1.3 Mechanism and structure of DNA polymerases
1.3.2 Two metal ion mechanism
The condensation reaction of 2’-deoxynucleoside triphosphate with 3’ end of the primer of the primer/template complex in the active center requires two divalent metal ions. For the accurate synthesis in the case of DNA polymerase I, two magnesium ions are necessary, which are coor-dinated by aspartates. In the error prone PCR, the magnesium ions are replaced with manganese ions, which leads to lower specificity and more mismatches. The proposed mechanism of nu-cleotide addition[104] is in analogy to the reverse nearly identical mechanism of the 3’-5’ ex-onuclease of DNA polymerase I.[105,106]One magnesium ion accompanies the incoming dNTP.
This magnesium ion is coordinated by the phosphates of the nucleotide and two conserved as-partates.[107] The second magnesium ion is required to condensate the incoming nucleotide to the 3’-OH end of the primer by nucleophilic substitution under release of pyrophoshate. In detail, the first metal ion (Fig. 1.8[1]) lowers the affinity of the 3’-OH for the hydrogen, facili-tating the 3’-O attack on theα-phosphate of the incoming dNTP. Both metal ions stabilize the structure and charge of the expected pentacovalent transition state and afterwards, the second metal ion (Fig. 1.8[2]) assists the leaving of the pyrophosphate.[107,108]A third metal ion (Fig.
1.8[3]) which has not been observed until now, possibly may bind to theβandγ phosphate.[104]
Figure 1.8: Hypothetical chemical mechanism for the polymerase reaction based on two metal ion catalysis analogous to that proposed for the 3’-5’ exonuclease reaction from Beese and Steitz.[105]It is proposed that the role of the catalytically essential carboxylates (Asp822, Glu883 and Asp705 in KF) is to bind two divalent metal ions as observed at the polymerase active site. One Mg2+[1] is hypothesized to promote the deprotonation of the 3’-OH of the primer strand analogous to the role of metal A[105,106]in the exonuclease active site. The other Mg2+[2]
could stabilize the formation of the pentacovalent transition state at theα-phosphate by facilitating the formation of a 90◦O-P-O bond angle. It could also facilitate the leaving of the pyrophosphate. A third Mg2+[3] may bind to theβandγphosphates (adapted from Steitz, 1993[104]).
by X-ray analysis.[109–111]The DNA polymerase I consists of 928 amino acids with 3 functional subunits. The N-terminal region (amino acids 1-324) includes the 5’-3’ exonuclease domain, the amino acids 324-517 build the middle region and include the 3’-5’ exonuclease domain, and the C-terminal region (amino acid 521-928) includes the DNA polymerase function. Those three domains are relatively independent from each other with regard to their tertiary structure and their enzymatic activities.[112,113] E. coliDNA polymerase I was digested by Klenowet. al with subtilisin in two fragments, the 35 kDa and 68 kDa fragment.[114] The 68 kDa fragment, also known after his discoverer as the Klenow fragment (KF), has DNA polymerase function and 3’-5’ exonuclease activity. Some examples for the family A repair enzymes are the Ther-mus aquaticusDNA polymerase I (Taq polymerase I), the Escherichia coli polymerase I (E.
colipolymerase I) and theBacillus stearothermophiluspolymerase I. Most of the polymerase I enzymes contain 5’-3’ exonuclease activity and 3’-5’ proofreading activity, while only the 5’-3’
exonuclease is required for viability, because it is necessary for the removal of RNA primers from Okazaki fragments generated during replicative DNA synthesis (see Chapter 1.2.1). The DNA polymerase activity is used to fill in the resulting gap. During repair, polymerase I en-zymes also fill in DNA gaps that result from the removal of a variety of DNA lesions.[40]
1.4.1 Taq DNA polymerase and the KlenTaq
The Taq DNA polymerase belongs to the repair enzymes of the family A polymerases.[80] It is involved in nucleotide excision repair and in processing Okazaki fragments which are gen-erated during lagging strand synthesis.[115] The Taq DNA polymerase is descended from the thermophilic organismThermus aquaticusand has, therefore, the specific characteristic of ther-mostability. The half-life ofTaqDNA polymerase is 9 minutes at97.5◦C. Due to this intrinsic property, theTaqDNA polymerase is used in the laboratory for the polymerase chain reaction (PCR).[45] It is a monomeric enzyme with 832 amino acids and with a molecular weight of 94 kDa.[116] Through subtilisin digestion, as mentioned in chapter 1.4, the 68 kDa Klenow frag-ment can be generated, known asKlenTaq. Several structural X-ray analyses ofKlenTaqshow, that the tertiary structure (Fig. 1.9), especially the polymerase domain, resembles the E. coli
Figure 1.9: Ribbon diagram of the open binary protein-p/t (A) and closed ternary protein-p/t-ddNTP (B) complex of Klenow fragment from TaqDNA polymerase I (Klentaq1). The structure can be compared with an open right hand viewed on thepalm(magenta) with thefingers (green) and thethumb(blue) subdomain, as well as the bound primer (gray) and the template (turquoise). The N-terminal domain starts in the yellow subdomain, and the O helix is shown in red (2KTQ and 3KTQ, adapted from Liet. al., 1998[124]).
