Basic Crystallography –

Data collection and processing

Louise N. Dawe, PhD
Wilfrid Laurier University
Department of Chemistry and Biochemistry
 References and Additional Resources
Faculty of Science, Bijvoet Center for Biomolecular Research,
Crystal and Structural Chemistry. ‘Interpretation of Crystal
Structure Determinations’ 2005 Course Notes:
http://www.cryst.chem.uu.nl/huub/notesweb.pdf

The University of Oklahoma: Chemical Crystallography Lab.

Crystallography Notes and Manuals.
http://xrayweb.chem.ou.edu/notes/index.html

Müller, P. Crystallographic Reviews, 2009, 15(1), 57-83.

Müller, Peter. 5.069 Crystal Structure Analysis, Spring 2010.

(Massachusetts Institute of Technology: MIT
OpenCourseWare), http://ocw.mit.edu/courses/chemistry/5-
069-crystal-structure-analysis-spring-2010/. License: Creative
Commons BY-NC-SA
X-ray Crystallography
 Data Collection and Processing
• Select and mount the crystal.
• Center the crystal to the center of the goniometer
circles (instrument maintenance.)
• Collect several images; index the diffraction spots;
refine the cell parameters; check for higher metric
symmetry
• Determine data collection strategy; collect data.
• Reduce the data by applying background, profile (spot-
shape), Lorentz, polarization and scaling corrections.
• Determine precise cell parameters.
• Collect appropriate information for an absorption
correction. (Index the faces of the crystal. A highly
redundant set of data is sufficient for an empirical
absorption correction.)
• Apply an absorption correction to the data.
(http://xrayweb.chem.ou.edu/notes/collect.html)
 Single crystal diffraction of X-rays

Principle quantum number

n = 1  K level
Note: The non SI unit Å is normally used. n = 2  L level
1 Å = 10-10 m n = 3  M level
etc…
L to K transitions produce 'Ka' emission
M to K transitions produce 'Kb' emission.
M to L transitions produce 'La' emissions.
There are several energy sublevels in the L, M, N levels so there are in fact 'Ka1'
and 'Ka2' peaks which are very close to one another in energy.
 Single crystal diffraction of X-rays
Each element has its own characteristic x-ray spectrum
• For Copper the characteristic
wavelengths (λ) are:
• Cu Kα1 = 1.540Å
• Cu Kα2 = 1.544Å
• Cu Kb = 1.392Å

I
• For Molybdenum they are:
• Mo Kα1 = 0.70932Å
• Mo Kα2 = 0.71354Å
• Mo Kb = 0.63225Å


• We use MoKα (avg.) radiation
• (λ) = 0.71073Å E
• Or CuKα (avg.)
• (λ) = 1.54178Å
A. Sarjeant
 Single crystal diffraction of X-rays
A large potential difference (ex. 50kV) is put
between a tungsten filament (cathode) and a
metal target (anode; ex. Molybdenum).

Electrons ejected from the filament ionize

electrons from the target material. When these
electrons drop back into the vacated energy
levels, they give off energy partially in the form
of electromagnetic radiation (and a lot of lot of
heat; the tube is water cooled.)

Different metal targets emit X-rays of different http://xray0.princeton.edu/~phil/Facility/Gui

wavelengths. des/Phillips_sealed_tube.jpg

Beryllium windows (toxic; do not touch!) are

relatively transparent to X-rays and let the X-
rays escape the evacuated tube.
 Single crystal diffraction of X-rays
Normally, X-ray lab users must become "authorized users“; these
users wear badges that monitor any exposure to radiation. This is
federally regulated.

Some general safety notes:

1. Know the expected path of the main X-ray beam. Always keep
all parts of your body outside of this path.

2. Whenever possible, keep the safety doors to the instrument

closed. For most modern instruments are safeties in place that
make it impossible for the X-ray shutter to be open at the same
time as the instrument doors.

3. No unauthorized personnel may defeat or override any safety

features
 Single crystal diffraction of X-rays
Some extra safety notes:

There is a serious hazard associated with possible electrical

shock. The X-ray generator is a highly-regulated DC power
supply that operates at an applied voltage of 50 kV, and 30-40
mA (this may vary with instrument and operator.)

The X-ray generator has several large capacitors. Even when the
instrument is turned off, these capacitors store sufficient power to
injure and possibly kill a person. All work on any X-ray generator
should be done only by personnel trained in high-voltage
electronics.

Never work above or below the generator cabinet.

