mad sad is simpler better
Download
Skip this Video
Download Presentation
MAD/SAD: Is simpler better?

Loading in 2 Seconds...

play fullscreen
1 / 36

MAD/SAD: Is simpler better? - PowerPoint PPT Presentation


  • 81 Views
  • Uploaded on

MAD/SAD: Is simpler better?. Howard Robinson Brookhaven Biology. Brookhaven Biology. We know that our crystallographic methods are able to provide us with remarkably large and complex structures. Brookhaven Biology.

loader
I am the owner, or an agent authorized to act on behalf of the owner, of the copyrighted work described.
capcha
Download Presentation

PowerPoint Slideshow about ' MAD/SAD: Is simpler better?' - ulric-logan


An Image/Link below is provided (as is) to download presentation

Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author.While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server.


- - - - - - - - - - - - - - - - - - - - - - - - - - E N D - - - - - - - - - - - - - - - - - - - - - - - - - -
Presentation Transcript
mad sad is simpler better

MAD/SAD:Is simpler better?

Howard Robinson

Brookhaven Biology

Brookhaven Biology

slide2

We know that our crystallographic methods are able to provide us with remarkably large and complex structures.

Brookhaven Biology

slide3

Often the electron density is accurate enough that one can find the precise position of atomsto give important chemical information.

Brookhaven Biology

slide4

MAD map of Zn Alcohol Dehydrogenase

(from Solve/Resolve, 2000)

9 hours

3 wavelengths

2.2Å

x12B

Brookhaven Biology

questions
Questions?
  • Why have reports of MAD phasing vanished from the literature?
  • Where are the Sulfur SAD phasing reports?
how do we determine these structures
How do we determine these structures?
  • Protein gives crystals
  • Crystals and x-rays give diffraction
  • Diffraction amplitudes plus phases give electron density
slide7

What’s more important,

Amplitudes or Phases

Jerome Karle

Herbert Hauptman

Herb’s amplitudes

Jerry’s phases

Jerry’s amplitudes

Herb’s phases

Randy Read,

used by permission

Brookhaven Biology

the goals of this lecture
The goals of this lecture
  • Describe various ways of determining diffraction phase
  • Discuss isomorphous replacement
  • Show effect of anomalous dispersion
  • Show parallels between MAD and Isomorphous Replacement
  • Describe usefulness of SAD
slide9

Some sources of intelligence about crystallography and phasing

(Go to wikipedia: x-ray crystallography)

Brookhaven Biology

slide10

If crystal diffraction is good enough, one can determine structure directly from diffraction amplitudes. Popularized for proteins by especially Chuck Weeks, Hauptman, and Sheldrick.

Direct

Methods

Brookhaven Biology

slide11

If the unknown molecule is similar enough to a structure that is already known, one can use Molecular Replacement to supply phases.

Compare diffraction intensities from the crystal with calculated intensities from a model positioned within unit cell, and then use the model’s phases and the crystal’s amplitudes to calculate a map

Brookhaven Biology

slide12

More generally, with large molecules like proteins, one must employ scattering from a few HEAVY atoms in a molecule to gain phase information.

  • Isomorphous Replacement – add a few heavy atoms, diffract with and without
  • MAD – add heavy atoms and exploit wavelength dispersion of anomalous and in-phase scattering
  • SAD – similar to MAD, but use only anomalous scattering at a single energy
slide13

We understand how the scattering from individual atoms combines to give a diffracted ray with predictable amplitude and phase. Note the precise angles and the color coding.

Scattering from

Bragg planes

Structure factors

Vector additive

Randy Read, used by permission

Brookhaven Biology

slide14

Look at what happens when one adds a single heavy atom. The two structure factors, FP and FPH can be used to solve for the phase. This is called Isomorphous Replacement.

Randy Read, used by permission

Brookhaven Biology

slide15

Perutz’s Fundamental Idea: Isomorphous Replacement

FP = S Fatoms

FPH = FP + FH

FH

We can approximate |FH| with |FPH - FP|. This often suffices for solving for the position of the heavy atom as if it were a small-molecule structure.

slide16

So for some particular reflection and a particular heavy atom, we can begin to find the phase:

Knowing the position of the heavy atom allows us to calculate FH. Then we use FP = FPH + (-)FH to show that the phase triangles close with a two-fold ambiguity, at G and at H. There are several ways to resolve the ambiguity.

slide17

One way to resolve the ambiguity is to use a second isomorphous heavy-atom derivative.

slide18

David Blow and Francis Crick, when they were postdocs for Max Perutz, deduced a neat statistical way to determine the “best” phase when the MIR or SIR indications are ambiguous. We call it Figure-of-merit weighting.

Randy Read, used by permission

Brookhaven Biology

slide19

In principle, if we reflect x-rays from the “back” of Bragg planes, the amplitude should be the same and the phase should have opposite sign from when we reflect from the “front.”

We call this

Friedel’s Law:

F(h) = F*(-h)

Randy Read, used by permission

Brookhaven Biology

slide20

What aboutFriedel’s Law: F(h) = F*(-h), here with a heavy atom?

Randy Read, used by permission

Brookhaven Biology

slide21

And what does this have to do with the

fluorescence scan near the Se K-edge?

Brookhaven Biology

slide22

Chooch analyzes the fluorescence scan and shows the elastic and inelastic scattering.

Brookhaven Biology

slide23

However, a heavy atom might scatter anomalously, then Friedel’s law fails.

In this case, the resonance between the electrons on the heavy atom and the x-rays causes a phase and amplitude shift. The symmetry of diffraction (from the front vs back of the Bragg planes) is broken. This can be measured and used.

slide24

One can see how to choose wavelengths to get large phase contrast for MAD phasing.

*

*

*

*

*

*

*

Maximum Real Signal

*

*

Maximum Imaginary Signal

*

*

*

Spectrum from Phizackerly, Hendrickson, et al. Study of Lamprey Haemoglobin.

slide25

This Multiwavelength Anomalous Diffraction method often gives very strong phase information and is the source of many new structures.

slide26

Synchrotron radiation is very available and useful in the 8 to 14 keV range, and possible outside that range. Quite a number of elements can be incorporated into macromolecules that resonate in that range.

Is only the bottom row

really useful?

Ethan Merritt, used by permission

Brookhaven Biology

slide28

MAD map of Zn Alcohol Dehydrogenase

(from Solve/Resolve)

9 hours

3 wavelengths

2.2Å

x12B

Brookhaven Biology

modern times
Modern times
  • Several things make data more accurate
    • Much better detectors
    • Well-collimated synchrotron beams
    • Improved software
  • There are powerful tools to improve approximate phases: Resolve, DM
  • We can use weaker signals to solve structures: Use one wavelength to acquire anomalous data and solve the structure from that: Single Anomalous Diffraction. Density modification improves phases.
slide30

SAD map of Zn Alcohol Dehydrogenase (from Solve/Resolve)

3 hours at

peak wavelength

2.2Å

Brookhaven Biology

slide31

5 minutes at x29.

How much here?

Brookhaven Biology

slide32

Too Much Radiation Damage

360 1 sec. 1 degree =

6 min exposure at x29

slide33

Do you need to be near an edge?

Fe anomalous map at 1.1A.

mad or sad
MAD or SAD?
  • Simplicity.
  • Other sources of phase information.
  • Managing radiation induced damage effects.
  • How about Sulfur phasing?
how about sulfur phasing
How about Sulfur phasing?
  • Damage to S and S-S.
  • S anomalous signal, optimal wavelength?
how about identifying metals
How about Identifying Metals?

Anomalous map above and below the Cu edge

ad