DNA polymerase I.[117–120] All polymerases have a common architectural framework consist-ing of three canonical subdomains termed thefingers,palmandthumbsubdomains as shown in Figure 1.9. The substrate for theTaqDNA polymerase is either a single stranded DNA (ssDNA) or a dsDNA. The catalytic activity and enzymatic capacity is determined by the pH-value, the KCl concentration as well as the concentration and kind of the essential divalent cations. The preferences for the essential divalent cations (in decreasing order) are: Mg2+, Mn2+, Co2+and Ni2+. TheTaqDNA polymerase exhibits a higher specific activity (units/mg) and a higher KM
value for dNTPs than the E. coli DNA polymerase I.[54,121–123] The mesophilic E. coli DNA polymerase I has the optimal reaction temperature between 22-37◦Cand the thermophilicTaq DNA polymerase at 65-75◦C.
1.4.2 Motif C from the KlenTaq
The accurateness of incorporation of the canonical nucleotides over noncanonical ones is im-portant for biotechnological applications and depends on the intrinsic selectivity of the DNA polymerase. The crucial criteria for DNA polymerase selectivity is widely believed to be the size and shape of the active site.[72]It is believed, that high-fidelity DNA polymerases edit the
the nucleobase through the minor groove. Size augmentation by chemical modification of the 2’-deoxyribose of the 3’-terminal nucleotide results in a significantly decreased primer exten-sion by family A DNA polymerases, which indicates that there are close contacts between the enzyme and the DNA substrate in this motif. Further, it was found that mutations in motif C can affect DNA polymerase mismatch- extension efficiency.[127,128]Randomization of the three amino acids in motif C from the wildtype Gln782-Val783-His784 (QVH) led to active mutants with an increased mismatch-extension selectivity. This increased mismatch-extension selectiv-ity was caused by a hydrophobic substitution from QVH to Lys782-Val783-Lys784 (LVL).[128]
This is consistent with the replacement of the polar His to Ala, which results in the same ef-fect.[127]
Figure 1.10: A: Structure of the Klenow fragment fromTaqDNA polymerase I complexed with p/t and ddNTP in the closed conformation (PDB ID:3KTQ). Highlighted in black are six motifs that are conserved within family A DNA polymerases. B: Detailed view of motif C. The last nucleotide pair of the 3’-primer terminus and the incoming dNTP pairing to its cognate nucleobase in the template strand are shown (drawn by myself using 3KTQ and PyMOL[129]and published in Strerath et al.[ 125 ]).
Selection of theKlenTaqby screening a library of arbitrarily randomized mutants for high activ-ity in combination with lesion bypass activactiv-ity leads to a mutant with a single cationic exchange in position 747 from the hydrophobic methionine in the wild-type to the cationic lysine in the M747K mutation.[130]To gain further insights into the mechanistic origins, the structure of the mutant was determined by X-ray crystallography. Based on the crystallization protocol of the wild-type (PDB ID:1KTQ) from Korolevet al.[120], crystallization conditions were tested. After initial difficulties, crystals of the open form ofKlenTaq M747K were obtained without DNA and could be analyzed by X-ray diffraction. Several diffracting crystals of the open form were tested on the ESRF in Grenoble, France and on the SLS in Villigen, Switzerland. Two inde-pendent crystallization conditions resulted in well diffracting crystals. The first condition was similar to the Tris HCl buffered condition of the open wild-type (PDB ID:1KTQ) with a pH be-tween pH 9.3 and pH 9.6 and a second condition was similar to the HEPES buffered condition of the closed tertiary wild-type structure (PDB ID:3KTQ) with a pH between pH 7.3 and pH 7.8 (Fig. 2.4 and Fig. 2.6). The best protein crystal was obtained with the Tris HCl condition and diffracted to 3.0 ˚A in the monoclinic space group P21 with unit cell parameters of a= 94.0 A, b= 84.8 ˚˚ A, c= 258.8 ˚A,α=γ= 90.0 ˚ ,β= 90.1 ˚ . The structure was solved by Molecular Re-placement using the wild-type protein structure as target (PDB ID:1KTQ) and refined to Rf ac= 23.9; Rf ree= 27.6. Analysis of the refined structure shows that Nz from the lysine side chain of the single mutation (M747K) in the open form is relatively flexible. This is indicated by the low density around the Nz end of the lysine 747 side chain compared to the clear density of the main chain of this residue.
