6xHis‐tagged (N‐terminal) WT VirB8.  Purification & Crystallization 

 

Purification 

Steps 1 and 2 are performed by lab assistants.  

1. Take a single colony or swab from a monoclonal stock of WT VirB8 in BL21(DE3)star and grow overnight culture on Kanamycin 50 selective LB broth at 37°C, 200 rpm.  2. Sub‐inoculate the culture ~ 1/100 into LB broth supplemented with Kanamycin 50. Grow 1 L of in two 2L flask at 200 rpm, 37°C until they reach an OD600 of 0.6‐0.8 then induce with 0.5 mM IPTG and drop the temperature to 25°C for overnight induction.   3. Harvest the cells at 5000 rpm for 15 min at 4°C. The cells can then be lysed by manual grinding with alumine, 4 X 2 minutes. Lysis buffer, up to 50 mL, is used to rinse the mortar and other equipment. 

4. The cell lysate (50 mL) is then centrifuged at 13000 rpm for 45 min at 4°C. The lysate should then be checked for viscosity before applying to the Nickel affinity column. If the solution is viscous, DNase I treatment (10 mg/ml stock) should be carried out by incubating the cell‐free lysate with DNaseI for 30 min to 1 hr on ice.   

5. During the centrifugation, prepare the His‐tag column: (possibly already done by lab assistants) 

i. remove the stopper and connect the column to the system “drop by drop” to avoid introducing air into the system 

ii. if a new column, remove the snap‐off end at the column outlet iii. Wash out the ethanol with 3‐5 CV of water 

        iv.  Equilibrate with at least 5 CV of binding buffer (recommended flow rate for 5ml      column 5ml/min with maximum pressure of 0.3mPa 

6. Once ready the lysate can be applied to the prepared Ni affinity His‐tag column (GE Healthcare) using sample pump (for best results use flow rate of 0.5‐5ml/min during sample application) 

7. Wash with binding buffer (generally at least 5‐10 CV) until the absorbance reaches a steady baseline or no material remains in the effluent (maintain a flow rate of 5‐10ml/min for 5ml column for washing) 

8. Elute with elution buffer using a linear gradient.  For a linear gradient elution, 10‐20 CVs is usually sufficient.  Maintain flow rate at 5‐10ml/min for elution. 

9. Collect fractions. The 6xHis‐tagged VirB8 should elute in a broad peak.  

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10. The protein can be dialysed versus 20 mM Tris, 200 mM NaCl (pH 7.6) and concentrated to 16‐20 mg/ml. Trays can then be set down for crystallography. 

11. Regenerate the column by washing it with at least 5 CVs of binding buffer.  The column is ready for a new purification of the same protein. 

  Note: The column does not need to be stripped and recharged between each purification   if the same protein is to be purified.  It should be sufficient to strip and recharge it after    approximately two to five purifications. 

 

Crystallization  

1. Fill the crystallization trays' wells according to the following instructions : 

      Salt concentration 

  0.775 M  0.800 M  0.825 M  0.850 M  0.875 M  0.900 M  0.925 M 

1 M Na2HPO4 

20 μL  20 μL  20 μL  20 μL  20 μL  20 μL  20 μL 

2 M K2HPO4 

194 μL  200 μL  206 μL  212 μL  218 μL  224 μL  230 μL 

H2O  286 μL  280 μL  274 μL  268 μL  262 μL  256 μL  250 μL 

Total  500 μL  500 μL  500 μL  500 μL  500μL  500 μL  500 μL 

  

2. Form 4 uL drops on the tray's caps by mixing 2 uL of protein + 2 uL of mother liquor from the well under standard hanging drop methods. 

 

3. Hermetically close the wells with the caps, place the tray in a temperature‐controlled environment (23 °C)  

 

3. VirB8 crystals should grow within 2 days. 

 

Stripping and Recharging the His‐tag column: 

 1. Strip the chromatography media by washing with at least 5 to 10 column volumes of 

stripping buffer. 2. Wash with at least 5 to 10 column volumes of binding buffer. 3. Immediately wash with 5 to 10 column volumes of distilled water. 4. Recharge the water‐washed column by loading 0.5 column volumes of 0.1 M NiSO4 in 

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distilled water onto the column. 5. Wash with 5 column volumes of distilled water, and 5 column volumes of binding buffer 

(to adjust pH) before storage in 20% ethanol. Salts of other metals, chlorides, or sulfates may also be used. 