 Single crystal diffraction of X-rays
Lights up Sample
when
shutter is
open Beamstop
(literally!)

Mo X-ray
tube CCD
Detector

Graphite
monochro
mator

Goiniometer

Collimator – Attenuates X-ray beam diameter

 Single crystal diffraction of X-rays

Monochromator

Collimator

Mo or Cu Source

 = 1.5418 Å

 = 0.7107 Å
 “Garbage In = Garbage out” (P. Müller, 2009)

• Your structure refinement will only be as good as

the data that you collect

• Four things to consider:

• Your crystal
• Your instrument
• How you collect your data
• How you treat your data post-collection
 Choosing a Crystal

• Upcoming lecture on crystal growth

• Earlier lecture on qualities to look for in a good crystal

• Worth spending time carefully looking for the best

possible crystal using a polarized microscope

• Limitations:
• Crystals that desolvate readily and are not amenable to
prolonged examination
• The “best” crystal may not be representative of the bulk
sample.
 Crystal Mounting

• Normally crystals are selected to be smaller than

the diameter of the beam to ensure a constant
volume of irradiated matter

• Crystals can be cut to size (with some practice)

https://www.bruker.com/fileadm
in/user_upload/8-PDF-Docs/X-
rayDiffraction_ElementalAnalysis
/SC-
XRD/Webinars/Bruker_AXS_Gro
wing_Mounting_Single_Crystals_
Webinar_201011026.pdf

• Critically examine a few initial images

 Crystal Mounting

• Other considerations
• Tools for mounting
• The actual mount
• Oil, epoxy, UV-curing

• Data Collection Temperature

• Low temperature (ex. 100 K) to minimize thermal
vibrations
• Constant temperature (even if collected close to RT, use
of a low temperature device to maintain a constant
temperature throughout experiment)
 Crystal Mounting

https://www.bruker.com/fileadmin/user_upload/8-PDF-Docs/X-
rayDiffraction_ElementalAnalysis/SC-
XRD/Webinars/Bruker_AXS_Growing_Mounting_Single_Crystals_Webinar_201011026.pdf
Experiment geometry

A. Sarjeant
Eulerian Geometry

A. Sarjeant
Kappa Geometry



dx

2

A. Sarjeant
A. Sarjeant
 Single crystal diffraction of X-rays
Recall: The diffraction pattern does not depend on translation, but does rotate if
the lattice is rotated.

The following video shows the images from an X-ray diffraction data collection:
http://ruppweb.org/data/vta1.wmv
 Instrumental Optimization

• Regular maintenance
• Correctly aligned

• How do you know?

• Stable test crystal that is regularly collected, with
comparison to previous results.
• When in doubt about your own instrument, recollect the
test crystal.
 Data Collection Strategy: Maximum Resolution
• Reflection intensities are generally weaker at higher
resolutions, but high angle data contains important
structural information.

• IUCr generally
recommends a
Minimum resolution of
0.54 Å.

(How does this

relate to Bragg’s Law?)
 Data Collection Strategy: Maximum Resolution

Problem: The Acta Cryst standard for 2 collections is a

minimum cut-off of 53o. Why do you think that is?

Solution: Employing Bragg’s law with  = 0.7107 Å (Mo-Ka

radiation) and  = 26.5o:
 0.7107 A 0.7107 A
d     0.803 A
d 2sin  2sin(26.5 )o 2(0.4462)  0.803 A
0.7107 oA 0.7107 A
2sin  2sin(26.5 ) 2(0.4462) o
The normal range of X-H bonds is ~0.80-0.95 A. At 53 these
separations can be resolved.
The normal range of X-H bonds is ~0.80-0.95 A. At 53o these
separations can be resolved.
 CH495 Dr. L. Dawe Fall 2014

Bragg’s Equation
2 = 50.7o ( = 0.7107 Å)
• See previous example
• This should lead to a
publishable result.

2 = 17o ( = 0.7107 Å) 2 = 41.6o ( = 0.7107 Å)

• Old protein structures • Small molecule solution
• No distinct atomic possible.
positions can be identified • Refinement of atomic
positions will have large
associated errors.

Reprinted from Interpretation of Crystal Structure Determinations. Copyright 2005 Huub Jooijman, Bijvoet
Center for Biomolecular Research and Structural Chemistry, Utrecht Univeristy.
 Data Collection Strategy: Data Completeness

• Data completeness is the data actually collected

compared to what is the unique data for the given
crystal symmetry.