2.2 Introduction
KlenTaq M747K was discovered by C. Gloecker[130] by screening a library of arbitrarily ran-domized mutants. He found one out of 736 PCR active variants of KlenTaq which was able to bypass DNA lesions significantly more efficiently than the wild-type enzyme[59](Fig. 2.1).
ThisKlenTaqcontains a single hydrophilic substitution from M to K at position 747. Moreover, KlenTaqM747K shows lesion bypass activity on stabilized abasic sites (Fig. 2.1A), 8-oxoA and 8-oxoG damaged templates (Fig. 2.1B) while the wild-type stops at those DNA damages. From
Figure 2.1: DNA synthesis with lesion bypass ofKlenTaqM747K. A) Experiments with the stabilized abasic site (AP). 5’-32P-labeled primer-template complexes were incubated with equally high amounts ofKlenTaqwild-type (WT) andKlenTaqM747K (M747K), respectively and all four dNTPs. P: primer alone; A: template containing a dA residue at position marked with an X; AP: template containing a lesion at the position marked with an X. B) Experiments with oxidized purines. Reactions were conducted as described for (A) with the exception that oxidized lesions were introduced at position X (adapted from Gloeckneret al.[59]).
structures of the wild-type (3KTQ) it is known that amino acid M747 is near the template DNA and is in contact with the 2‘-deoxyribose of the template of the previously incorporated base pair. Interestingly, the discovery of this mutation was not unique. There was another group, which found a substitution at this position in combination with a second substitution, namely E742K and M747K which is able to use RNA instead of DNA as template.[131] Steady state measurements from C. Gloecker[130] showed, thatKlenTaqM747K has a slightly lower selec-tivity for extension of mismatches compared with the wild-type. The relative efficiency for the extension of mismatch primers (dC/dT) and (dT/dT) is twice that for wild-type. Opposite a
Figure 2.2: Generation of UV-damaged DNA and PCR amplification. A) Irradiation of double-stranded plasmid DNA with UV-light in varying doses. B) PCR amplification of an approximately 1.8-kb fragment using the UV-damaged DNA as the template. For all reactions identical conditions were employed in PCR. Left lane: DNA marker; right lane: PCR performed in the absence of target DNA. Analysis was performed by agarose gel electrophoresis and ethidium bromide staining (adapted from Gloeckneret al.[59]).
where lesion bypass activity is reported for DNA polymerases, it is associated with relatively low specific activity and selectivity in processing non-damaged templates. However,KlenTaq M747K shows high activity and selectivity in combination with lesion bypass activity.[130]Even the error rate for incorporation of the noncanonical nucleotide is of the same magnitude for the mutant compared with the wild-type (8.8x10−5 for the wild-type and 11.5x10−5 for the Klen-TaqM747K). In both cases it is typical for theTaqpolymerases that there are more transitions (purine-purine exchange [A<->G] or pyrimidine-pyrimidine exchange [C<->T]) than transver-sions (purine-pyrimidine, or pyrimidine-purine).[132,133] Moreover, this mutant is able to use DNA that was damaged by UV-light as a target for PCR amplification contrary to the wild-type which is not able to proceed over such damages (Fig. 2.2A and B).[130] UV-light damage pro-duces several different DNA damages (as mentioned in chapter 1.1.2). Most of these damages are pyrimidine dimers and bulky adducts. Those compounds normally block DNA replication
and are removed by nucleotide excision repair.
Recently, DNA polymerases were discovered which are able to make trans-lesion synthesis (TLS). Those TLS polymerases are grouped in the Y-family polymerases.[134–139] This makes them useful for PCR amplifications for damaged DNA and forensic applications. To understand how this single mutation interacts in theKlenTaqM747K and leads to the discussed differences in substrate tolerance and influence on the dNTP incorporation, the atomic structure of the mutant was determined by X-ray crystallography.
