Structure Determination and Refinement

The objective of the Midwest Center for Structural Genomics (MCSG) is to develop and optimize new, rapid, integrated methods for highly cost-effective determination of protein structures through X-ray crystallography using a third-generation synchrotron. Some of the goals are to develop standard multiwavelength anomalous diffraction (MAD) protocols and optimize anomalous dispersion strategies for rapid structure determination using synchrotron radiation, increase the speed of structure determination using parallel approaches, and minimize manual intervention in model building and structure refinement.


| Application of anomalous diffraction for automated protein structure determination |

Since 2000, anomalous scattering for protein structure determination has become the most important means to automation. The multi- and singlewavelength anomalous dispersion (MAD/SAD) method of protein structure determination is becoming a routine technique in protein crystallography. Ultrafast MAD/SAD data collection is now possible and, with the widespread use of selenium in the form of selenomethionine for phase determination, the method is being used extensively. Recent developments in crystallographic software are complementing these advances, paving the way for rapid protein structure determination. The SAD/MAD method has matured to the point that it has outpaced MIR as the method of choice for determining the structure of novel proteins up 350 kDa in size. Although SAD/MAD relies on a small signal, it has advantages over the MIR method all data can usually be measured from a single crystal cooled to liquid-N2 temperature, for example, to obtain very accurate phase information. The isomorphism of the SAD/MAD data collected from a single crystal is perhaps the key contributor to successful phase determination.

The MCSG is using synchrotron-based x-ray crystallography almost exclusively and all diffraction data are collected at synchrotron facilities (with exception of phasing with sulphur which is done using home source). The SBC 19-ID and 19-BM beamlines can easily meet stringent experimental requirements for SAD/MAD data collection, which include accurate and reproducible wavelength tunability and stability, accurate measurement of diffraction intensities using electronic detectors, and stable cryosystems for maintaining crystals at -100K.


| Synchrotron beamlines |

Modern synchrotron protein crystallography facilities provide tunable and stable sources of x-rays in the 6-20 keV range. There are numerous advantages to using dedicated synchrotron beamlines for protein crystallography:
  • The very high beam flux density improves the efficiency and quality of data, allowing the use of very small crystals.
  • The highly brilliant x-ray beams improve spatial resolution, facilitating the determination of very large structures.
  • The wavelength is tunable, which is critical for phase determination using SAD/MAD techniques.
  • The crystals are damaged less by the radiation when controlled x-ray dose, application of cryotemperatures, and precise x-ray wavelengths for data collection can be combined, as they can be with a dedicated synchrotron beamline.
  • The background is lower and the signal-to-noise ratios higher for smaller synchrotron beams. Dedicated beamlines at third-generation undulator sources have dramatically increased the number of successful MAD/SAD experiments.

For alFor all proteins containing methionine, we produce SeMet derivative. This approach has proven to be the most cost effective (>94% of MCSG structures have been phased with selenium atoms). For proteins that fail we consider alternative phasing strategies (other rational derivatives such as Br and Xe and co-crystallization with metals (Rb, Zn, Hg, lanthanides)). Our results show that an anomalous diffraction signal from a single SeMet residue is usually sufficient for structure determination. The MCSG pipeline allows a structure to be solved from one wavelength during data collection for a second wavelength.


| Data collection |

A macromolecular crystallographic experiment is a compromise between the best possible protocol and the various limitations encountered at the beamline. Advances in hardware as well as in data integration and phasing software have resulted in rapid evolution of experimental protocols. The ability to analyze and back up a fast stream of data produced by a multi-module CCD is now the rate-limiting step that must be improved. At Structural Biology Center beamlines the HKL3000, the advanced crystallographic software, is integrated with the beamline-control system using an SBCcollect server. Combinations of features, including exceptional beamline optics, high-speed CCD detector, and HKL3000 data analysis can accommodate the most demanding protein crystallography experiments, such as collecting diffraction from weakly scattering crystals and from crystals with very long unit cells.

The Graphical Command Center (GCC) of HKL3000 organizes data collection and forwards information for display, indexing, strategy considerations, simulation, refinement, integration, scaling, and phasing, merging these tasks into a continuous data collection process. A Gigabit internet with higher bandwidth will allow the web to be used for remote control of synchrotron experiments.

