Physics 570 Michael Martynowycz

The Structural Basis for Specificity in Human ABO(H) Blood Group Biosynthesis Patenaude et al.

Two glycotransferases of differing sugar donors: GTA, GTB

Differ by four residues Only one found to be critical Needed for H-antigen binding Structures resolve binding sites

Why X-Rays?

Recall Braggs Law 2 =

Average bond length ~1-2 X-Rays generally ~1

Comparatively, visible light is ~5500 X-Rays are in a resolution sweet spot

for biological samples

Protein Crystallography

Purification Crystallization Mounting Data Collection Data Processing Structure Solution Structure Analysis

Ref. 8

Pre-Work Protein extracted from E. coli

Purified via affinity columns

Crystallized at 18 via hanging drop diffusion Evaporates the sample to needed concentration

Sample placed within an X-Ray chamber

Controlled temperature and vacuum required

Ref. 6

The X-Rays (Data Collection)

Overwhelmingly elastic scattering

Requires formation of ordered domains Symmetry groups describing a primitive unit

cell, c222 in this case Ultimately resolve the electron density, (,, )

Ref. 4

X-Ray Optics of Crystallography

Recall that for X-Rays, = 1 +

= 22

= 2

Optical phenomena

arise from difference in path length, seen here as r

Ref. 2

Crystal Reflection Path difference can

be seen as: = =

Geometry yields:

= 2 sin The phase

difference is then given by: = 2

= 2

= 2

Ref. 2

Electron density If is scattered to , from the source at , then:

= 2 is the electron

density at that point This can be

integrated over a finite volume to get the total amplitude and phase:

= 2

Which can be Fourier Transformed in order to obtain the total electron density:

= 2

What is measured? We do not directly

measure amplitude, we measure intensity

2 Phase information is

lost, and must be reconstructed later

Reconstruction is possible due to periodicity of the lattice

We may say the transformed amplitude(unit) is then:

= 2

If we translate each D by some a, then:

() 2=0 = ()

Protein structures The method described

does not work directly Heavy molecules

attached to the larger in set locations

Diffraction patters are taken with and without the heavy molecules and solved as a superposition

Ref. 2

Current methodology The modern approach

is to tune the X-Rays to the absorption edge of the heavy particles

This results in a known phase and amplitude shift

Essentially the same idea as isomorphous replacement, but much faster

Ref. 2

The result Intensity gathered by

a charge-coupled device(CCD) detector

Initial data passed to HKL2000 (software) to prepare data

Rendering done by ARP/wARP (software)

Final rendering done by SETOR, and placed on the Protein Data Bank PDB.org

Ref. 1

References Online sources

[1]Nature Structural Biology, Vol 9, nu 9, sept 2002

[2]Encyclopedia of Optical Engineering, Robert M. Sweet

[3]Elements of modern X-Ray Physics 2nd ed, Wiley 2012, Als-Nielsen, McMorrow

[4]http://en.wikipedia.org/wiki/X-ray_crystallography

[5]http://web.pdx.edu/~pmoeck/phy381/Topic5a-XRD.pdf

[6]http://www.bio.davidson.edu/courses/molbio/molstudents/spring2003/kogoy/protein.html

[7]http://www.proteincrystallography.org/

[8]http://www.pdb.org/pdb/home/home.do

http://en.wikipedia.org/wiki/X-ray_crystallographyhttp://en.wikipedia.org/wiki/X-ray_crystallographyhttp://web.pdx.edu/~pmoeck/phy381/Topic5a-XRD.pdfhttp://web.pdx.edu/~pmoeck/phy381/Topic5a-XRD.pdfhttp://web.pdx.edu/~pmoeck/phy381/Topic5a-XRD.pdfhttp://www.bio.davidson.edu/courses/molbio/molstudents/spring2003/kogoy/protein.htmlhttp://www.bio.davidson.edu/courses/molbio/molstudents/spring2003/kogoy/protein.htmlhttp://www.bio.davidson.edu/courses/molbio/molstudents/spring2003/kogoy/protein.htmlhttp://www.bio.davidson.edu/courses/molbio/molstudents/spring2003/kogoy/protein.htmlhttp://www.proteincrystallography.org/http://www.proteincrystallography.org/http://www.pdb.org/pdb/home/home.dohttp://www.pdb.org/pdb/home/home.do

Final PresentationThe Structural Basis for Specificity in Human ABO(H) Blood Group BiosynthesisPatenaude et al.Why X-Rays?Protein CrystallographyPre-WorkThe X-Rays (Data Collection)X-Ray Optics of CrystallographyCrystal ReflectionElectron densityWhat is measured?Protein structuresCurrent methodologyThe resultSlide Number 14