2.3 Material and Methods
2.3.1 Bacterial strains
TheE. coliBL21-Gold(DE3) was used for heterologous overproduction of theKlenTaqM747K.
This expression system, harboring the KlenTaq M747K sequence in the pASK37plus vector, purchased from IBA was taken over from C. Gloeckner.[130] Between the N-terminal His6 -tag and the protein-coding sequence is a Factor Xa cleavage site in the pASK37plus. The vector contains a tet promoter inducible with anhydrotetracycline for cytoplasmic expression.
A second expression system was used inE. coliBL21-Gold(DE3) with the plasmid pGDR11 (a pQE31 (Qiagen) derivative harboring thelacIq gene[140] obtained from J. Nesper) with a N-terminal His6-tag. Expression of the pGDR11 is under T5- promoter control of the phage T5 and regulated through the lac promoter.
2.3.2 Expression and Purification
Heterologous expression inE. coliBL21-Gold(DE3) was done in 10 x 0.8 l scale Luria Bertani (LB) medium (10 g/l tryptone, 5 g/l yeast extract, 10 g/l NaCl, pH 7.0) with 100 µg/ml car-benicillin for pASK37plus or with 100 µg/ml ampicillin for the pGDR11. The medium was inoculated with 1:100 (v/v) overnight culture and cells were grown at 37◦Cin a shaking in-cubator at 220 rpm. After cell growth reached the mid-exponential phase (OD600= 0.5-0.8), protein expression was induced with anhydrotetracyclin (AHT) in the pASK37plus to an end-concentration of 200µM and with 1 mM IPTG in the pGDR11. After 4 h expression, the cells were harvested by centrifugation at 5000 x g for 10 min. Subsequently, pelleted cells were lysated for 30 min at 35◦C and 250 rpm using 30 ml lysisbuffer (10 mM Tris HCl pH 9.0, 300 mM NaCl, 2.5 mM MgCl2, 0.1 % Triton X100, 1 mM Benzamidin, 1 mM PMSF, 2 mg/ml lysozyme) per l cell culture. The lysate was heat denatured at 75◦Cfor 50min to precipitate heat labile enzymes. After cooling down to room temperature, the solution was ultracentrifuged at 50 000 x g for 1 h. The supernatant was filtered through a sterile filter (cut off size: 50µm)
Figure 2.3: SDS-PAGE of expression and Ni-purification ofKlenTaqM747K. Arrow indicates position of the purified protein. M: Marker M12;
1: before induction; 2: 1 h expression; 3: 3 h expression; 4: before induction; 5: 1 h expression; 6: 3 h expression; 7: after heat denaturation;
8: Supernatant of Ni-slurry 9: first Ni-slurry washing step; 10: second Ni-slurry washing step; 11: elution with 0.5 M imidazole.
from the Ni affinity purification was concentrated to 2 ml and applied to a pre-equilibrated (50 mM Tris HCl, 20 mM MgCl2, pH 9.0) gel filtration Superdex 200 (160 ml, GE Healthcare) to remove the imidazole and change the buffer. Pure protein elutes after 70 ml and the protein was concentrated to 7 mg/ml using centrifuge filtration devices (Vivaspin 50 kDa, Vivascience).
Expression could be optimized using vector pGDR11 (Chapter 9.3.2) instead of using the lower yield vector pASK37plus. In detail, out of 8 l cell culture, approximately 12 mg protein could be achieved with pGDR11 with the same purity as the 3 mg yield from pASK37plus.
2.3.3 Crystallization and data collection
Crystallization trials were carried out. Therefore, the protein was concentrated to 7 mg/ml and 20 mg/ml and crystallization screens were prepared using rational design based on the condition of the open 1KTQ[120] form. A screen was prepared using the published condition (100 mM Tris HCl, pH 9.0, 50 mM MgCl2, 6 % (w/v) PEG 3350) by varying pH from pH 8.2 to pH 9.5 (Fig. 2.4). First crystals were achieved with a protein concentration of 20 mg/ml in the hanging drop method using 100 mM Tris HCl, pH 9.5, 50 mM MgCl2, and 12 % PEG 3350 as reservoir (Fig. 2.4A). In fact, this condition is at the end of the screen so a second refined rational design
screen (Fig. 2.4B) was prepared with extended steps regarding PEG 3350 concentration and with different MgCl2 concentrations. In all conditions of the second screen appeared crystals after one week with a suitable size of 300 x 80 x 10 µm (Fig. 2.5B, C) in a hexagonal plate shape. Additionally, another condition from Li et al. 2001[141] for the tertiary form (0.1 M Hepes, pH 7.5, 20 mM MnCl2, 0.1 M Na-Acetate, 10 % (w/v) PEG 4000) was tested and a screen was prepared (Fig. 2.6A).
