolution: 1.95 Å
  • R-Value Free: 0.201 
  • R-Value Work: 0.169 
  • R-Value Observed: 0.170 

wwPDB Validation   3D Report Full Report


Ligand Structure Quality Assessment 


This is version 1.2 of the entry. See complete history


Literature

Multiparameter screening on SlipChip used for nanoliter protein crystallization combining free interface diffusion and microbatch methods.

Li, L.Du, W.Ismagilov, R.F.

(2010) J Am Chem Soc 132: 112-119

  • DOI: https://doi.org/10.1021/ja908558m
  • Primary Citation of Related Structures:  
    3KJR

  • PubMed Abstract: 

    This paper describes two SlipChip-based approaches to protein crystallization: a SlipChip-based free interface diffusion (FID) method and a SlipChip-based composite method that simultaneously performs microbatch and FID crystallization methods in a single device. The FID SlipChip was designed to screen multiple reagents, each at multiple diffusion equilibration times, and was validated by screening conditions for crystallization of two proteins, enoyl-CoA hydratase from Mycobacterium tuberculosis and dihydrofolate reductase/thymidylate synthase from Babesia bovis, against 48 different reagents at five different equilibration times each, consuming 12 microL of each protein for a total of 480 experiments using three SlipChips. The composite SlipChip was designed to screen multiple reagents, each at multiple mixing ratios and multiple equilibration times, and was validated by screening conditions for crystallization of two proteins, enoyl-CoA hydratase from Mycobacterium tuberculosis and dihydrofolate reductase/thymidylate synthase from Babesia bovis. To prevent cross-contamination while keeping the solution in the neck channels for FID stable, the plates of the SlipChip were etched with a pattern of nanowells. This nanopattern was used to increase the contact angle of aqueous solutions on the surface of the silanized glass. The composite SlipChip increased the number of successful crystallization conditions and identified more conditions for crystallization than separate FID and microbatch screenings. Crystallization experiments were scaled up in well plates using conditions identified during the SlipChip screenings, and X-ray diffraction data were obtained to yield the protein structure of dihydrofolate reductase/thymidylate synthase at 1.95 A resolution. This free-interface diffusion approach provides a convenient and high-throughput method of setting up gradients in microfluidic devices and may find additional applications in cell-based assays.


  • Organizational Affiliation

    Department of Chemistry and Institute for Biophysical Dynamics, The University of Chicago, 929 East 57th Street, Chicago, Illinois 60637, USA.


Macromolecules
Find similar proteins by:  (by identity cutoff)  |  3D Structure
Entity ID: 1
MoleculeChains Sequence LengthOrganismDetailsImage
Dihydrofolate reductase/thymidylate synthase
A, B
511Babesia bovis T2BoMutation(s): 0 
EC: 2.1.1.45
UniProt
Find proteins for A7ASX7 (Babesia bovis)
Explore A7ASX7 
Go to UniProtKB:  A7ASX7
Entity Groups  
Sequence Clusters30% Identity50% Identity70% Identity90% Identity95% Identity100% Identity
UniProt GroupA7ASX7
Sequence Annotations
Expand
  • Reference Sequence
Small Molecules
Ligands 3 Unique
IDChains Name / Formula / InChI Key2D Diagram3D Interactions
NAP
Query on NAP

Download Ideal Coordinates CCD File 
C [auth A],
I [auth B]
NADP NICOTINAMIDE-ADENINE-DINUCLEOTIDE PHOSPHATE
C21 H28 N7 O17 P3
XJLXINKUBYWONI-NNYOXOHSSA-N
NHE
Query on NHE

Download Ideal Coordinates CCD File 
D [auth A]2-[N-CYCLOHEXYLAMINO]ETHANE SULFONIC ACID
C8 H17 N O3 S
MKWKNSIESPFAQN-UHFFFAOYSA-N
GOL
Query on GOL

Download Ideal Coordinates CCD File 
E [auth A],
F [auth A],
G [auth A],
H [auth A],
J [auth B]
GLYCEROL
C3 H8 O3
PEDCQBHIVMGVHV-UHFFFAOYSA-N
Experimental Data & Validation

Experimental Data

  • Method: X-RAY DIFFRACTION
  • Resolution: 1.95 Å
  • R-Value Free: 0.201 
  • R-Value Work: 0.169 
  • R-Value Observed: 0.170 
  • Space Group: P 21 21 21
Unit Cell:
Length ( Å )Angle ( ˚ )
a = 83.113α = 90
b = 97.559β = 90
c = 152.83γ = 90
Software Package:
Software NamePurpose
HKL-2000data collection
MOLREPphasing
REFMACrefinement
HKL-2000data reduction
HKL-2000data scaling

Structure Validation

View Full Validation Report



Ligand Structure Quality Assessment 


Entry History 

Revision History  (Full details and data files)

  • Version 1.0: 2009-11-17
    Type: Initial release
  • Version 1.1: 2011-07-13
    Changes: Advisory, Refinement description, Version format compliance
  • Version 1.2: 2023-09-06
    Changes: Data collection, Database references, Derived calculations, Refinement description