Note: It is important to wash with binding buffer as the last step to obtain the correct pH before storage. Washing with buffer before applying the metal ion solution may cause unwanted precipitation.     

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6xHis-tagged (N-terminal) WT VirB8. Crystal Mounting & Diffraction

Specialized instrumentation generating X-rays, such as generators or particles accelerators, are used to obtain X-ray diffraction data from a crystal. The crystal is mounted for measurements so that it may be held in the X-ray beam and rotated. A modern approach is to scoop the crystal up in a tiny loop, made of nylon or plastic and attached to a solid rod, which is then flash-frozen with liquid nitrogen. This freezing reduces the radiation damage caused by the X-rays. The loop is then mounted on a goniometer, which allows it to be positioned accurately within the X-rays beam and rotated. Since both the crystal and the beam are often very small, the crystal must be exactly centered within the beam.

Protein Crystallography deals with crystals about 0.1 mm in size and smaller; crystal mounting thus requires very highly coordinated and precise movements and reasonable practice.

Under the microscope

1. Examine the crystallization plate under a microscope. Crystals should be the largest possible, and have a thick, 3-dimensional rod shape.

2. Select the well giving the best looking crystals, and open it by unscrewing the cap. Place the cap, drops up, under the microscope.

3. Using the micro-knife, remove the sticky ''skin'' covering the drops, while at the same time try to keep the crystals from adhering to the skin. Close the cap once done.

4. Fill the aluminum bath with liquid nitrogen and put the pliers holding an empty loop vial into the bath, allowing the vial to fill up with liquid nitrogen. Check on the liquid nitrogen level regularly, it must always cover the vial.

5. Prepare your cryo-protection solutions in drops on a microscope glass plate, according to the following instructions :

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Mother liquor (μL)

Glycerol (μL)

Total Volume (μL)

Final concentration (%)

Solution 1 18 2 20 10

Solution 2 16 4 20 20

Solution 3 13 7 20 35

6. Select a nylon micro-loop with an aperture that matches the size of the crystal. Use a loop that is slightly larger than your crystal, so that the crystal doesn't rest on the edge of the loop.

7. Scoop out a crystal using the loop and plunge it into the first cryo-protection solution. After 5 to 10 seconds, fish out your crystal and put it into the second cryo-protection solution. Repeat for the third cryo-protection solution. The crystal will hence be plunged into drops of progressively higher cryoprotectant concentrations.

8. Scoop out your crystal from the last drop, put the loop on the magnetic wand, and flash-cool your crystal by putting the loop in its liquid nitrogen-filled cap. Use the magnetic wand and leave the loop and cap in the liquid nitrogen bath.

9. Using the pliers, secure the magnetic head of the loop on the goniometer of the X-ray generator. Remove carefully the cap with the pliers, without touching the actual loop containing your crystal.

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On the generator

1. Check the Cryosystem first : the temperature must be 100°K.

2. On the Current board, the High Voltage must be 40 kV and the Current 25 mA. When leaving the generator idle for extended periods of time, adjust the High Voltage to 25 kV and the Current to 5 mA.

3. The computer controls the goniometer as well as the X-ray beam.

BCP : Crystal Centering using the goniostat

a) in Mode, select Master

b) in Tools, select Manual and then Optical

c) open up the Video

d) using the screwdriver and the A&B commands, center the beam on your crystal

e) in Manual Control, select Restore

f) Close

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APEX2 : Data collecting

a) Enter the Login and Password

b) in Open, select Protein

c) in Collect, select Experiment

leave the Hostname blank, and select Connect

d) in Setup :

i. enter the appropriate Filename

ii. enter 120 seconds under Default time

iii. enter 0.5 degrees under Default width

e) in Colones :

i. under Operation, select Phi Scan

ii. under Active, select Yes

iii. under Distance, enter 80

iv. under Theta and Omega, enter 0

v. under Phi, enter 0

.....

Fill in three lines with the exact same parameters, only modifying the Phi value : 0, 45, and 90 degrees.

4. Manually place the detector at the 80 cm mark.

5. Open the X-rays shutter by selecting Shutter 1 Open on the dial box.

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6. Hit Execute.