PowerPoint Presentation PDF

The highly integrated HKL3000 system allows cross-talk with other programs and subsequent off-line data analysis. The goniostat/detector-control software plays a key role in the HKL3000/SBC system, integrating a high-flux beam, ultra-fast CCD detector, and goniostat and data processing/analysis software. This control system is changing protein crystallography experiments from a series of single-task experiments linked together manually, to a single, multi-step, integrated and streamlined process. The final product, a high-quality electron density map and an initial structural model, is obtained faster, more efficiently, and at a lower cost.


| Toward complete automation of protein structure determination |

The MCSG has developed semi-automated approaches to structure determination that is implemented in HKL3000 package. Our goal is to provide the experimenter with near-real-time feedback by integrating the x-ray diffraction experiment with a rapid evaluation of data quality and structure solution. This approach has already improved the success rate of x-ray diffraction experiments at the synchrotron beamlines and significantly increased the efficiency of structure determination. We have built a system to analyze data and carry out the various computational steps needed for crystallographic structure determination using alternative data-processing paths. The HKL3000 integrates data collection, reduction, phasing, and model building — significantly accelerating structure determination and reducing the number of data sets required for a single structure determination. This system makes parallel attempts to solve the structure using different algorithms and exploring different approaches and is combined with relational databases and linked to external web resources. This HKL3000 converts diffraction data to an interpretable electron density map, and for smaller structures, into an initial model. Automated model building is done using the SOLVE/RESOLVE and ARP/wARP program suites. The system is interfaced with MCSG’s SGPDB and with PDB, Swissprot, and other generally available databases. The current version of our semi-automatic system has solved over 300 new structures.


| Beamline & End Station Schematic |



| Detectors & Gonistat |

19 BM
19 ID


SBC-3 CCD X-ray Detector
  1. 3 x 3 mosaic CCD detector
  2. Active area: 210 x 210 mm2
  3. Pixel sizes: 0.079 mm (unbinned mode)
    and 0.159 mm (2 x 2 binned mode)
  4. Unbinned images: 3072 pixels x 3072 pixels
    (18.5 MB)
  5. 2 x 2 Binned images: 1536 pixels x 1536
    pixels (4.6 MB)
  6. Specifications - coming soon
Fluorescence Detectors
    1. Model XR-100CR
    2. 3 K cps for linear detection
    3. Energy resolution ~186 eV resolution
      at 5.9 keV
    1. 30-40 K cps for linear detection
    2. Energy resolution 55% PHR at 5.9 keV
Kappa Goniostat
  1. Omega, Kappa, and Phi rotations
  2. Motorized X, Y, and Z translation



ADSC Quantum 315
  1. 3 x 3 mosaic CCD detector
  2. Active area: 315 x 315 mm2
  3. Pixel sizes: 0.051 mm (unbinned mode)
    and 0.102 mm (2 x 2 binned mode)
  4. Unbinned images: 6144 pixels x 6144
    pixels (75.6 MB)
  5. 2 x 2 Binned images: 3072 pixels x 3072 pixels
    (18.9 MB)
  6. Specifications - coming soon
Fluorescence Detectors
    1. Model XR-100CR
    2. 3 K cps for linear detection
    3. Energy resolution ~186 eV at 5.9 keV
    1. 30-40 K cps for linear detection
    2. Energy resolution 55% PHR at 5.9 keV
Kappa Goniostat
  1. Omega, Kappa, and Phi rotations
  2. Motorized Z translation


| Limits |

Limits for 19-BM



  • Photon Energy (keV)


lower limit


upper limit


  • Energy Bandwidth


ΔE/E @ 13.5 keV

2.5 x 10-4

  • Minimum Focus Achieved (mm)






  • Maximum Accepted Divergence from Source (mrad)


vertical @ 12.0 keV


horizontal @ 12.0 keV


  • Beam Position Stability at Focus (mm)

< 0.010

Limits for 19-ID



  • Photon Energy (keV)


lower limit


upper limit (Si 111 crystal)