1 2 3 4 5 6
pH 8.2 pH 8.5 pH 8.7 pH 9.0 pH 9.3 pH 9.5
100mM Tris 100mM Tris 100mM Tris 100mM Tris 100mM Tris 100mM Tris A 50mM MgCl2 50mM MgCl2 50mM MgCl2 50mM MgCl2 50mM MgCl2 50mM MgCl2
4% PEG3350 4% PEG3350 4% PEG3350 4% PEG3350 4% PEG3350 4% PEG3350 100mM Tris 100mM Tris 100mM Tris 100mM Tris 100mM Tris 100mM Tris B 50mM MgCl2 50mM MgCl2 50mM MgCl2 50mM MgCl2 50mM MgCl2 50mM MgCl2
6% PEG3350 6% PEG3350 6% PEG3350 6% PEG3350 6% PEG3350 6% PEG3350 100mM Tris 100mM Tris 100mM Tris 100mM Tris 100mM Tris 100mM Tris C 50mM MgCl2 50mM MgCl2 50mM MgCl2 50mM MgCl2 50mM MgCl2 50mM MgCl2
9% PEG3350 9% PEG3350 9% PEG3350 9% PEG3350 9% PEG3350 9% PEG3350 100mM Tris 100mM Tris 100mM Tris 100mM Tris 100mM Tris 100mM Tris D 50mM MgCl2 50mM MgCl2 50mM MgCl2 50mM MgCl2 50mM MgCl2 50mM MgCl2
12% PEG3350 12% PEG3350 12% PEG3350 12% PEG3350 12% PEG3350 12% PEG3350
A
1 2 3 4 5 6
pH 9.4 pH 9.4 pH 9.5 pH 9.5 pH 9.6 pH 9.6
100mM Tris 100mM Tris 100mM Tris 100mM Tris 100mM Tris 100mM Tris A 20mM MgCl2 50mM MgCl2 20mM MgCl2 50mM MgCl2 20mM MgCl2 50mM MgCl2
12% PEG3350 10% PEG3350 12% PEG3350 10% PEG3350 12% PEG3350 10% PEG3350 100mM Tris 100mM Tris 100mM Tris 100mM Tris 100mM Tris 100mM Tris B 50mM MgCl2 50mM MgCl2 50mM MgCl2 50mM MgCl2 50mM MgCl2 50mM MgCl2
12% PEG3350 12% PEG3350 12% PEG3350 12% PEG3350 12% PEG3350 12% PEG3350 100mM Tris 100mM Tris 100mM Tris 100mM Tris 100mM Tris 100mM Tris C 75mM MgCl2 50mM MgCl2 75mM MgCl2 50mM MgCl2 75mM MgCl2 50mM MgCl2
12% PEG3350 14% PEG3350 12% PEG3350 14% PEG3350 12% PEG3350 14% PEG3350 100mM Tris 100mM Tris 100mM Tris 100mM Tris 100mM Tris 100mM Tris D 100mM MgCl2 50mM MgCl2 100mM MgCl2 50mM MgCl2 100mM MgCl2 50mM MgCl2
12% PEG3350 16% PEG3350 12% PEG3350 16% PEG3350 12% PEG3350 16% PEG3350
B
Figure 2.4: Example of a rational design crystallization screen: [A] Initial and [B] refined crystal screen fromKlenTaqM747K. Crystals appeared in A in condition D6 and in all conditions in B.
Figure 2.5: Crystals fromKlenTaqM747K (crystal plate 072), A: first hit at 100 mM Tris HCl pH 9.5, 50 mM MgCl2, 12 % PEG 3350, Size:
160µm x 70µm, B: Refinement screen of condition A: Size: 800µm x 300µm, Condition: 100 mM Tris HCl, 20 mM MgCl2, 12 % PEG 3350, pH 9.6. Space group P21, 3.0 ˚A, a=93.1 ˚A, b=84.1 ˚A, c=256.1 ˚A;α=γ= 90.0˚ ,β = 98.8˚ , 6 monomers per ASU C: Different crystals of the refinement screen, cracked on the down left hand side.