7. Select Instrument and then Status to monitor the experiment.

Important Note : Use of liquid nitrogen requires certain important safety practices :

1. Always wear your lab coat, long sleeves, long trousers and closed shoes when manipulating liquid nitrogen.

2. Always use eye goggles. 3. Always wear one pair of latex or nitrile gloves on top of a pair of white cold gloves. 4. Never operate in a closed space, as liquid nitrogen vapors can cause asphyxiation by

chasing the oxygen away. 5. Never pour liquid nitrogen in a sink or any canalization, since it can cause freezing and

breaking.

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6xHis-tagged (N-terminal) WT VirB8. Diffraction data processing, Phasing, Refinement & Molecular Visualization

The final quality of X-Ray data is related to the data processing procedure, which includes integration of the crystallographic data and scaling. Between experimental X-ray data and protein structure is the structure solution procedure and phasing. Structure solution can be based on many different techniques, depending on the data available. The next stage of protein crystallography is structure refinement, an iterative process. Finally, protein structure representation with subsequent analysis of all features related with this particular structure is the main goal of all protein crystallography studies.

Diffraction data processing HKL2000 ; Mosflm ; XDS

1. Reflections : when a crystal is mounted and exposed to an intense beam of X-rays, it scatters the X-rays into a pattern of spots or reflections that can be observed and recorded. The relative intensities of these spots provide the information to determine the arrangement of molecules within the crystal in atomic detail.

2. High and low resolution : the peaks at small angles correspond to low-resolution data, whereas those at high angles represent high-resolution data; thus, an upper limit on the eventual resolution of the structure can be determined from the first few images. Some measures of diffraction quality can be determined at this point, such as the mosaicity of the crystal and its overall disorder, as observed in the peak widths.

3. Completeness and Symmetry : one image of spots is insufficient to reconstruct the whole crystal; it represents only a small slice of the full Fourier transform. To collect the complete information, the crystal must be rotated step-by-step through 180°, with an image recorded at every step; however, if the crystal has a higher symmetry, a smaller angular range such as 90° or 45° may be recorded.

4. Indexing : data processing begins with indexing the reflections. This means identifying the dimensions of the unit cell and which image peak corresponds to which position in reciprocal space. A byproduct of indexing is the determination of the symmetry of the crystal.

5. Integration : having assigned symmetry, the data is then integrated. This converts the hundreds of images containing the thousands of reflections into a single file.

6. Merging and Scaling : a full data set may consist of hundreds of separate images taken at different orientations of the crystal. It is essential to merge and scale these various images, that is, to identify which peaks appear in two or more images (merging) and to scale the relative images so that they have a consistent intensity scale (scaling).

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Phasing Experimental : SHARP ; CNS ; SOLVE ; PHASER ...

Molecular Replacement : Amore ; CNS ; Phenix ...

1. The position of each diffraction 'spot' is governed by the size and shape of the unit cell, and the inherent symmetry within the crystal. The intensity of each diffraction 'spot' is recorded, and this intensity is proportional to the square of the structure factor (Fhkl) amplitude. The structure factor is a complex number containing information relating to both the amplitude and phase of a wave. In order to obtain an interpretable electron density map ρ (x, y, z), both amplitude and phase must be known. The phase cannot be directly recorded during a diffraction experiment: this is known as the phase problem. Initial phase estimates can be obtained in a variety of ways, but we will use only Molecular Replacement as part of our experiment.

2. MR tries to find the model which fits best experimental intensities among known structures.

MR relies upon the existence of a previously solved protein structure which is homologous (similar) to our unknown structure from which the diffraction data is derived. As for our protein of interest VirB8, a native structure has already been deposited in the Protein Data Bank.

i. open a web browser and go to

http://www.pdb.org/pdb/explore/explore.do?structureId=2BHM

ii. in Download Files, select PDB file (text), and save the file

iii. in the ssh window, enter kate 2BHM.pdb

iv. remove all the HETATM entries from the pdb file, and close Kate

v. we are going to use the PHENIX program to run our Molecular Replacement. In the terminal window, enter the following command :

>phenix.refine 2BHM.pdb ...mtz strategy=rigid_body refinement.main.number_of_macro_cycles=5 prefix=ref1

vi. after a few seconds, the program abruptly stops and gives the following message :

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vii. recall the last command by using the ↑ arrow, and add the suggested command :

>phenix.refine 2BHM.pdb ...mtz strategy=rigid_body refinement.main.number_of_macro_cycles=5 prefix=ref1 ... --overwrite

Structure Refinement CCP4 ; CNS ; Phenix ; Coot ; O

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Once an electron density map becomes available, atoms may be fitted into the map using computer graphics to give an initial structural model of the protein. The quality of the electron density map and structural model may be improved through iterative structural refinement, but will ultimately be limited by the resolution of the diffraction data.