  • Energy Bandwidth (eV)


nonresonant crystallography @ 10.4 keV


resonant @ 6.5 keV


resonant @ 20.0 keV


  • Harmonic Contamination (%)


  • Minimum Focus Achieved (mm)






  • Maximum Accepted Divergence from Source (mrad)


vertical @ 12.0 keV


horizontal @ 12.0 keV


  • Beam Position Stability at Focus (mm)

< 0.010

  • Maximum Specimen to Detector Distance with A-frame (mm)



| Sample Mounting |

The Kappa goniostat phi Z translation allows the crystal height to be adjusted by changing the position of the Kappa goniostat crystal mounting pins may be mounted directly onto the magnet from a cryo-vial. (NO cryo-tongs are required). The Kappa goniostat has a x-y translation stage for the alignment of crystals (It does not accept the standard gomiometer head)

Cryo-pins we currently recomend for mounting on the Kappa head using magnetic mounts may be a maximum of 24.5 mm, magnetic base to protein crystal, inclusive.


Other sizes or styles or heights may be able to be accommodated, including

  1. Yale style
  2. Harvard style
  3. Hampton style tall
  4. Hampton style short
  5. 1/8 brass pin


| Biochemistry Laboratory |

Laboratory space is available for the use of SBC Users.  Access is gained via the User Badge and a key-card reader mounted at one of the laboratory doors. Please note that only BioSafety Level One (BSL-1) experiments will typically be approved. A few projects requiring BSL-2 facilities may be allowed at SBC beamlines, pending further review. Projects requiring either BSL-3 or BSL-4 facilities cannot be conducted at the SBC facilities. Information regarding these classification definitions may be found at (link): Biosafety in Microbiological and Biomedical Laboratories.

A small workbench area will be available for use during data collection time. Due to the presence of other scheduled Users, access to the laboratory may only be available immediately before and after the assigned data collection time.

A walk-in, +5° C cold room is available. One biochemistry workbench area is available in the cold room, and is shared by all 19ID and 19BM Users.

One fume hood is present. Work involving either freezing of crystals in liquid-propane or heavy-atom compounds must be performed in this fume hood.

Microscopes are available for viewing crystals.  Two microscopes are available for 19ID; one in the biochemistry laboratory, one inside the beamline experimental hutch. Two additional, analogous microscopes are available for beamline 19BM. A fifth microscope is available in the cold room (+5° C). Preferential access to these microscopes is given to User groups actively collecting data at the beamlines. All microscopes are locked down in position, and may not be moved.

Limited laboratory equipment is available for general use by all SBC Users. Although SBC staff try to maintain equipment in good working order, please be aware that heavy use may lead to repairs and unavailability of the equipment during your assigned beamtime. As always, if you feel the success of your experiment will depend upon the availability of consumable items, please bring your own supplies with you from your home institution.


Equipment presently available:
Consumables that may be available, depending upon funding and time constraints: Equipment that is NOT available:

Dewars; for freezing crystals and transferring frozen crystals

Racks, tweezers, and canes; for assisting
in freezing crystals and transferring frozen crystals

Hampton Research supplies
- of any sort!

Analytical Balance; 0.0 – 10.0 g range

Disposable tips; 10µl, 200µl, 1000µl

Loops, pins, crystal-mounting tools

Pan Balance; 0.0 – 200.0 g range

Glass microscope slides


Microfuge; +5° C; single speed, approximately 10,000 rpm; NOT suitable for concentration-type membranes

Microfuge tubes; 0.50ml, 1.50ml

Bunsen burners; or any other source of open flame

Vortex mixer; +5 ° C

pH paper; few strips

Aspirators; or any other source of biochemistry laboratory vacuum

pH meter

Magnetic stirrer bars; few sizes


Hot plate; with stirrer function

Aluminum foil


Stirrer plate; no hot-plate function

Plastic wrap


Milli-Q water; limited quantities

Ethanol; 95%


Ice machine; access to APS
supplied ice (+0 ° C)



Paper tape





| Cryocrystallography Equipment |

The sector 19 beamlines use an Oxford Cryosystem or Oxford Instruments Cryojet for crystal cooling. It is routinely operated at 100K but can be set to a lower value if required; this gives a temperature of approximately 105-110K at the crystal. We also have available for backup a MSC cryosystem and a Siemans LT2.