To prevent the crystals from secondary radiation damage, cryocrystallography is used and therefore, the crystals were frozen in liquid nitrogen and measured at 100 K. To avoid water crystallization, which would lead to ice rings and damage the crystal lattice, different cryopro-tectans were tested:
Cryoconditions had to be established, where water at 100 K is in vitreous state so that it does not diffract the X-rays, while the protein crystal is still well ordered. This was tested by using a ro-tating anode generator (Schneider, Offenburg, Germany) equipped with a Proteum Pt135detector (Bruker AXS, AG Welte). Sequential soaking of the crystals with reservoir solutions containing increasing glycerol concentration was tried. In the optimized concentration, the concentration was increased in 5% (w/v) steps until 20 % (w/v) glycerol were reached. Different crystals of theKlenTaqM747K were measured either on beamline ID14-4, ID23-2 or ID29-1 on the ESRF in Grenoble, France or the beamline X06SA at the SLS in Villigen, Switzerland. In Figure 2.7, the diffraction pattern of one frame from theKlenTaq M747K is shown, which diffracted to 2.7 ˚A on the beamline ID29-1 ESRF, in Grenoble. Data reduction was done with XDS[142],
1 2 3 4 5 6
pH 7.3 pH 7.4 pH 7.5 pH 7.6 pH 7.7 pH 7.8
0.1M Hepes 0.1M Hepes 0.1M Hepes 0.1M Hepes 0.1M Hepes 0.1M Hepes 0.1M NaAc 0.1M NaAc 0.1M NaAc 0.1M NaAc 0.1M NaAc 0.1M NaAc A 20mM MnCl2 20mM MnCl2 20mM MnCl2 20mM MnCl2 20mM MnCl2 20mM MnCl2
8% PEG4000 8% PEG4000 8% PEG4000 8% PEG4000 8% PEG4000 8% PEG4000 0.1M Hepes 0.1M Hepes 0.1M Hepes 0.1M Hepes 0.1M Hepes 0.1M Hepes 0.1M NaAc 0.1M NaAc 0.1M NaAc 0.1M NaAc 0.1M NaAc 0.1M NaAc B 20mM MnCl2 20mM MnCl2 20mM MnCl2 20mM MnCl2 20mM MnCl2 20mM MnCl2
10% PEG4000 10% PEG4000 10% PEG4000 10% PEG4000 10% PEG4000 10% PEG4000 0.1M Hepes 0.1M Hepes 0.1M Hepes 0.1M Hepes 0.1M Hepes 0.1M Hepes 0.1M NaAc 0.1M NaAc 0.1M NaAc 0.1M NaAc 0.1M NaAc 0.1M NaAc C 20mM MnCl2 20mM MnCl2 20mM MnCl2 20mM MnCl2 20mM MnCl2 20mM MnCl2
12% PEG4000 12% PEG4000 12% PEG4000 12% PEG4000 12% PEG4000 12% PEG4000 0.1M Hepes 0.1M Hepes 0.1M Hepes 0.1M Hepes 0.1M Hepes 0.1M Hepes 0.1M NaAc 0.1M NaAc 0.1M NaAc 0.1M NaAc 0.1M NaAc 0.1M NaAc D 20mM MnCl2 20mM MnCl2 20mM MnCl2 20mM MnCl2 20mM MnCl2 20mM MnCl2
14% PEG4000 14% PEG4000 14% PEG4000 14% PEG4000 14% PEG4000 14% PEG4000
A
B
Figure 2.6: A: Crystal Screen fromKlenTaqM747K, Hepes condition (0.1 M Hepes, 0.1 M Na-Acetate (NaAc), 20 mM MnCl2, 10 % (w/v) PEG 4000, pH 7.3) , B: Crystal ofKlenTaqM747K in Hepes condition from A, condition B1 (0.1M Hepes, 0.1M Na-Acetate, 20mM MnCl2, 10 % (w/v) PEG 4000 pH 7.3), Space group P21, 2.8 ˚A, a= 93.2 ˚A, b= 84.7 ˚A, c= 130.7 ˚A,α=γ= 90.0˚ ,β= 101.5˚ , 3 monomers per ASU.
data analysis using the CCP4i interface.[143]The structure was solved with Molecular
data analysis using the CCP4i interface.[143]The structure was solved with Molecular