1. when Phenix has finished, you should see ...

2. to visualize your results :

i. in the command line, enter :

> coot &

ii. in Coot, select File and then Open Coordinates ...

iii. in the pop-up window, click on Filter and select ref1_001.pdb

iv. select File again, and this time click on Auto Open MTZ ...

v. in the pop-up window, click on Filter and select ref1_001_map_coeffs.mtz

vi. select Display Manager, and click on Scroll on the second line of the pop-up window. Using the mouse scroll, adjust the FoFc map to 3.23 sigma level (upper right-hand corner of the window). Repeat for the first line of the pop-up window, adjusting the sigma level of the 2FoFc map to 0.91. Close the window when done.

vii. use the mouse (middle button click on selected atoms or Ctrl+right click) to move about the structure.

viii. close Coot.

3. in the terminal window, enter

> ls

> cd ..

> mkdir ref2

> cd ref2

> cp ../ref1_001.pdb .

> cp ../ref1_001_data.mtz .

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> phenix.refine ref1_001.pdb ref1_001_data.mtz ordered_solvent=true refinement.main.number_of_macro_cycles=5 prefix=ref2

Phenix is now using the ref1_001.pdb structure, resulting from the Molecular Replacement phasing, and refining it against the reflection data contained in the ref1_001_data.mtz file.

4. when Phenix is done, use Coot to visualize your newly refined structure :

i. in the command line, enter

> coot

ii. in Coot, select File and then Open Coordinates ...

iii. in the pop-up window, click on Filter and select ref2_001.pdb

iv. select File again, and this time click on Auto Open MTZ ...

v. in the pop-up window, click on Filter and select ref2_001_map_coeffs.mtz

vi. select Display Manager, and click on Scroll on the second line of the pop-up window. Using the mouse scroll, adjust the FoFc map to 3.23 sigma level. Repeat for the first line of the pop-up window, adjusting the sigma level of the 2FoFc map to 0.91. Close the window when done.

vii. use the mouse (click with the middle button on selected atoms or Ctrl+right click) to move about the structure.

Note : the ordered_solvent command allowed Phenix to search and find water molecules, which are represented as little red crosses in Coot. It is important to remember that any given protein crystal is composed of 40 to 60 % water.

viii. close Coot.

Structural Coordinates & Molecular Visualization O ; Coot ; PyMOL

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Once a crystal structure is determined, the information is communicated in the form of an atomic coordinates file, that can be visualized with several programs. Molecular Visualization is the process of interpreting visual images of molecules. You have already used Coot in order to see and modify your protein structure as part of the refinement process; we are now going to use the PyMOL program to better understand the aim and scope of Structural Biology.

1. to start PyMOL, use the command :

> pymol &

i. the graphic user interface (GUI) is comprised of 2 windows : the PyMOL Viewer :

and the toolbar, called the The PyMOL Molecular Graphics System

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ii. to open a PDB file, you can either enter the command in PyMOL :

PyMOL> load fichier.pdb

or click on File, Open, and select you PDB file

iii. to run the visualization script prepared to help you, enter :

PyMOL> @proteinview.pml

or click on File, Run and select proteinview.pml

iv. take a little time to play with PyMOL. You should realize that a left mouse click allows you to turn the molecule, a right-click to zoom on it, and a middle one to center on an atom.

v. a left-click on an atom will allow you to select a whole residue, which will then be visible in the right hand menu as (sele), and will be highlighted in the sequence.

vi. a right-click on an atom will bring out a pop-up window

vii. to better visualize the molecule's secondary structure, click on H next to ...., and on lines. Then, click on S and on cartoon.

2. we will now learn how to make a movie in PyMOL :

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i. re-initialize the session by entering :

PyMOL> reinitialize

or by selecting File, and Reinitialize

ii. run the visualization script again.

iii.

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