• Software will allow you to determine a data

collection strategy to yield 100% completeness.

• Some crystallographers have developed their own

collection strategies (based on presumed low
symmetry and experience.)
 Data Collection Strategy: I/s
• Average measured intensity/estimated noise

• Ideally should be as
high as possible (~10
throughout the data
set)

• Values less than 2

are essentially noise

• Decisions about where

to cut off your resolution?
 Data Collection Strategy: Multiplicity of
Observations
• Multiplicity of Observation (MoO) refers to
multiple measurements of the same, or
symmetry equivalent, reflection, obtained from a
different crystal orientation.
• Higher values of MoO should yield better
statistics
• Higher symmetry crystals require less images to
obtain equivalent MoO to lower symmetry
crystals
• One approach is to collect all crystals as though
they were triclinic (over-estimating symmetry
can yield incomplete data.)
 Processing
• Modifications to measured I(hkl) are required to
correct for geometry of measurement

• Essential to yield high quality accurate data for

solution and refinement.

• Some correction factors include:

• Lorentz factor (accounts for time required for a Bragg
reflection to cross the surface of the sphere of reflection)
• Polarization factor (polarization of the incident X-ray
beam)
• Absorption (intensity of measure reflections is reduced by
the absorption of X-rays by the crystal)
 Processing: Corrections
For a small crystal completely bathed in a uniform beam of
radiation, the integrated intensity, I, is given by:

I = Io (re)2 (Lp/A) (λ/Ω) (F/V)2 λ2υ

The quantity re = e2/mc2 = 2.82 × 10-13 cm is the classical radius

of an electron. V is the unit cell volume; υ is the volume of the
crystal. Ω is the angular velocity of the sample as the peak
moves through the Ewald sphere. Correction terms include the
Lorentz correction, L, the polarization correction, p, and the
absorption correction, A.

http://xrayweb.chem.ou.edu/notes/collect.html#correction
 Processing: Absorption Corrections
The absorption of X rays follows Beer's Law:

I / Io = exp(-μ × t)

where I = transmitted intensity, Io = incident intensity, t = thickness of material, μ

= linear absorption coefficient of the material. The linear absorption coefficient
depends on the composition of the substance, its density, and the wavelength of
the radiation. Since μ depends on the density of the absorbing material, it is
usually tabulated as the related function mass absorption coefficient μm = (μ /
ρ).

The linear absorption coefficient is then calculated from the formula:

μ = ρ ∑ (Pn / 100) × (μ / ρ) = ρ ∑ (Pn / 100) × μm

where the summation is carried out over the n atom types in the cell, and Pn is
the percent by mass of the given atom type in the cell.

http://xrayweb.chem.ou.edu/notes/collect.html#corrections
 Processing: Absorption Corrections
• Crystals were ground or cut to be approximately spherical in order to
minimize unequal absorption effects

• Numerical absorption corrections require accurate information about

crystal shape by way of indexing a crystal’s faces. Analytical absorption
corrections are accomplished by mathematically dividing the sample
into very small pieces and calculating the transmittance for each piece
of the crystal for each reflection measured.This can be difficult for
crystals with many closely spaced faces. The hkl indices of faces and
their distances from the center of the crystal are required. Less
common now, but still used for very strongly absorbing materials and
charge density studies.

• Modern semi-empirical methods are based on measurement of

equivalent reflections and work well when there is a high multiplicity of
observations. By comparing the intensities from the redundant
measurements, an absorption surface for the sample is calculated.

• http://xrayweb.chem.ou.edu/notes/collect.html#corrections
 Data Collection Strategy: Merging Residuals
• Symmetry-equivalent intensity data are merged using the
following relationship
F2 = ∑ ωj Fj2 / ∑ ωj
where the summations are over the set of symmetry-equivalent
data. In this formula, weights can be either from statistics (ω =
1/σ(F2)) or unit values .
(http://xrayweb.chem.ou.edu/notes/collect.html#merge)

• When comparing unique data to total data collected, there

are a variety of residuals that can be calculated as a
measure of internal data consistency

• For example: _diffrn_reflns_av_R_equivalents with is the

residual for symmetry-equivalent reflections used to
calculate the average intensity.

• Lower merging R-factors indicate better datasets (would like

to see less than 10% over the entire range of resolution)
Where can I find this info?
 Something fun and marginally related

A complete collection of at least one crystal structure

for all of the 230 space groups:

https://crystalsymmetry.wordpress.com/230-2/