Cryo-equipment available for users:

  1. Dewars and racks to assist is the holding of vials and mounting of crystals
  2. Forcepts to hold cryo vials
  3. Storage dewars for the long term storage of crystals (upon special request)


| Crystallography Software |

A number of common crystallographic software packages are available at the Structure Biology Center.

Most programs are located in /net/prog1.  At any Linux computer (at the prompt) type:  ls  /net/prog1

Command scripts are in your path for the following programs:


For: Type at the prompt:
(case sensitive)
  Some useful web sites :

GUI interfaces










  Uppsala Software Factory    

HKL2000 guide
















xfit or xtalmgr


command line suites**




  X-ray Anomalous Scattering









  European Bioinformatics Institute




some USF programs




















| Computing Resources |

SBC maintains a network of high end, dual processor Linux workstations that are used for beamline control, data acquisition, data processing and structure determination.  These resources are distributed into two beamline control areas (one for 19ID and one for 19BM) and four data processing work areas (two on 19ID and two on 19BM).

In each beamline control area SBC also maintains a desktop PC running Windows XP Professional.  These systems are used primarily as an option for performing data backup to Windows compatible firewire disks.

A high speed Network Attached Storage device provides over 4 Terabytes of storage per beamline.



 Selected related publications:


Borek D, Ginell SL, Cymborowski M, Minor W, Otwinowski Z.
The many faces of radiation-induced changes. J Synchrotron Radiat. 2007 Jan;14(Pt 1):24-33.


Borek D, Minor W, Otwinowski Z (2003)
Measurement errors and their consequences in protein crystallography.
Acta Crystallogr D Biol Crystallogr, 59, 2031-8 Times cited: 7. [PubMed]


Korolev S, Dementieva I, Sanishvili R, Minor W, Otwinowski Z, Joachimiak A (2001)
Using surface-bound rubidium ions for protein phasing.
Acta Crystallogr D Biol Crystallogr, 57, 1008-12 Times cited: 11. [PubMed]

Minor W, Cymborowski M, Otwinowski Z, Chruszcz M (2006)
HKL-3000: the integration of data reduction and structure solution--from diffraction images to an initial model in minutes.
Acta Crystallogr D Biol Crystallogr, 62, 859-66 Times cited: 13. [PubMed]


Minor W, Tomchick D, Otwinowski Z (2000)
Strategies for macromolecular synchrotron crystallography.
Structure Fold Des, 8, R105-10 Times cited: 26. [PubMed]


Otwinowski Z, Borek D, Majewski W, Minor W (2003)
Multiparametric scaling of diffraction intensities.
Acta Crystallogr A, 59, 228-34 Times cited: 82. [PubMed]

Rosenbaum G, Alkire RW, Evans G, Rotella FJ, Lazarski K, Zhang RG, Ginell SL,
Duke N, Naday I, Lazarz J, Molitsky MJ, Keefe L, Gonczy J, Rock L, Sanishvili R,
Walsh MA, Westbrook E, Joachimiak A. The Structural Biology Center 19ID undulator beamline:
facility specifications and protein crystallographic results. J Synchrotron Radiat. 2006 Jan;13(Pt 1):30-45.


Walsh MA, Dementieva I, Evans G, Sanishvili R, Joachimiak A.
Taking MAD to the extreme: ultrafast protein structure determination.
Acta Crystallogr D Biol Crystallogr. 1999 Jun;55(Pt 6):1168-73.


Walsh MA, Evans G, Sanishvili R, Dementieva I, Joachimiak A. MAD data collection - current trends.
Acta Crystallogr D Biol Crystallogr. 1999 Oct;55(Pt 10):1726-32.

Walsh MA, Otwinowski Z, Perrakis A, Anderson PM, Joachimiak A (2000)
Structure of cyanase reveals that a novel dimeric and decameric arrangement
of subunits is required for formation of the enzyme active site.
Structure, 8, 505-14 Times cited: 19. [PubMed] [PDB] [PDB]


For a more exhaustive list of publications see the MCSG publications website.